CN118382693A

CN118382693A - Genetically modified cells for allogeneic cell therapy to reduce immediate blood-mediated inflammatory responses

Info

Publication number: CN118382693A
Application number: CN202280067035.XA
Authority: CN
Inventors: S·施雷弗
Original assignee: Sana'a Biotechnology Co
Current assignee: Sana'a Biotechnology Co
Priority date: 2021-08-11
Filing date: 2022-08-11
Publication date: 2024-07-23

Abstract

Provided herein are engineered cells for allogeneic cell therapy that contain one or more modifications, such as genetic modifications. In some embodiments, the engineered cell is a low immunogenicity cell. In some embodiments, the engineered cells comprise reduced CD142 expression and/or are administered in combination with an anticoagulant.

Description

Genetically modified cells for allogeneic cell therapy to reduce immediate blood-mediated inflammatory responses

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application No. 63/232,162 filed on 8-11 of 2021 and U.S. provisional patent application No. 63/353,527 filed on 17 of 6-6 of 2022, the contents of each of which are incorporated herein by reference in their entirety for all purposes.

Reference electronic sequence Listing

The contents of the electronic sequence Listing (18615205240 SEQLIST. Xml; size: 41,567 bytes; and date of creation: 2022, 8, 9) are incorporated herein by reference in their entirety.

Technical Field

In certain aspects, the disclosure relates to engineered cells containing one or more modifications (such as genetic modifications) for use in allogeneic cell therapy. In some embodiments, the engineered cell is a low immunogenicity cell.

Disclosure of Invention

The sensitivity of the recipient to donor alloantigens is a problem faced by clinical transplantation therapies, including cell therapies. For example, the propensity of the immune system of the transplant recipient to reject allogeneic material greatly reduces the potential efficacy of transplant therapies and reduces the possible positive effects associated with such treatments. There remains a need for improved allogeneic cells to treat a variety of conditions and disorders. Thus, there remains a need for new approaches, compositions and methods for generating allogeneic cell-based therapies that avoid detection by the recipient's immune system.

In some aspects, provided herein is an engineered cell comprising a modification that (I) increases expression of one or more tolerogenic factors, (II) decreases expression of CD142, and (iii) decreases expression of one or more MHC class I molecules and/or one or more MHC class II molecules, wherein the increased expression of (I) and the decreased expression of (II) and (iii) are relative to a cell of the same cell type that does not comprise the modification. In some embodiments, the modification in (iii) reduces expression of one or more MHC class I molecules and one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules).

In some embodiments, the one or more tolerogenic factors are selected from the group consisting of: CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDOl, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8 and SERPINB9 and any combination thereof. In some embodiments, the one or more tolerogenic factors are selected from the group consisting of CD47, PD-L1, HLA-E or HLA-G, CCL21, FASL, SERPINB9, CD200, MFGE8, and any combination thereof. In some embodiments, at least one of the one or more tolerogenic factors is CD47. In some embodiments, at least one of the one or more tolerogenic factors is PD-L1. In some embodiments, at least one of the one or more tolerogenic factors is HLA-E. In some embodiments, at least one of the one or more tolerogenic factors is HLA-G.

In some of any of the embodiments, the one or more tolerogenic factors are selected from the group consisting of: CD47; HLA-E; CD24; PD-L1; CD46; CD55; CD59; CR1; a MANF; A20/TNFAIP3; HLA-E and CD47; CD24, CD47, PD-L1, and any combination thereof; HLA-E, CD, CD47, and PD-L1, and any combination thereof; CD46, CD55, CD59, and CR1, and any combination thereof; HLA-E, CD46, CD55, CD59 and CR1 and any combination thereof; HLA-E, CD24, CD47, PDL1, CD46, CD55, CD59, and CR1, and any combination thereof; HLA-E and PDL1; HLA-E, PDL1 and A20/TNFAIP, and any combination thereof; HLA-E, PDL1 and MANF, and any combination thereof; HLA-E, PDL1, A20/TNFAIP and MANF, and any combination thereof; CD47, PD-L1, HLA-E, HLA-G, CCL21, FASL, SERPINB9, CD200, MFGE8, and any combination thereof.

In some of any of the embodiments, the modification is selected from the group consisting of decreasing MHC I and/or MHC II expression; reducing expression of CD 142; increase expression of CD47, and optionally CD24 and PD-L1; and modifications that increase the expression of CD46, CD55, CD59 and CR 1.

In some of any of the embodiments, the modification is selected from the group consisting of reducing expression of an MHC class I molecule; reducing expression of CD 142; reducing TXNIP expression; modifications that increase the expression of PD-L1 and HLA-E, and optionally A20/TNFAIP3 and MANF.

In some of any of the embodiments, the modification is selected from the group consisting of increasing the expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200, and MFGE 8; and modifications that reduce the expression of CD 142.

In some embodiments, the modification is selected from the group consisting of decreasing MHC I and/or MHC II expression; and a modification that increases expression of CD 47.

In some embodiments, any of the above modifications are present in the provided engineered cells along with one or more additional edits that increase or decrease gene expression in the cells. In some embodiments, any one or more further modifications may be modifications ：B2M、TAP I、NLRC5、CIITA、HLa-a、HLA-B、HLA-C、HLA-DP、HLA-DM、HLA-DOA、HLA-DOB、HLA-DQ、HLA-DR、RFX5、RFXANK、RFXAP、NFY-A、NFY-B、NFY-C、CTLA-4、PD-1、IRF1、CD142、MIC-A、MIC-B. that reduce expression (such as disrupting, inactivating, or knocking out expression) in some embodiments, any one or more further modifications may be modifications that reduce expression of a protein involved in oxidative stress or ER stress: TRAC, TRB, CD142, ABO, CD38, PCDH11Y, NLGN Y and/or RHD. In some embodiments, proteins involved in oxidative stress or ER stress include thioredoxin interacting proteins (TXNIP), PKR-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE 1 α), and DJ-1 (PARK 7).

In some aspects, provided herein is an engineered cell comprising a modification that (i) increases the expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD, CD200, and MFGE8, and (ii) decreases the expression of CD142, wherein the increased expression of (i) and the decreased expression of (ii) are relative to a cell of the same cell type that does not comprise the modification. In some embodiments, the engineered cells further comprise one or more modifications that increase expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD55, wherein the increased expression of one or more complement inhibitors is relative to cells of the same cell type that do not comprise the modification. In some embodiments, the modification that increases expression comprises increased surface expression and/or the modification that decreases expression comprises decreased surface expression. In some embodiments, the modification that increases expression of one or more complement inhibitors comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and/or an exogenous polynucleotide encoding CD 55. In some embodiments, the one or more complement inhibitors are CD46 and CD59, optionally wherein the modification comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59. In some embodiments, the one or more complement inhibitors are CD46, CD59, and CD55, optionally wherein the modification comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55.

In some embodiments, any modification herein that increases expression may be one or more modifications that increase the gene activity of an endogenous gene, such as one or more modifications of an endogenous promoter or enhancer of the gene or the introduction of a heterologous promoter. In some cases, the heterologous promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse Mammary Tumor Virus (MMTV) promoter, HIV LTR promoter, moloney virus (moloney virus) promoter, epstein Barr virus (Epstein barr virus) (EBV) promoter, and Rous sarcoma virus (Rous sarcoma virus) (RSV) promoter, and UBC promoter.

In some embodiments, the exogenous polynucleotide encoding CD46 encodes an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO. 3. In some embodiments, the exogenous polynucleotide encoding CD46 encodes the sequence set forth in SEQ ID NO. 3. In some embodiments, the exogenous polynucleotide encoding CD59 encodes an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO. 5 and exhibits complement inhibitory activity. In some embodiments, the exogenous polynucleotide encoding CD59 encodes the sequence set forth in SEQ ID NO. 5. In some embodiments, the exogenous polynucleotide encoding CD55 encodes an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO. 8 and exhibits complement inhibitory activity. In some embodiments, the exogenous polynucleotide encoding CD55 encodes the sequence set forth in SEQ ID NO. 8. In some embodiments, the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, and/or the exogenous polynucleotide encoding CD55 are each operably linked to a promoter.

In some embodiments, the modification that increases expression of CD47 comprises an exogenous polynucleotide encoding a CD47 protein. In some embodiments, the exogenous polynucleotide encoding CD47 encodes an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO. 1 and reduces innate immune killing of the engineered cells. In some embodiments, the exogenous polynucleotide encoding CD47 encodes the sequence set forth in SEQ ID NO. 1. In some embodiments, the exogenous polynucleotide encoding CD47 is operably linked to a promoter.

In some embodiments, the engineered cell comprises a polycistronic transgene comprising two or more exogenous polynucleotides selected from the group consisting of: one or more exogenous polynucleotides encoding one or more tolerogenic factors, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding a CD55 polypeptide. In some embodiments, each polynucleotide is isolated by an IRES or self-cleaving peptide. In some embodiments, each polynucleotide of the polycistronic transgene is operably linked to the same promoter.

In some embodiments, the polycistronic transgene comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59. In some embodiments, the polycistronic transgene comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55. In some embodiments, the polycistronic transgene further comprises an exogenous polynucleotide encoding CD 47. In some embodiments, the polycistronic transgene is a first transgene and the engineered cell comprises a separate transgene comprising a polynucleotide encoding CD 47.

In some embodiments, the engineered cell comprises a transgene comprising a polynucleotide encoding CD 47. In some embodiments, the engineered cell comprises a first transgene and a second transgene, wherein the first transgene and the second transgene each comprise one or more exogenous polynucleotides selected from the group consisting of an exogenous polynucleotide encoding CD47, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding a CD55 polypeptide, and wherein the first transgene and the second transgene are monocistronic or polycistronic transgenes.

In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter for HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter for HIV, moloney virus promoter, epstein Barr Virus (EBV) promoter and Rous Sarcoma Virus (RSV) promoter.

In some embodiments, the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, and/or the exogenous polynucleotide encoding CD55 polypeptide is integrated into the genome of the engineered cell. In some embodiments, the exogenous polynucleotide encoding CD47 is integrated into the genome of the engineered cell. In some embodiments, integration is by non-targeted insertion into the genome of the engineered cell, optionally by introducing an exogenous polynucleotide into the cell using a lentiviral vector. In some embodiments, the integration is by targeted insertion into a target genomic locus of the cell. In some embodiments, the target genomic locus is a B2M locus, a CIITA locus, a CD142 locus, a TRAC locus, or a TRBC locus. In some embodiments, the target genomic locus is selected from the group consisting of: CCR5 locus, CXCR4 locus, PPP1R12C (also known as AAVS 1) locus, albumin locus, SHS231 locus, CLYBL locus and ROSA26 locus. In some embodiments, the exogenous polynucleotide encoding CD47 is integrated into a first target genomic locus, the exogenous polynucleotide encoding CD46 is integrated into a second target genomic locus, and the polynucleotide encoding CD59 is integrated into a third target genomic locus. In some embodiments, the exogenous polynucleotide encoding CD55 is integrated into the fourth target genomic locus. In some embodiments, at least two of the first, second, and third target genomic loci are the same locus. In some embodiments, at least two of the first, second, third, and fourth target genomic loci are the same locus. In some embodiments, the first, second, and third target genomic loci are the same locus. In some embodiments, the first, second, third, and fourth target genomic loci are the same locus. In some embodiments, each of the first, second, and third target genomic loci are different loci. In some embodiments, the first, second, third, and fourth target genomic loci are different loci.

In some embodiments, the modification that reduces expression of CD142 reduces CD142 protein expression. In some embodiments, the modification eliminates CD142 gene activity. In some embodiments, the modification comprises inactivation or disruption of both alleles of the CD142 gene. In some embodiments, the modification comprises inactivation or disruption of all CD142 coding sequences in the cell. In some embodiments, the inactivation or disruption comprises an indel in the CD142 gene. In some embodiments, the modification is a frameshift mutation or deletion of a stretch of contiguous genomic DNA of the CD142 gene. In some embodiments, the CD142 gene is knocked out. In some embodiments, the modification is by nuclease-mediated genome editing. In some embodiments, nuclease-mediated genome editing is by a Zinc Finger Nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas combination targeting the CD142 gene, optionally wherein Cas is selected from Cas9 or Cas12. In some embodiments, nuclease-mediated genome editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site within the CD142 gene, optionally wherein the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising the gRNA and a Cas protein.

In some embodiments, the modification that reduces expression of one or more MHC class I molecules reduces expression of one or more MHC class I molecule proteins. In some embodiments, the modification that reduces expression of one or more MHC class I molecules comprises reduced expression of B2M. In some embodiments, the modification that reduces expression of one or more MHC class I molecules comprises reduced protein expression of B2M. In some embodiments, the modification eliminates B2M gene activity. In some embodiments, the modification comprises inactivation or disruption of both alleles of the B2M gene. In some embodiments, the modification comprises inactivation or disruption of all B2M coding sequences in the cell. In some embodiments, the inactivation or disruption comprises an indel in the B2M gene. In some embodiments, the modification is a frameshift mutation or deletion of a stretch of contiguous genomic DNA of the B2M gene. In some embodiments, the B2M gene is knocked out. In some embodiments, the modification is by nuclease-mediated gene editing. In some embodiments, nuclease-mediated gene editing is by a Zinc Finger Nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas combination targeting the B2M gene, optionally wherein Cas is selected from Cas9 or Cas12.

In some embodiments, nuclease-mediated gene editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site within the B2M gene. In some embodiments, the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising a gRNA and a Cas protein.

In some embodiments, the modification that reduces expression of one or more MHC class II molecules reduces expression of one or more MHC class II molecule proteins. In some embodiments, the modification that reduces expression of one or more MHC class II molecules comprises reduced expression of CIITA. In some embodiments, the modification that reduces expression of one or more MHC class II molecules comprises reduced protein expression of CIITA. In some embodiments, the modification eliminates CIITA. In some embodiments, the modification comprises inactivation or disruption of both alleles of the CIITA gene. In some embodiments, the modification comprises inactivation or disruption of all CIITA coding sequences in the cell. In some embodiments, the inactivation or disruption comprises an indel in the CIITA gene. In some embodiments, the indels are frameshift mutations or deletions of a stretch of contiguous genomic DNA of the CIITA gene. In some embodiments, the CIITA gene is knocked out.

In some embodiments, the engineered cell is a human cell or an animal cell. In some embodiments, the engineered cell is a human cell. In some embodiments, the engineered cell is a pig (pig/gardine) cell, a cow (cow/bovine) cell, or a sheep (shaep/ovine) cell. In some embodiments, the cell is a blood-exposed cell type or a cell type capable of differentiating into a blood-exposed cell type.

In some embodiments, the engineered cell is a differentiated cell derived from a pluripotent stem cell or progeny thereof. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells.

In some embodiments, the engineered cell is a primary cell isolated from a donor subject. In some embodiments, the donor subject is healthy or not suspected of having a disease or disorder at the time the donor sample is obtained from the individual donor.

In some embodiments, the engineered cells are selected from the group consisting of beta islet cells, B cells, T cells, NK cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, glial progenitor cells, nerve cells, cardiac cells, and blood cells (e.g., plasma cells or platelets). In some embodiments, the engineered cell is an endothelial cell. In some embodiments, the engineered cell is an epithelial cell. In some embodiments, the engineered cell is a T cell. In some embodiments, the engineered cell is an NK cell. In some embodiments, the engineered cell comprises a Chimeric Antigen Receptor (CAR). In some embodiments, the engineered cell is a beta islet cell. In some embodiments, the engineered cell is a hepatocyte. In some embodiments, the engineered cell is a pluripotent stem cell. In some embodiments, the engineered cell is an induced pluripotent stem cell. In some embodiments, the engineered cell is an embryonic stem cell. In some embodiments, the cell is ABO blood group O. In some embodiments, the cell is rhesus factor negative (Rh-). In some embodiments, the cell comprises a functional ABO a allele and/or a functional ABO B allele. In some embodiments, the cell is rhesus factor positive (rh+).

In some aspects, provided herein is a method of generating an engineered cell, the method comprising: a. reducing or eliminating expression of one or more MHC class I molecules and/or one or more MHC class II molecules in a cell; b. reducing expression of CD142 in the cell; increasing expression of tolerogenic factors in cells.

In some embodiments, the one or more tolerogenic factors are selected from the group consisting of: CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDOl, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8 and SERPINB9 and any combination thereof.

In some embodiments, the one or more tolerogenic factors are selected from the group consisting of CD47, PD-L1, HLA-E or HLA-G, CCL21, FASL, SERPINB9, CD200, MFGE8, and any combination thereof. In some embodiments, at least one of the one or more tolerogenic factors is CD47. In some embodiments, at least one of the one or more tolerogenic factors is PD-L1. In some embodiments, at least one of the one or more tolerogenic factors is HLA-E. In some embodiments, at least one of the one or more tolerogenic factors is HLA-G. In some embodiments, the method comprises reducing expression of one or more MHC class I molecules and one or more MHC class II molecules.

In some aspects, provided herein is a method of generating a low-immunogenicity cell, comprising: a. increase the expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200 and MFGE8 in the cell and b.

In some embodiments, the method further comprises increasing expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD55 in the cell. In some embodiments, reduced expression comprises reduced surface expression and/or increased expression comprises increased surface expression. In some embodiments, increasing expression of one or more complement inhibitors comprises introducing into the cell an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and/or an exogenous polynucleotide encoding CD 55. In some embodiments, the one or more complement inhibitors are CD46 and CD59, optionally wherein increasing expression of the one or more complement inhibitors comprises introducing an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59. In some embodiments, the one or more complement inhibitors are CD46, CD59, and CD55, optionally wherein increasing expression of the one or more complement inhibitors comprises introducing an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55. In some embodiments, the exogenous polynucleotide encoding CD46 encodes an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO. 3 and exhibits complement inhibitory activity. In some embodiments, the exogenous polynucleotide encoding CD46 encodes the sequence set forth in SEQ ID NO. 3. In some embodiments, the exogenous polynucleotide encoding CD59 encodes an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO. 5 and exhibits complement inhibitory activity. In some embodiments, the exogenous polynucleotide encoding CD59 encodes the sequence set forth in SEQ ID NO. 5. In some embodiments, the exogenous polynucleotide encoding CD55 encodes a sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO. 8 and exhibits complement inhibitory activity. In some embodiments, the exogenous polynucleotide encoding CD55 encodes the sequence set forth in SEQ ID NO. 8. In some embodiments, the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, and/or the exogenous polynucleotide encoding CD55 are each operably linked to a promoter.

In some embodiments, the modification that increases expression of CD47 comprises an exogenous polynucleotide encoding a CD47 protein. In some embodiments, the exogenous polynucleotide encoding CD47 encodes a sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO. 1 and reduces innate immune killing of the engineered cells. In some embodiments, the exogenous polynucleotide encoding CD47 encodes the sequence set forth in SEQ ID NO. 1. In some embodiments, the exogenous polynucleotide encoding CD47 is operably linked to a promoter.

In some embodiments, the method comprises introducing into the cell a polycistronic transgene comprising two or more exogenous polypeptides selected from the group consisting of: one or more exogenous polynucleotides encoding one or more tolerogenic factors, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding a CD55 polypeptide. In some embodiments, each polynucleotide is isolated by an IRES or self-cleaving peptide. In some embodiments, the two or more exogenous polynucleotides are selected from the group consisting of an exogenous polynucleotide encoding CD47, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding a CD55 polypeptide. In some embodiments, each polynucleotide of the polycistronic transgene is operably linked to the same promoter.

In some embodiments, the polycistronic transgene comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59. In some embodiments, the polycistronic transgene comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55. In some embodiments, the polycistronic transgene further comprises an exogenous polynucleotide encoding CD 47. In some embodiments, the engineered cell comprises a separate transgene comprising a polynucleotide encoding CD 47. In some embodiments, the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, and/or the exogenous polynucleotide encoding CD55 is integrated into the genome of the engineered cell.

In some embodiments, the exogenous polynucleotide encoding CD47 is integrated into the genome of the engineered cell. In some embodiments, integration is by non-targeted insertion into the genome of the engineered cell, optionally by introducing an exogenous polynucleotide into the cell using a lentiviral vector. In some embodiments, the integration is by targeted insertion into a target genomic locus of the cell, optionally wherein the targeted insertion is by nuclease-mediated gene editing using homology directed repair.

In some embodiments, the target genomic locus is a harbor locus, a B2M locus, a CIITA locus, a CD142 locus, a TRAC locus, or a TRBC locus. In some embodiments, the target genomic locus is selected from the group consisting of: CCR5 locus, CXCR4 locus, PPP1R12C (also known as AAVS 1) locus, albumin locus, SHS231 locus, CLYBL locus and ROSA26 locus. In some embodiments, the target genomic locus is a safe harbor locus. In some embodiments, nuclease-mediated gene editing is by a Zinc Finger Nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas combination targeted to the target genomic locus, optionally wherein Cas is selected from Cas9 or Cas12. In some embodiments, nuclease-mediated gene editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to a target sequence of a target genomic locus and a homology directed repair template comprising an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, an exogenous polynucleotide encoding CD55, and/or an exogenous polynucleotide encoding CD 47. In some embodiments, the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising a gRNA and a Cas protein.

In some embodiments, reducing expression of CD142 reduces CD142 protein expression. In some embodiments, reducing expression of CD142 comprises introducing a modification that reduces activity of the CD142 gene. In some embodiments, the modification that reduces the activity of the CD142 gene comprises inactivation or disruption of both alleles of the CD142 gene. In some embodiments, the modification that reduces the activity of the CD142 gene comprises inactivation or disruption of all CD142 coding sequences in the cell. In some embodiments, the inactivation or disruption comprises an indel in the CD142 gene or a deletion of a stretch of contiguous genomic DNA of the CD142 gene. In some embodiments, the indels are frameshift mutations. In some embodiments, the CD142 gene is knocked out. In some embodiments, the modification that reduces CD142 gene activity is introduced by nuclease-mediated gene editing. In some embodiments, nuclease-mediated gene editing is by a Zinc Finger Nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas combination targeting the CD142 gene, optionally wherein Cas is selected from Cas9 or Cas12. In some embodiments, nuclease-mediated gene editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site within the CD142 gene. In some embodiments, the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising a gRNA and a Cas protein.

In some embodiments, reducing expression of one or more MHC class I molecules comprises introducing modifications that reduce expression of one or more MHC class I molecule proteins. In some embodiments, the modification that reduces expression of one or more MHC class I molecule proteins comprises reduced expression of B2M. In some embodiments, the modification that reduces the expression of one or more MHC class I molecule proteins comprises reduced protein expression of B2M. In some embodiments, the modification that reduces expression of one or more MHC class I molecule proteins reduces B2M gene activity. In some embodiments, the modification that reduces expression of one or more MHC class I molecules comprises inactivation or disruption of both alleles of the B2M gene. In some embodiments, the modification that reduces expression of one or more MHC class I molecule proteins comprises inactivation or disruption of all B2M coding sequences in the cell. In some embodiments, the inactivation or disruption comprises an indel in the B2M gene or a deletion of a stretch of contiguous genomic DNA of the B2M gene. In some embodiments, the indels are frameshift mutations. In some embodiments, the B2M gene is knocked out. In some embodiments, the modification that reduces the expression of one or more MHC class I molecule proteins is by nuclease-mediated gene editing. In some embodiments, nuclease-mediated gene editing is by a Zinc Finger Nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas combination targeting the B2M gene, optionally wherein Cas is selected from Cas9 or Cas12. In some embodiments, nuclease-mediated gene editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site within the B2M gene. In some embodiments, the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising a gRNA and a Cas protein.

In some embodiments, reducing expression of one or more MHC class II molecules comprises introducing a modification that reduces expression of one or more MHC class II molecule proteins. In some embodiments, the modification that reduces expression of one or more MHC class II molecule proteins comprises reduced expression of CIITA. In some embodiments, the modification that reduces the expression of one or more MHC class II molecule proteins comprises reduced protein expression of CIITA. In some embodiments, modifications that reduce expression of one or more MHC class II molecule proteins reduce CIITA gene activity. In some embodiments, the modification that reduces expression of one or more MHC class II molecule proteins comprises inactivation or disruption of both alleles of the CIITA gene. In some embodiments, the modification comprises inactivation or disruption of all CIITA coding sequences in the cell. In some embodiments, the inactivation or disruption comprises an indel in the CIITA gene or a deletion of a stretch of contiguous genomic DNA of the CIITA gene. In some embodiments, the indels are frameshift mutations. In some embodiments, the CIITA gene is knocked out.

In some embodiments, the cell is a human cell or an animal cell. In some embodiments, the animal cell is a pig (pig/pig) cell, a cow (cow/cow) cell, or a sheep (shaep/ovine) cell. In some embodiments, the engineered cell is a human cell. In some embodiments, the cell is a blood-exposed cell type or a cell type capable of differentiating into a blood-exposed cell type. In some embodiments, the cell is a primary cell isolated from a donor subject. In some embodiments, the low immunogenicity cell is a differentiated cell derived from a pluripotent stem cell, and the method further comprises differentiating the pluripotent stem cell. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the hypoimmunogenic cells are selected from the group consisting of beta islet cells, B cells, T cells, NK cells, glial progenitor cells, neural cells, cardiac cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, and blood cells (e.g., plasma cells or platelets). In some embodiments, the engineered cell is a beta islet cell. In some embodiments, the engineered cell is a hepatocyte.

In some of any of the embodiments of the methods of producing an engineered cell provided herein, the engineered cell comprises an exogenous polynucleotide encoding a suicide gene or suicide switch. In some embodiments, the suicide gene is selected from the group consisting of: cytosine deaminase (CyD), herpes virus thymidine kinase (HSV-Tk), inducible cysteine proteinase 9 (iCaspase 9) and rapamycin activated cysteine proteinase 9 (rapaCasp). In some embodiments, the suicide gene or suicide switch and the gene associated with the suicide gene or safety switch are expressed by a bicistronic cassette integrated into the genome of the engineered cell. In some embodiments, the suicide gene or suicide switch and the one or more tolerogenic factors are expressed by a bicistronic cassette integrated into the genome of the engineered cell. In some embodiments, the bicistronic cassette is integrated by non-targeted insertion into the genome of the engineered cell. In some embodiments, the bicistronic cassette is integrated by targeted insertion into a target genomic locus of an engineered cell. In some embodiments, the one or more tolerogenic factors is CD47.

In some aspects, provided herein is an engineered cell produced according to the methods described herein. In some embodiments, the engineered cell or progeny or differentiated cells derived from the engineered cell are capable of escaping NK cell-mediated cytotoxicity upon administration to a recipient patient. In some embodiments, the engineered cells or progeny or differentiated cells derived from the engineered cells are protected from cell lysis of mature NK cells after administration to a recipient patient. In some embodiments, the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce an immune response to the cell after administration to a recipient patient. In some embodiments, the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce a systemic inflammatory response to the cell after administration to a recipient patient. In some embodiments, the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce a localized inflammatory response to the cell after administration to a recipient patient.

In some embodiments, the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce complement pathway activation upon administration to a recipient patient. In some embodiments, the engineered cells or progeny or differentiated cells derived from the engineered cells do not induce clotting after administration to a recipient patient. In some embodiments, the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce an immediate blood-mediated inflammatory response upon administration to a recipient patient. In some embodiments, the cells are contacted with blood after administration to a recipient patient.

In some of any of the embodiments of the engineered cells provided herein, the engineered cells comprise an exogenous polynucleotide encoding a suicide gene or suicide switch. In some embodiments, the suicide gene or suicide switch is selected from the group consisting of: cytosine deaminase (CyD), herpes virus thymidine kinase (HSV-Tk), inducible cysteine proteinase 9 (iCaspase 9) and rapamycin activated cysteine proteinase 9 (rapaCasp). The suicide gene or suicide switch and the gene associated with the suicide gene or safety switch are expressed by a bicistronic cassette integrated into the genome of the engineered cell. In some embodiments, the suicide gene or suicide switch and the one or more tolerogenic factors are expressed by a bicistronic cassette integrated into the genome of the engineered cell. In some embodiments, the bicistronic cassette is integrated by non-targeted insertion into the engineered cell genome, optionally by introducing an exogenous polynucleotide into the cell using a lentiviral vector. In some embodiments, the bicistronic cassette is integrated by targeted insertion into a target genomic locus of a cell, optionally wherein the targeted insertion is by nuclease-mediated gene editing using homology directed repair. In some embodiments, the one or more tolerogenic factors is CD47.

In some aspects, provided herein is an engineered cell population comprising a plurality of engineered cells described herein.

In some embodiments, the plurality of engineered primary cells are derived from cells pooled from more than one donor subject. In some embodiments, each of the more than one donor subjects is a healthy subject or is not suspected of having a disease or disorder when the donor sample is obtained from the donor subject.

In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise the modification.

In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise an exogenous polynucleotide encoding CD 47.

In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise an exogenous polynucleotide encoding CD 46.

In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise an exogenous polynucleotide encoding CD 59.

In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise an exogenous polynucleotide encoding CD 55.

In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise one or more alterations that inactivate both alleles of the B2M gene.

In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise one or more alterations that inactivate both alleles of the CIITA gene.

In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise reduced expression of CD142 relative to unmodified or wild-type cells.

In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise one or more alterations that inactivate both alleles of the CD142 gene.

In some aspects, provided herein is a composition comprising a population as described herein.

In some aspects, provided herein is a composition comprising a population of engineered beta islet cells, wherein the engineered beta islet cells comprise: (i) a transgene comprising an exogenous polynucleotide encoding CD47, (ii) inactivation or disruption of both alleles of the CD142 gene, and (iii) inactivation or disruption of both alleles of the B2M gene.

In some embodiments, the engineered β -cell comprises inactivation or disruption of both alleles of the CIITA gene.

In some aspects, provided herein is a composition comprising an engineered population of hepatocytes, wherein the engineered hepatocytes comprise: (i) a transgene comprising an exogenous polynucleotide encoding CD47, (ii) inactivation or disruption of both alleles of the CD142 gene, and (iii) inactivation or disruption of both alleles of the B2M gene.

In some embodiments, the engineered hepatocyte comprises inactivation or disruption of both alleles of the CIITA gene. In some embodiments, the transgene is a polycistronic transgene, and wherein the transgene further comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59.

In some embodiments, the beta islet cells or liver cells further comprise a polycistronic transgene, wherein the polycistronic transgene comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59. In some embodiments, the transgene is introduced at the target genomic locus site by nuclease-mediated gene editing using homology directed repair. In some embodiments, the inactivation or disruption is by nuclease-mediated gene editing. In some embodiments, nuclease-mediated gene editing is by a Zinc Finger Nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas combination targeted to the target genomic locus, optionally wherein Cas is selected from Cas9 or Cas12.

In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient is a buffer solution, such as saline.

In some embodiments, the composition is formulated in a serum-free cryopreservation medium comprising a cryoprotectant. In some embodiments, the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (volume/volume). In some embodiments, the cryoprotectant is or is about 10% DMSO (volume/volume). In some embodiments, the composition is sterile.

In some embodiments of any of the compositions disclosed herein, the engineered cells in the population of engineered cells comprise an exogenous polynucleotide encoding a suicide gene or suicide switch. In some embodiments, the suicide gene or suicide switch is selected from the group consisting of: cytosine deaminase (CyD), herpes virus thymidine kinase (HSV-Tk), inducible cysteine proteinase 9 (iCaspase 9) and rapamycin activated cysteine proteinase 9 (rapaCasp). In some embodiments, the suicide gene and the gene associated with the suicide gene or safety switch are expressed by a bicistronic cassette integrated into the genome of the engineered cells in the engineered cell population. In some embodiments, the suicide gene or suicide switch and the exogenous CD47 are expressed by a bicistronic cassette integrated into the genome of the engineered cell. In some embodiments, the bicistronic cassette is integrated by non-targeted insertion into the genome, optionally by introducing the exogenous polynucleotide into an engineered cell in an engineered cell population using a lentiviral vector. In some embodiments, the bicistronic cassette is integrated by targeted insertion into a target genomic locus of an engineered cell in the engineered cell population, optionally wherein the targeted insertion is by nuclease-mediated gene editing using homology directed repair.

In some aspects, provided herein is a container comprising any of the compositions described herein. In some embodiments, the container is a sterile bag. In some embodiments, the pouch is a cryopreservation compatible pouch.

In some aspects, provided herein is a method of treating a disease, disorder, or cell defect in a patient in need thereof, the method comprising administering to the patient an effective amount of a population described herein or a composition described herein.

In some embodiments, the method further comprises administering to the patient an anticoagulant that reduces coagulation.

In some aspects, provided herein is a method of treating a disease, disorder, or cell defect in a patient in need thereof, the method comprising: (a) administering to the patient an effective amount of: a cell population comprising a plurality of engineered cells, wherein the engineered cells comprise a modification that (i) increases expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD 55; (ii) Increasing expression of one or more tolerogenic factors, and (iii) decreasing expression of one or more MHC class I molecules and/or one or more MHC class II molecules, wherein the increased expression of (I) and (II) and the decreased expression of (iii) are relative to a cell of the same cell type that does not comprise the modification; and (b) administering to the patient an anticoagulant that reduces coagulation.

In some embodiments, the one or more tolerogenic factors are selected from the group consisting of: CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDOl, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8 and SERPINB9.

In some embodiments, the one or more tolerogenic factors is CD47.

In some aspects, provided herein is a method of treating a disease, disorder, or cell defect in a patient in need thereof, the method comprising: (a) administering to the patient an effective amount of: a cell population comprising a plurality of engineered cells, wherein the engineered cells comprise a modification that (i) increases expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD 55; and (ii) increasing expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD, CD200, and MFGE8, wherein the increased expression of (i) and (ii) is relative to a cell of the same cell type that does not comprise the modification; and (b) administering to the patient an anticoagulant that reduces coagulation.

In some embodiments, the population is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

In some embodiments, the population and the anticoagulant are administered simultaneously or sequentially.

In some embodiments, the anticoagulant is heparin. In some embodiments, heparin is plain heparin. In some embodiments, heparin is low molecular weight heparin. In some embodiments, heparin is soluble heparin.

In some embodiments, heparin is immobilized on the surface of cells prior to administration of the cells to a patient. In some embodiments, the anticoagulant is melagatran (melagatran) or LMW-DS. In some embodiments, the anticoagulant is N-acetylcysteine (NAC). In some embodiments, the anticoagulant is alpha-1 antitrypsin (AAT) and/or activated protein C.

In some embodiments, the disorder or disease is selected from the group consisting of: diabetes, cancer, angiogenesis disorders, ocular diseases, thyroid diseases, skin diseases and liver diseases.

In some embodiments, the cell defect is associated with diabetes, or the cell therapy is used to treat diabetes, optionally wherein the diabetes is type I diabetes. In some embodiments, the cell population is a population of islet cells (including beta islet cells). In some embodiments, the islet cells are selected from the group consisting of islet progenitor cells, immature islet cells, and mature islet cells.

In some embodiments, the cell defect is associated with a vascular disorder or disease, or the cell therapy is used to treat a vascular disorder or disease. In some embodiments, the population of cells is a population of endothelial cells.

In some embodiments, the cell deficiency is associated with autoimmune thyroiditis, or the cell therapy is used to treat autoimmune thyroiditis. In some embodiments, the cell population is a thyroid progenitor cell population. In some embodiments, the cell defect is associated with a liver disease, or the cell therapy is used to treat a liver disease. In some embodiments, the liver disease comprises liver cirrhosis. In some embodiments, the population of cells is a population of hepatocytes or hepatic progenitors. In some embodiments, the cell defect is associated with a corneal disease, or the cell therapy is used to treat a corneal disease. In some embodiments, the corneal disease is Fuchs dystrophy (Fuchs dystophy) or congenital genetic endothelial dystrophy.

In some embodiments, the cell population is a population of corneal endothelial progenitor cells or a population of corneal endothelial cells. In some embodiments, the cell deficiency is associated with kidney disease, or the cell therapy is used to treat kidney disease. In some embodiments, the cell population is a kidney precursor cell or a kidney cell population.

In some embodiments, cell therapy is used to treat cancer. In some embodiments, the cancer is selected from the group consisting of: b-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous lymphoblastic leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

In some embodiments, the cell population is a T cell population or NK cell population. In some embodiments, the cells are expanded and cryopreserved prior to administration. In some embodiments, the administration population comprises an intravenous injection, an intramuscular injection, an intravascular injection, or a transplant population. In some embodiments, the population is transplanted via renal capsule transplantation or intramuscular injection. In some embodiments, the population is derived from a donor subject, wherein the HLA type of the donor does not match the HLA type of the patient. In some embodiments, the population is derived from a donor, wherein the blood group of the donor does not match the blood group of the patient and the blood group of the donor is not O-type. In some embodiments, the population is derived from a donor, wherein the blood group of the donor is rhesus factor (Rh) positive and the blood group of the patient is Rh negative. In some embodiments, the patient's serum comprises antibodies to Rh.

In some embodiments, the population is a population of human cells and the patient is a human patient. In some embodiments, the population of cells comprises a functional ABO a allele and/or a functional ABO B allele. In some embodiments, the population of cells presents ABO type a antigens and the patient's serum comprises anti-a antibodies. In some embodiments, the population of cells presents ABO type B antigens and the patient's serum comprises anti-B antibodies. In some embodiments, the population of cells presents ABO type a and type B antigens and the patient's serum comprises anti-a and/or anti-B antibodies. In some embodiments, the population of cells expresses Rh factor and the patient's serum comprises anti-Rh antibodies.

In some embodiments, the method comprises administering one or more immunosuppressants to the patient. In some embodiments, one or more immunosuppressants have been administered to a patient. In some embodiments, the one or more immunosuppressants are small molecules or antibodies. In some embodiments, the antibody binds to one or more receptors or ligands selected from the group consisting of: p75、MHC、CD2、CD3、CD4、CD7、CD28、B7、CD40、CD45、IFN-γ、TNF-α、IL-4、IL-5、IL-6R、IL-6、IGF、IGFR1、IL-7、IL-8、IL-10、CD11a、CD58, of the IL-2 receptor and antibodies that bind to any of its ligands. In some embodiments, the one or more immunosuppressants are selected from the group consisting of: cyclosporin (cyclosporine), azathioprine (azathioprine), mycophenolic acid (mycophenolic acid), mycophenolic acid ester (mycophenolate mofetil), corticosteroids, prednisone (prednisone), methotrexate, gold salts, sulfasalazine (sulfasalazine), antimalarials, bucquinar (brequinar), leflunomide (leflunomide), mizoribine (mizoribine), 15-deoxyspergualin (15-deoxyspergualine), 6-mercaptopurine, cyclophosphamide, rapamycin (rapamycin), tacrolimus (tacrolimus) (FK-506), OKT3, anti-thymocyte globulin, thymopentapeptide (thymopentin) (thymosin-alpha) and immunosuppressive antibodies. In some embodiments, the one or more immunosuppressants comprise cyclosporine. In some embodiments, the one or more immunosuppressants comprise mycophenolate mofetil. In some embodiments, the one or more immunosuppressants comprise a corticosteroid. In some embodiments, the one or more immunosuppressants comprise cyclophosphamide. In some embodiments, the one or more immunosuppressants comprise rapamycin. In some embodiments, the one or more immunosuppressants comprise tacrolimus (FK-506). In some embodiments, the one or more immunosuppressants comprise anti-thymocyte globulin.

In some embodiments, the one or more immunosuppressants are one or more immunomodulators. In some embodiments, the one or more immunomodulatory agents are small molecules or antibodies. In some embodiments, the antibody binds to one or more receptors or ligands selected from the group consisting of: p75、MHC、CD2、CD3、CD4、CD7、CD28、B7、CD40、CD45、IFN-γ、TNF-α、IL-4、IL-5、IL-6R、IL-6、IGF、IGFR1、IL-7、IL-8、IL-10、CD11a、CD58, of the IL-2 receptor and antibodies that bind to any of its ligands.

In some embodiments, one or more immunosuppressants are administered to the patient prior to or already administered to the patient. In some embodiments, the one or more immunosuppressants are administered to the patient or have been administered to the patient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to administration of the engineered cells. In some embodiments, the patient is administered or has been administered one or more immunosuppressants at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more prior to administration of the engineered cells. In some embodiments, one or more immunosuppressants are or have been administered to a patient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after administration of the engineered cells. In some embodiments, the one or more immunosuppressants are administered to the patient or have been administered to the patient at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more after administration of the engineered cells. In some embodiments, the one or more immunosuppressants are administered to the patient or have been administered to the patient on the same day as the first administration of the engineered cells. In some embodiments, the one or more immunosuppressants are administered to the patient or have been administered to the patient after administration of the engineered cells. In some embodiments, the one or more immunosuppressants are administered to the patient or have been administered to the patient after the administration of the first and/or second administration of the engineered cells. In some embodiments, the one or more immunosuppressants are administered to the patient or have been administered to the patient prior to the administration of the first and/or second administration of the engineered cells. In some embodiments, the one or more immunosuppressants are administered to the patient or have been administered to the patient at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to the administration of the first and/or second administration of the engineered cells.

In some embodiments, the one or more immunosuppressants are administered to the patient or have been administered to the patient at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more prior to the administration of the engineered cells to the first and/or second administration. In some embodiments, the one or more immunosuppressants are administered to the patient or have been administered to the patient at least 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the administration of the first and/or second administration of the engineered cells. In some embodiments, the one or more immunosuppressants are administered to the patient or have been administered to the patient at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more after the administration of the engineered cells for the first and/or second administration. In some embodiments, the one or more immunosuppressants are administered at a lower dose to reduce immune rejection of modified immunogenic cells that do not comprise engineered cells than the dose of the one or more immunosuppressants administered.

In some embodiments, the engineered cell is capable of controlled killing of the engineered cell. In some embodiments, the engineered cell comprises a suicide gene or suicide switch. In some embodiments, the suicide gene or suicide switch induces controlled cell death in the presence of a drug or prodrug or after activation by a selective exogenous compound. In some embodiments, the suicide gene or suicide switch is an inducible protein capable of inducing apoptosis of the engineered cell. In some embodiments, the inducible protein capable of inducing apoptosis in the engineered cell is a cysteine protease protein. In some embodiments, the cysteine protease protein is cysteine protease 9. In some embodiments, the suicide gene or suicide switch is selected from the group consisting of: cytosine deaminase (CyD), herpes virus thymidine kinase (HSV-Tk), inducible cysteine proteinase 9 (iCaspase 9) and rapamycin activated cysteine proteinase 9 (rapaCasp). In some embodiments, the suicide gene or suicide switch is activated to induce controlled cell death after administration of one or more immunosuppressants to the patient. In some embodiments, the suicide gene or suicide switch is activated to induce controlled cell death prior to administration of the one or more immunosuppressants to the patient. In some embodiments, the suicide gene or suicide switch is activated to induce controlled cell death after the engineered cells are administered to the patient. In some embodiments, the suicide gene or suicide switch is activated to induce controlled cell death if it has cytotoxicity or other negative consequences for the patient.

In some embodiments, the method comprises administering an agent that allows depletion of engineered cells in the population of engineered cells. In some embodiments, the agent that allows depletion of the engineered cell is an antibody that recognizes a protein expressed on the surface of the engineered cell. In some embodiments, the antibody is selected from the group consisting of antibodies that recognize CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR 8. In some embodiments, the antibody is selected from the group consisting of: mo Geli bead mab, AFM13, MOR208, octuzumab, rituximab, oxcarbatuzumab, rituximab-Rllb, tobrauximab, RO5083945 (GA 201), cetuximab, hul4.18k322a, hul4.18-IL2, hul3F 8, rituximab, c.60c3-Rllc, and biological analogs thereof. In some embodiments, the method comprises administering an agent that recognizes one or more tolerogenic factors on the surface of the engineered cell. In some embodiments, the engineered cells are engineered to express one or more tolerogenic factors. In some embodiments, the one or more tolerogenic factors is CD47.

In some embodiments, the method further comprises administering one or more additional therapeutic agents to the patient. In some embodiments, one or more additional therapeutic agents have been administered to the patient. In some embodiments, the method comprises monitoring the efficacy of the treatment of the method. In some embodiments, the method comprises monitoring the prophylactic efficacy of the method. In some embodiments, the method is repeated until a desired inhibition of one or more disease symptoms occurs.

In some aspects, provided herein is a combination comprising an engineered population of cells described herein and an anticoagulant or cell coating that reduces blood coagulation.

In some aspects, provided herein are combinations comprising: (a) A cell population comprising a plurality of engineered cells, wherein the engineered cells comprise a modification that (i) increases expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD 55; (ii) Increasing expression of CD47, and (iii) decreasing expression of one or more MHC class I molecules and/or one or more MHC class II molecules, wherein increased expression of (I) and (II) and decreased expression of (iii) are relative to a cell of the same cell type that does not comprise the modification; and (b) an anticoagulant.

In some embodiments, the anticoagulant is selected from the group consisting of heparin, an antithrombin activator, a factor II (fhi) inhibitor, a factor VII (fhi) inhibitor, and a factor X (fX) inhibitor.

In some embodiments, the anticoagulant is heparin.

In some embodiments, heparin is plain heparin.

In some embodiments, heparin is low molecular weight heparin.

In some embodiments, heparin is soluble heparin.

In some embodiments, the anticoagulant is melagatran or LMW-DS.

In some embodiments, the anticoagulant is N-acetylcysteine (NAC).

In some embodiments, the anticoagulant is alpha-1 antitrypsin (AAT) and/or activated protein C.

In some embodiments, the anticoagulant is an antibody directed against CD 142.

In some aspects, provided herein is a kit comprising a composition described herein.

Drawings

FIGS. 1A to 1B show B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion} measured by flow cytometry; CD47tg human induced pluripotent stem cells (hiPSC) (fig. 1A) and slave B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion}; the expression levels of HLA class I (HLA-I), HLA class II (HLA-II) and CD47 of the endothelial cells differentiated by CD47tg hiPSC (hiEC) demonstrated that the cells were deficient in HLA-I and HLA-II expression and had increased CD47 expression.

FIGS. 2A through 2B illustrate B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion}; CD47tg hiPSC (fig. 2A) and B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion}; surface expression levels of CD46, CD55 and CD59 in CD47tg hiEC (fig. 2B).

FIGS. 3A-3B show B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion} in an ABO incompatible Complement Dependent Cytotoxicity (CDC) assay; CD47tg hiPSC (fig. 3A) and B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion}; killing of CD47tg hiEC (fig. 3B).

FIGS. 4A-4B illustrate representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion} in an ABO incompatible CDC assay; CD47tg cd46+ +. +hiPSC cloning [ ], fig. 4A) and representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion}; CD47tg CD46 +. +hiEC killing of clone (FIG. 4B).

FIGS. 5A-5B illustrate representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion} in an ABO incompatible CDC assay; CD47tg cd55++ hiPSC clone (fig. 5A) and representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion}; killing of CD47tg cd55++ hiEC clone (fig. 5B).

FIGS. 6A-6B illustrate representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion} in an ABO incompatible CDC assay; CD47tg CD59+ +. +hiPSC cloning [ ], fig. 6A) and representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion}; killing of CD47tg CD59++ hiEC clone (fig. 6B).

FIGS. 7A-7B illustrate representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion} in an ABO incompatible CDC assay; CD47tg cd46++/cd55++ hiPSC clone (fig. 7A) and representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion}; killing of CD47tg cd46++/cd55++ hiEC clone (fig. 7B).

FIGS. 8A-8B illustrate representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion} in an ABO incompatible CDC assay; CD47tg CD55++. CD59+ + and +hiPSC cloning and representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion}; CD47tg CD55++/CD59+. Killing of++ hiEC clones.

FIGS. 9A-9B illustrate representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion} in an ABO incompatible CDC assay; CD47tg CD46++/CD59++ + hiPSC cloning (FIG. 9A) and representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion}; survival of CD47tg CD46++/CD59++ hiEC clone (FIG. 9B).

FIGS. 10A-10B illustrate representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion} in an ABO incompatible CDC assay; CD47tg cd46++/cd55+/cd59++ hiPSC clone (fig. 10A) and representative B2M ^{Indel of insertion / Indel of insertion};CIITA^{Indel of insertion / Indel of insertion}; survival of CD47tg CD46++/CD55++/CD59++ hiEC clone (FIG. 10B).

Fig. 11 shows CDC assay results of endothelial cells derived from human ipscs in the absence of ABO incompatible serum (survival control).

Detailed Description

Provided herein are methods and compositions for reducing and/or avoiding the effects of immune system responses to allografts. To overcome the problem of immune rejection of cell-derived and/or tissue grafts, disclosed herein is an engineered immune evading cell (e.g., an engineered primary low-immunogenicity cell) or population or pharmaceutical composition thereof, which represents a viable source of any transplantable cell type. The engineered cells disclosed herein provide for the recognition of a reduced immune system of a recipient subject, regardless of the genetic composition of the subject or any existing response in the subject to one or more previous allografts, previous autologous Chimeric Antigen Receptor (CAR) T-rejections, and/or other autologous or allogeneic therapies in which the transgene is expressed. Engineered cells may include, but are not limited to, beta islet cells, B cells, T cells, NK cells, retinal pigment epithelial cells, glial progenitor cells, endothelial cells, hepatocytes, thyroid cells, skin cells, and blood cells (e.g., plasma cells or platelets).

In some aspects, provided herein are methods and compositions for reducing or avoiding an immediate blood-mediated inflammatory response (IBMIR) associated with cell transplantation therapies. In some cases, IBMIR occurs immediately after islet implants (e.g., islets that do not contain CD142 modifications to reduce CD142 expression and/or activity) are exposed to recipient blood. For example, IBMIR is a major cause of tissue loss in clinical islet allografts, beginning with exposure to blood after islet cell infusion into the portal vein. It is estimated that up to 60% of islets are lost within one week after transplantation. At worst, IBMIR causes portal thrombosis, liver infarction, and portal hypertension. IBMIR typically occurs immediately after islet implants (e.g., islets that do not contain CD142 modifications for reducing CD142 expression and/or activity) are exposed to the recipient's blood. \

In some embodiments, provided herein is a type of engineered cell (e.g., beta islet cells, hepatocytes, and other cells) that typically initiates IBMIR when contacted with blood during common transplantation methods, such as intravenous infusion of cells or intramuscular injection of cells. In some embodiments, the engineered cells comprise reduced or eliminated expression of CD142, CD142 also known as factor III, tissue Factor (TF), thrombin, platelet tissue factor, or factor III, which is a membrane receptor in the coagulation pathway that contributes to priming IBMIR. In some embodiments, the engineered cells comprise reduced expression of CD142 and increased expression of one or more tolerogenic factors and/or reduced expression of one or more MHC class I molecules and/or one or more MHC class II molecules. In some embodiments, cells comprising the modifications described herein (including reduced or eliminated expression of CD 142) survive, are implanted, and function after transplantation. In some embodiments, the cells exhibit enhanced survival and/or enhanced engraftment and/or long-term function as compared to cells that do not comprise a modification to CD 142. In some embodiments, the cells are administered via intravenous infusion, intramuscular injection, or renal capsule transplantation.

In some embodiments, the engineered cells described herein further comprise increased expression and/or overexpression of one or more complement inhibitors. In some embodiments, the one or more complement inhibitors are selected from the group consisting of CD46, CD59, and CD55. In some embodiments, the engineered cells comprise increased expression of a combination of two or more complement inhibitors, such as increased expression of CD46 and CD59 or increased expression of CD46, CD59, and CD55. In some embodiments, the engineered cells provided herein are protected from complement-mediated cytotoxicity. In some embodiments, the engineered cells (e.g., over-expressing CD46 and CD 59) are protected from complement reactions that occur concurrently with IBMIR. In some embodiments, the engineered cells are protected from complement reactions that occur independently of IBMIR.

In some aspects, provided herein are methods, compositions, and combinations for reducing or avoiding an immediate blood-mediated inflammatory response (IBMIR) associated with cell transplantation therapies. In some cases, the methods herein comprise administering a combination of an engineered cell described herein and an anticoagulant combination. In some embodiments, the cells administered in combination with the anticoagulant do not comprise reduced expression of CD 142. In some embodiments, the anticoagulant is administered concurrently with cell transplantation to prevent rapid IBMIR. In some embodiments, the anticoagulant is administered at the time of implantation (e.g., the anticoagulant may be administered intravenously at the same time as the engineered cell population described herein is administered). In some embodiments, the anticoagulant is also administered before or after the transplantation therapy. In some embodiments, the anticoagulant may be administered between 24 hours and 12 hours, between 12 hours and 6 hours, between 6 hours and 3 hours, between 3 hours and 1 hour, or within 1 hour before or after implantation. In some embodiments, the anticoagulant is administered concurrently with the treatment.

In some embodiments, the engineered cells described herein further comprise increased expression and/or overexpression of one or more complement inhibitors. In some embodiments, the one or more complement inhibitors are selected from the group consisting of CD46, CD59, and CD55. In some embodiments, the engineered cells comprise increased expression of a combination of two or more complement inhibitors, such as increased expression of CD46 and CD59 or increased expression of CD46, CD59, and CD55. In some embodiments, the engineered cells provided herein are protected from complement-mediated cytotoxicity. In some embodiments, the engineered cells (e.g., over-expressing CD46 and CD 59) are protected from Complement Dependent Cytotoxicity (CDC) due to IBMIR. In some embodiments, the engineered cells are protected from CDC occurring independently of IBMIR.

The engineered cells provided herein utilize expression of tolerogenic factors, and may also modulate (e.g., reduce or eliminate) expression (e.g., surface expression) of one or more MHC class I molecules and/or one or more MHC class II molecules. In some embodiments, genome editing techniques that utilize rare-cutting endonucleases (e.g., CRISPR/Cas, TALENs, zinc finger nucleases, meganucleases, and homing endonuclease systems) are also used to reduce or eliminate expression of critical immune genes in human cells (e.g., by deleting genomic DNA of the critical immune genes). In certain embodiments, genome editing techniques or other gene regulation techniques are used to insert tolerance-inducing (tolerogenic) factors (e.g., CD 47) in human cells, thereby producing engineered cells that can evade immune recognition after implantation into a recipient subject. Thus, the engineered cells provided herein exhibit modulated expression (e.g., reduced or eliminated expression) of one or more genes and factors affecting one or more MHC class I molecules and/or one or more MHC class II molecules and modulated expression (e.g., over-expression) of tolerogenic factors (such as CD 47) and provide for reduced recognition of the immune system of a recipient subject. In some embodiments, the engineered cells provided herein exhibit modulated expression (e.g., reduced expression) of CD 142. In some embodiments, the engineered cells provided herein exhibit modulated expression (e.g., increased expression) of one or more complement inhibitors selected from CD46, CD59, and CD 55.

In some aspects, the engineered cells provided herein exhibit reduced innate immune cell rejection and/or adaptive immune cell rejection (e.g., low-immunogenicity cells). For example, in some embodiments, the engineered cells exhibit reduced sensitivity to NK cell-mediated lysis and/or macrophage phagocytosis. In some embodiments, the engineered cells can be used as a source of universally compatible cells or tissues (e.g., universal donor cells or tissues) that are transplanted into a recipient subject with little to no need for an immunosuppressant. Such low-immunogenicity cells retain cell-specific features and characteristics after transplantation.

Also provided herein are methods for treating a disorder comprising administering an engineered cell (e.g., an engineered primary cell) that evades immune rejection in an MHC mismatched allogeneic receptor. In some embodiments, the engineered cells produced by any of the methods described herein evade immune rejection when repeatedly administered (e.g., transplanted or implanted) to MHC mismatched allogeneic recipients.

The practice of particular embodiments will employ, unless indicated to the contrary explicitly, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology, which are within the skill of the art, many of which are described below for purposes of illustration. Such techniques are well explained in the literature. See, e.g., sambrook et al, molecular Cloning: A Laboratory Manual (3 rd edition, 2001); sambrook et al, molecular Cloning: ALaboratory Manual (2 nd edition, 1989); maniatis et al, molecular Cloning: ALaboratory Manual (1982); ausubel et al Current Protocols in Molecular Biology (John Wiley and Sons,2008, 7, update );Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience;Glover,DNA Cloning:A Practical Approach,, volumes I and II (IRL Press,Oxford,1985);Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Transcription and Translation(B.Hames and s.higgins, 1984); perbal, A PRACTICAL Guide to Molecular Cloning (1984); harlow and Lane,Antibodies,(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1998)Current Protocols in Immunology Q.E.Coligan,A M.Kruisbeek,D.H.Margulies,E.M.Shevach and W.Strober, 1991); annual Review of Immunology; and monographs such as those on the ADVANCES IN Immunology journal.

All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in a patent, application, published application and other publication, which is incorporated by reference herein, the definition set forth herein takes precedence over the definition set forth herein.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present disclosure. The following description illustrates the present disclosure and, of course, should not be construed as in any way limiting the scope of the invention described herein.

I. Definition of the definition

Unless defined otherwise, all technical, symbolic and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ease of reference, and the inclusion of such definitions herein should not be construed to represent a significant difference from the commonly understood meaning in the art.

The term "about" as used herein when referring to a measurable value, such as an amount or concentration, is intended to encompass a 20%, 10%, 5%, 1%, 0.5% or even 0.1% change in the specified amount. As used herein, including in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more". It is to be understood that the aspects and variations described herein include embodiments that "consist of" and/or "consist essentially of" such aspects and variations.

As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

As used herein, the term "exogenous" with respect to a polypeptide or polynucleotide is intended to mean that the molecule in question is introduced into a cell of interest. Exogenous molecules, such as exogenous polynucleotides, may be introduced, for example, by introducing the exogenous molecule encoding the nucleic acid into the genetic material of the cell (such as by integration into the chromosome) or as non-chromosomal genetic material (such as a plasmid or expression vector). Thus, when used in reference to expression of a coding nucleic acid, the term refers to introduction of the coding nucleic acid into a cell in an expressible form. In some cases, an "exogenous" molecule is a molecule, construct, factor, etc., that is not normally present in a cell, but can be introduced into the cell by one or more genetic, biochemical, or other methods.

The term "endogenous" refers to a reference molecule, such as a polynucleotide (e.g., a gene), or polypeptide, that is present in a natural or unmodified cell. For example, when used in reference to expression of an endogenous gene, the term refers to expression of a gene encoded by an endogenous nucleic acid contained within a cell and not introduced exogenously. "Gene" includes DNA regions encoding a gene product, as well as all DNA regions regulating the production of a gene product, whether or not such regulatory sequences are adjacent to coding and/or transcriptional sequences. Thus, genes include, but are not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, border elements, origins of replication, matrix attachment sites, and locus control regions. The sequence of a gene is typically present at a fixed chromosomal location or locus on a chromosome in a cell.

The term "locus" refers to a fixed location on a chromosome where a particular gene or genetic marker is located. Reference to a "target locus" refers to a particular locus of a desired gene, where it is desired to target genetic modifications, such as gene editing or integration of an exogenous polynucleotide.

The term "expression" in reference to a gene or "gene expression" refers to the conversion of information contained in a gene into a gene product. The gene product may be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or may be a protein produced by mRNA translation. Gene products also include RNA modified by processes such as capping, polyadenylation, methylation and editing, as well as proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation and glycosylation. Thus, reference to expression or gene expression includes expression of a protein (or polypeptide) or expression of a transcribable product of a gene, such as mRNA. Protein expression may include intracellular expression or surface expression of the protein. Typically, expression of a gene product (such as an mRNA or protein) is at a level that is detectable in a cell.

As used herein, "detectable" expression level means a level detectable by standard techniques known to the skilled artisan and including, for example, differential display, RT (reverse transcriptase) coupled Polymerase Chain Reaction (PCR), northern blot and/or rnase protection analysis, and immunoaffinity based protein detection methods such as flow cytometry, ELISA or western blot. The extent of expression level need only be large enough to be visualized or measured via standard characterization techniques.

As used herein, the terms "increased expression," "enhanced expression," or "overexpression" mean any form of expression other than expression in a primary or source cell that does not contain modifications for regulating expression of a particular gene (e.g., wild-type expression levels (which may also be non-expressed or expression is not measurable)). Reference herein to "increased expression", "enhanced expression" or "overexpression" means an increase in gene expression and/or, in the case of a polypeptide, an increase in the level of the polypeptide and/or an increase in the activity of the polypeptide relative to the level in a cell of original origin that does not contain the modification, such as a cell of original origin prior to engineering to introduce the modification, such as an unmodified cell or a wild-type cell. The increase in expression, polypeptide level or polypeptide activity may be at least 5%, 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90% or 100% or even more. In some cases, the increase in expression, polypeptide level, or polypeptide activity may be at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, or more.

The term "low immunogenicity" refers to cells that are less prone to immune rejection by a subject into which such cells are transplanted. For example, such a low-immunogenicity cell may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or less susceptible to immune rejection by a subject into which such a cell is transplanted relative to a similar cell that does not contain the modification, such as an unaltered or unmodified wild-type cell. Typically, the hypo-immunogenic cells are allogeneic to the subject, and the hypo-immunogenic cells evade immune rejection in MHC-mismatched allogeneic recipients. In some embodiments, the hypoimmunogenic cells are protected from T cell-mediated adaptive immune rejection and/or innate immune cell rejection.

The low immunogenicity of a cell can be determined by assessing the immunogenicity of the cell (such as the ability of the cell to elicit an adaptive and/or innate immune response). Such immune responses may be measured using assays recognized by those skilled in the art.

The term "tolerogenic factors" as used herein includes immunosuppressive factors or immunomodulatory factors that regulate or affect the ability of cells to be recognized by the immune system of a host or recipient subject after administration, transplantation or implantation. Typically, tolerogenic factors are factors that induce immune tolerance to the engineered primary cells such that the engineered primary cells are not targeted (such as rejected) by the host immune system of the recipient. Thus, the tolerogenic factors may be hypoimmunity factors. Examples of tolerogenic factors include immune cell inhibitory receptors (e.g., CD 47), proteins that bind to immune cell inhibitory receptors, checkpoint inhibitors, and other molecules that reduce innate or adaptive immune recognition.

The terms "decrease (decrease)", "reduced", "decrease (reduction)" and "decrease (decrease)" are all generally used herein to mean a statistically significant amount of decrease. However, for the avoidance of doubt, "reduced" and "reduced (decrease)" mean at least a 10% reduction from the reference level, for example at least about 20% reduction from the reference level, or at least about 30% reduction, or at least about 40% reduction, or at least about 50% reduction, or at least about 60% reduction, or at least about 70% reduction, or at least about 80% reduction, or at least about 90% reduction, or up to and including 100% reduction (i.e., a level that is not present as compared to the reference sample), or any reduction between 10-100%.

The terms "increase (increase)" or "enhancement" or "activation" are used herein to generally mean increasing by a statistically significant amount; for the avoidance of any doubt, the term "increased", "increased" (increase) "or" enhanced "or" activated "means at least a 10% increase from a reference level, for example at least about 20% increase from a reference level, or at least about 30% increase from a reference level, or at least about 40% increase from a reference level, or at least about 50% increase from a reference level, or at least about 60% increase from a reference level, or at least about 70% increase from a reference level, or at least about 80% increase from a reference level, or at least about 90% increase from a reference level, or at least about 3 times increase from a reference level, or at least about 4 times increase from a reference level, or at least about 5 times increase from a reference level, or at least about 10 times increase from a reference level, or any increase between 2 times and 10 times increase from a reference level.

As used herein, the term "modification" refers to any change or alteration in a cell that affects gene expression in the cell. In some embodiments, the modification is a genetic modification that directly alters a gene encoding a protein product or regulatory element thereof in the cell (such as by gene editing, mutagenesis, or by genetic engineering of an exogenous polynucleotide or transgene).

As used herein, "indels" refers to mutations resulting from insertions, deletions, or combinations thereof of nucleotide bases in the genome. Thus, indels typically insert or delete nucleotides from the sequence. As will be appreciated by those skilled in the art, unless the length of the insertion deletion is a multiple of three, the insertion deletion in the coding region of the genomic sequence will result in a frameshift mutation. The CRISPR/Cas systems of the present disclosure can be used to induce indels of any length in a target polynucleotide sequence.

In some embodiments, the alteration is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one of the nucleotides. The CRISPR/Cas systems of the present disclosure can be used to induce indels or point mutations of any length in a target polynucleotide sequence.

As used herein, "knockout" includes deletion of all or part of the target polynucleotide sequence in a manner that interferes with the function of the target polynucleotide sequence. For example, knockout can be achieved by altering a target polynucleotide sequence by inducing an indel in the target polynucleotide sequence in a functional domain (e.g., a DNA binding domain) of the target polynucleotide sequence. Based on the details described herein, one of skill in the art will readily understand how to use the CRISPR/Cas system of the present disclosure to knock out a target polynucleotide sequence or a portion thereof.

In some embodiments, the alteration results in a knockout of the target polynucleotide sequence or portion thereof. Knocking out target polynucleotide sequences or portions thereof using the CRISPR/Cas systems of the present disclosure can be used in a variety of applications. For example, for research purposes, the target polynucleotide sequence in the knocked-out cells may be performed in vitro. For ex vivo purposes, the target polynucleotide sequence in the knockout cell can be used to treat or prevent a disorder associated with expression of the target polynucleotide sequence (e.g., by knocking out mutant alleles in the cell ex vivo and introducing those cells comprising the knockout mutant alleles into the subject).

"Knock-in" herein means the process of adding genetic functions to a host cell. This results in increased levels of knocked-in gene products (e.g., RNA or encoded protein). As will be appreciated by those skilled in the art, this can be accomplished in a variety of ways, including adding one or more additional copies of the gene to the host cell or altering regulatory components of the endogenous gene to increase expression of the protein. This can be achieved by modifying the promoter, adding a different promoter, adding an enhancer or modifying other gene expression sequences.

In some embodiments, the alterations or modifications described herein result in reduced expression of the target or selected polynucleotide sequence. In some embodiments, the alterations or modifications described herein result in reduced expression of the target or selected polypeptide sequence.

In some embodiments, the alterations or modifications described herein result in increased expression of the target or selected polynucleotide sequence. In some embodiments, the alterations or modifications described herein result in increased expression of the target or selected polypeptide sequence.

"Modulation" of gene expression refers to a change in the level of gene expression. Modulation of expression may include, but is not limited to, gene activation and gene suppression. Modulation may also be complete, i.e., where gene expression is completely inactivated or activated to wild-type levels or higher; or it may be partial, wherein gene expression is partially reduced or partially activated to a portion of wild-type levels.

The terms "operatively connected (operatively linked)" or "operatively connected (operably linked)" are used interchangeably with respect to the juxtaposition of two or more components, such as sequential elements, wherein the components are arranged such that both components function properly and allow the possibility that at least one component may mediate a function imposed on at least one other component. For example, a transcriptional regulatory sequence (such as a promoter) is operably linked to a coding sequence if it controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. The transcriptional regulatory sequences are typically operably linked to the coding sequence in a cis-form, but need not be immediately adjacent thereto. For example, enhancers are transcriptional regulatory sequences that are operably linked to a coding sequence even though they are discontinuous.

As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to a series of amino acid residues (i.e., a polymer of amino acid residues) joined by peptide bonds, and are not limited to a minimum length. Such polymers may contain natural or unnatural amino acid residues or combinations thereof, and include, but are not limited to, peptides, polypeptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Thus, proteins or polypeptides include those having modified amino acids (e.g., phosphorylated, glycosylated, etc.) and amino acid analogs. This definition encompasses full-length polypeptides or proteins and fragments thereof. The term also includes substances whose modification, for example post-translational modification of one or more residues, such as methylation, phosphorylation, glycosylation, sialylation or acetylation.

Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be interpreted as a inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual values within the range. For example, where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure and subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. In some embodiments, two opposite and open-ended ranges of characteristics are provided, and in such descriptions, it is contemplated that combinations of these two ranges are provided herein. For example, in some embodiments, a characteristic of greater than about 10 units is described, and a characteristic of less than about 20 units is described (such as in another sentence), thus, a range of about 10 units to about 20 units is described herein.

As used herein, "subject" or "individual" is an interchangeable term, which is a mammal. In some embodiments, "mammal" includes humans, non-human primates, domestic and farm animals, as well as zoo, sports or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc. In some embodiments, the subject or individual is a human. In some embodiments, the subject is a patient known or suspected of having a disease, disorder, or condition.

As used herein, the terms "treating" and "treatment" include administering to a subject an effective amount of a cell described herein such that at least one symptom of a disease in the subject is reduced or the disease is ameliorated, e.g., a beneficial or desired clinical result. For the purposes of this technology, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "treatment" may refer to prolonged survival compared to the expected survival without treatment. Thus, those skilled in the art recognize that treatment may improve a disease condition, but may not be a complete cure for the disease. In some embodiments, after treatment of the disease, one or more symptoms of the disease or disorder are reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

For the purposes of this technique, beneficial or desired clinical results of disease treatment include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

A "vector" or "construct" is capable of transferring a gene sequence to a target cell. In general, "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer a gene sequence to a target cell. Thus, the term includes cloning and expression vectors and integration vectors. Methods for introducing vectors or constructs into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and/or viral vector-mediated transfer.

Engineered cells and methods of engineering cells

Provided herein are engineered cells comprising modifications that regulate expression of CD 142. In some embodiments, the modification reduces or eliminates expression of CD 142. In some embodiments, the modification that reduces expression of CD142 reduces CD142 protein expression. In some embodiments, the modification eliminates CD142 gene activity. In some embodiments, the modification comprises inactivation or disruption of both alleles of the CD142 gene. In some embodiments, the modification comprises inactivation or disruption of all CD142 coding sequences in the cell. In some embodiments, the inactivation or disruption comprises an indel in the CD142 gene. In some embodiments, the modification is a frameshift mutation or deletion of a stretch of contiguous genomic DNA of the CD142 gene. In some embodiments, the CD142 gene is knocked out.

In some embodiments, the engineered cells provided further contain one or more modifications that increase expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, CD55, and combinations thereof. In some embodiments, the modification that increases expression comprises increased surface expression and/or the modification that decreases expression comprises decreased surface expression. In some embodiments, the modification that increases expression of one or more complement inhibitors comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and/or an exogenous polynucleotide encoding CD 55. In some embodiments, the one or more complement inhibitors are CD46 and CD59, optionally wherein the modification comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59. One or more complement inhibitors are CD46, CD59, and CD55, optionally wherein the modification comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55. In some embodiments, the engineered cell comprises a polycistronic vector comprising two or more exogenous polypeptides selected from the group consisting of: one or more exogenous polynucleotides encoding one or more tolerogenic factors, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding a CD55 polypeptide. In some embodiments, each polynucleotide is isolated by an IRES or self-cleaving peptide.

In some embodiments, the engineered cells provided further contain modifications of one or more target polynucleotide sequences that regulate expression of one or more MHC class I molecules, one or more MHC class II molecules, or one or more MHC class I molecules and one or more MHC class II molecules.

In some embodiments, the engineered cells provided further comprise a modification that increases expression of one or more tolerogenic factors. In some embodiments, the tolerogenic factors are one or more of the following: DUX4, B2M-HLA-E, CD, CD52, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc receptor, IL15-RF and H2-M3 or any combination thereof. In some embodiments, the modification that increases expression of one or more tolerogenic factors is or includes increased expression of CD 47. In some embodiments, the modification that increases expression of one or more tolerogenic factors is or includes increased expression of PD-L1. In some embodiments, the modification that increases expression of one or more tolerogenic factors is or includes increased expression of HLA-G. In some embodiments, the modification that increases expression of one or more tolerogenic factors is or includes increased expression of HLA-G. In some embodiments, the modification that increases expression of one or more tolerogenic factors is or includes increased expression of CCL21, PD-L1, fasL, serpin b9, H2-M3 (HLA-G), CD47, CD200, and Mfge.

In some embodiments, the cell comprises one or more genomic modifications that reduce expression of one or more MHC class I molecules and a modification that increases expression of CD 47. In other words, the engineered cells comprise exogenous CD47 protein and exhibit reduced or silenced surface expression of one or more MHC class I molecules. In some embodiments, the cell comprises one or more genomic modifications that reduce expression of one or more MHC class II molecules and a modification that increases expression of CD 47. In some cases, the engineered cells comprise exogenous CD47 nucleic acids and proteins and exhibit reduced or silenced surface expression of one or more MHC class I molecules. In some embodiments, the cell comprises one or more genomic modifications that reduce or eliminate expression of one or more MHC class II molecules, and a modification that increases expression of CD 47. In some embodiments, the engineered cells comprise exogenous CD47 protein, exhibit reduced or silenced surface expression of one or more MHC class I molecules, and exhibit reduced or lack surface expression of one or more MHC class II molecules. In many embodiments, the cells are B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg cells.

In some embodiments, any gene editing technique can be used to reduce the expression of the one or more target polynucleotides or target proteins. In some embodiments, the gene editing techniques may include systems involving nucleases, integrases, transposases, recombinases. In some embodiments, gene editing techniques may be used for knockout or knockdown of genes. In some embodiments, gene editing techniques may be used to knock-in or integrate DNA into regions of the genome. In some embodiments, the gene editing technique mediates Single Strand Breaks (SSBs). In some embodiments, the gene editing technique mediates Double Strand Breaks (DSBs), including in combination with non-homologous end joining (NHEJ) or Homology Directed Repair (HDR). In some embodiments, the gene editing techniques may include DNA-based editing or guided editing. In some embodiments, the gene editing technique may include Programmable Addition (PASTE) via a site-specific targeting element.

In some embodiments, any gene editing technique can be used to increase the expression of the one or more target polynucleotides or target proteins. In some embodiments, the gene editing techniques may include systems involving nucleases, integrases, transposases, recombinases. In some embodiments, gene editing techniques may be used for modification to increase endogenous gene activity (e.g., by modifying or activating a promoter or enhancer operably linked to a gene). In some embodiments, gene editing techniques may be used to knock-in or integrate DNA into regions of the genome (e.g., to introduce constructs encoding target polynucleotides or target proteins, such as constructs encoding any of tolerogenic factors, CD55, CD46, CD59, or any other molecules described herein for increasing expression in engineered cells). In some embodiments, the gene editing technique mediates Single Strand Breaks (SSBs). In some embodiments, the gene editing technique mediates Double Strand Breaks (DSBs), including in combination with non-homologous end joining (NHEJ) or Homology Directed Repair (HDR). In some embodiments, the gene editing techniques may include DNA-based editing or guided editing. In some embodiments, the gene editing technique may include Programmable Addition (PASTE) via a site-specific targeting element.

In some embodiments, the gene editing technique is associated with base editing. Base Editors (BE) are typically fusions of Cas ("CRISPR-associated") domains and nucleobase modification domains (e.g., natural or evolved deaminase, such as cytidine deaminase, which includes apodec 1 ("apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1"), CDA ("cytidine deaminase"), and AID ("activation-induced cytidine deaminase") domains). In some cases, the base editor may also contain proteins or domains that alter cellular DNA repair processes to increase the efficiency and/or stability of the resulting single nucleotide changes.

In some aspects, currently available base editors include a cytidine base editor (e.g., BE 4) that converts target C.G to T.A and an adenine base editor (e.g., ABE 7.10) that converts A.T to G.C. In some aspects, cas9 targeted deamination was demonstrated for the first time to BE associated with a Base Editor (BE) system designed to induce base changes without introducing double-stranded DNA breaks. Further, the rat deaminase apodec 1 (rAPOBEC 1) fused to the inactivated Cas9 (dCas 9) was used successfully to convert cytidine upstream of PAM of sgRNA to thymidine. In some aspects, this first BE system is optimized by changing dCas9 to a "nickase" Cas 9D 10A that nicks on the deaminated cytidine opposing strand. Without being bound by theory, this is expected to initiate long patch Base Excision Repair (BER), where deaminated chains are preferentially used for template repair to produce U.A base pairs, which are then converted to T.A during DNA replication.

In some embodiments, the base editor is a nucleobase editor comprising a catalytically inactive first DNA-binding protein domain, a domain having base editing activity, and a second DNA-binding protein domain having nicking enzyme activity, wherein the DNA-binding protein domains are expressed on a single fusion protein or expressed separately (e.g., on separate expression vectors). In some embodiments, the base editor is a fusion protein comprising a domain having base editing activity (e.g., cytidine deaminase or adenosine deaminase) and two nucleic acid programmable DNA binding protein domains (napDNAbp), the first having nickase activity, and the second napDNAbp being catalytically inactive, wherein at least two napDNAbp are linked by a linker. In some embodiments, the base editor is a fusion protein comprising a DNA domain of a CRISPR-Cas (e.g., cas 9) having nickase activity (nCas; nCas 9), a catalytically inactive domain of a CRISPR-Cas protein (e.g., cas 9) (dCas; e.g., dCas 9) having nucleic acid programmable DNA binding activity, and a deaminase domain, wherein dCas is linked to nCas by a linker and dCas is immediately adjacent to the deaminase domain. In some embodiments, the base editor is an adenine to thymine or "ATBE" (or thymine to adenine or "TABE") transversion base editor. Exemplary base editors and base editor systems include any base editor and base editor system as described in patent publication number US20220127622、US20210079366、US20200248169、US20210093667、US20210071163、WO2020181202、WO2021158921、WO2019126709、WO2020181178、WO2020181195、WO2020214842、WO2020181193, which is hereby incorporated by reference in its entirety.

In some embodiments, the gene editing technique is target-initiated reverse transcription (TPRT) or "guided editing". In some embodiments, the guided editing mediates targeted insertions, deletions, all 12 possible base-to-base conversions, and combinations thereof in human cells without the need for DSBs or donor DNA templates.

Guided editing is a genomic editing method that uses a nucleic acid programmable DNA binding protein ("napDNAbp") working in conjunction with a polymerase (i.e., in the form of a fusion protein provided in trans with napDNAbp or otherwise) to write new genetic information directly to a designated DNA site, wherein the guided editing system is programmed with a guided editing (PE) guide RNA ("PEgRNA") that both designates the target site and templates the synthesis of the desired editing by engineering onto the guide RNA in the form of a replacement DNA strand (e.g., at the 5 'or 3' end, or at an internal portion of the guide RNA). The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence with the endogenous strand of the target site to be edited (except that it contains the desired edit). The endogenous strand of the target site is replaced by a newly synthesized replacement strand containing the desired editing by DNA repair and/or replication mechanisms. In some cases, guided editing may be considered a "search and replace" genome editing technique in that the guided editor searches for and locates the desired target site to be edited and encodes a replacement strand containing the desired editing, which simultaneously installs a strand of endogenous DNA that replaces the corresponding target site. For example, guided editing may be adapted for precise CRISPR/Cas-based genome editing to avoid double strand breaks. In some embodiments, the homologous protein is or encodes a Cas protein-reverse transcriptase fusion or related system to target a particular DNA sequence with a guide RNA, generate a single-stranded nick at the target site, and use the nick DNA as a primer for reverse transcription of an engineered reverse transcriptase template integrated with the guide RNA. In some embodiments, the guide editor protein is paired with two guide editing guide RNAs (pegrnas) that template synthesis of complementary DNA flaps on opposite strands of genomic DNA, resulting in replacement of endogenous DNA sequences between PE-induced nick sites with pegRNA-encoded sequences.

In some embodiments, the gene editing technique is associated with a guided editor that is a reverse transcriptase or any DNA polymerase known in the art. Thus, in one aspect, the guide editor can comprise Cas9 (or equivalently napDNAbp) programmed to target a DNA sequence by associating the DNA sequence with a specialized guide RNA (i.e., PEgRNA) containing a spacer sequence that anneals to a complementary proto-spacer in the target DNA. Such methods include Anzalone et al (doi.org/10.1038/s 41586-019-1711-4) or any of the methods disclosed in PCT publication Nos. WO2020191248, WO2021226558 or WO2022067130, which are incorporated herein in their entirety.

In some embodiments, the gene editing technique is via programmable addition of a site-specific targeting element (PASTE). In some aspects, a PASTE is a platform in which genomic insertion is guided via a CRISPR-Cas9 nickase fused to both a reverse transcriptase and a serine integrase. As described in Ioannidi et al (doi. Org/10.1101/2021.11.01.466786), PASTE does not generate a double strand break, but allows integration of sequences of about 36 kb. In some embodiments, the serine integrase may be any serine integrase known in the art. In some embodiments, the serine integrase has sufficient orthogonality that the PASTE can be used for multiple gene integration, integrating at least two different genes at least two genomic loci simultaneously. In some embodiments, the PASTE has editing efficiency comparable to or superior to integration based on homology-directed repair or non-homologous end joining, has activity in non-dividing cells, and fewer detectable off-target events.

In some embodiments, the engineered cell population elicits reduced levels of immune activation or no immune activation upon administration to a recipient subject. In some embodiments, the cells elicit reduced levels of systemic TH1 activation or do not elicit systemic TH1 activation in the recipient subject. In some embodiments, the cells elicit a reduced level of immune activation of Peripheral Blood Mononuclear Cells (PBMCs) or do not elicit immune activation of PBMCs in the recipient subject. In some embodiments, the cells elicit reduced levels of donor-specific IgG antibodies against the cells or do not elicit the donor-specific IgG antibodies after administration to the recipient subject. In some embodiments, the cells elicit reduced levels of IgM and IgG antibody production in the recipient subject that are directed against the cells or do not elicit such IgM and IgG antibody production. In some embodiments, the cells elicit a reduced level of cytotoxic T cell killing of the cells after administration to a recipient subject.

In some embodiments, the cells provided herein comprise a "suicide gene" or a "suicide switch. Suicide genes or suicide switches may be incorporated to act as "safety switches" that can cause the death of the engineered cells (e.g., primary engineered cells or cells differentiated from engineered pluripotent stem cells), such as after administration of the engineered cells to a subject and when the cells grow and divide in an undesired manner. The "suicide gene" ablation method includes suicide genes in gene transfer vectors, which suicide genes encode proteins that cause cell killing only when activated by a given compound. Suicide genes may encode enzymes that selectively convert non-toxic compounds to highly toxic metabolites. The result is a specific elimination of the cells expressing the enzyme. In some embodiments, the suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene and the trigger is ganciclovir. In other embodiments, the suicide gene is the E.coli cytosine deaminase (EC-CD) gene and the trigger is 5-fluorocytosine (5-FC) (Barese et al mol. Therapeutic.20 (10): 1932-1943 (2012), xu et al, cell Res.8:73-8 (1998), both of which are incorporated herein by reference in their entirety).

In other embodiments, the suicide gene is an inducible cysteine protease protein. The inducible cysteine protease protein comprises at least a portion of a cysteine protease protein capable of inducing apoptosis. In some embodiments, the inducible cysteine protease protein is iCasp9. Comprising the sequence of the human FK506 binding protein FKBP12 with the F36V mutation linked by a series of amino acids to the gene encoding human cysteine protease 9. FKBP12-F36V binds with high affinity to the small molecule dimerization API 903. Thus, the suicide function of iCasp9 in the present invention is triggered by the application of a dimerization Chemical Inducer (CID). In some embodiments, CID is small molecule drug API 903. Dimerization leads to rapid induction of apoptosis. ( See WO2011146862; stasi et al, N.Engl.J.Med 365;18 (2011); tey et al, biol. Blood Marrow Transmount.13:913-924 (2007), each of which is incorporated herein by reference in its entirety. )

In the event of cytotoxicity or other negative consequences to the recipient, the inclusion of a safety switch or suicide gene allows controlled cell killing, thereby increasing the safety of cell-based therapies, including therapies using tolerogenic factors.

In some embodiments, the safety switch may be incorporated (such as introduced) into an engineered cell provided herein to provide the ability to induce death or apoptosis of the engineered cell containing the safety switch, for example, when the cell grows and divides in an undesired manner or is overly toxic to the host. Thus, the use of safety switches enables one to conditionally eliminate abnormal cells in the body and may be a critical step in the clinical application of cell therapies. Safety switches and their use are described, for example, in Duzgune ≡ Origins of Suicide GENE THERAPY (2019); duzgune ≡3 (code), suicide Gene therapeutics, methods in Molecular Biology, vol.1895 (Humana Press, new York, NY) (for HSV-tk, cytosine deaminase, nitroreductase, purine nucleoside phosphorylase and horseradish peroxidase); zhou and Brenner Exp Hematol (11): 1013-1019 (2016) (for iCaspase); wang et al, blood 18 (5): 1255-1263 (2001) (for huEGFR); U.S. patent application publication number 20180002397 (for HER 1); and Philip et al, blood124 (8): 1277-1287 (2014) (for RQR 8).

In some embodiments, the safety switch may cause cell death in a controlled manner, e.g., in the presence of a drug or prodrug or after activation by a selective exogenous compound. In some embodiments, the safety switch is selected from the group consisting of: herpes simplex virus thymidine kinase (HSV-tk), cytosine deaminase (CyD), nitroreductase (NTR), purine Nucleoside Phosphorylase (PNP), horseradish peroxidase, inducible cysteine protease 9 (iCasp 9), rapamycin activated cysteine protease 9 (rapaCasp), CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA and RQRR 8.

In some embodiments, the safety switch may be a transgene encoding a product that has cell killing ability when activated by a drug or prodrug (e.g., by intracellular conversion of a non-toxic prodrug to a toxic metabolite). In these embodiments, cell killing is activated by contacting the engineered cells with a drug or prodrug. In some cases, the safety switch is HSV-tk, which converts Ganciclovir (GCV) to GCV-triphosphate, thereby interfering with DNA synthesis and killing dividing cells. In some cases, the safety switch is CyD or a variant thereof that converts the antifungal drug 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU) by catalyzing the hydrolytic deamination of cytosine to uracil. The 5-FU is further converted into potent antimetabolites by cellular enzymes (5-FdUMP, 5-FdUTP, 5-FUTP). These compounds inhibit thymidylate synthase and the production of RNA and DNA, leading to cell death. In some cases, the safety switch is NTR or a variant thereof, which may act on the prodrug CB 1954 via reduction of the nitro group to a reactive N-hydroxylamine intermediate that is toxic in proliferating and non-proliferating cells. In some cases, the safety switch is a PNP or variant thereof that can convert the prodrug 6-methylpurine deoxynucleoside or fludarabine to a metabolite that is toxic to both proliferating and non-proliferating cells. In some cases, the safety switch is horseradish peroxidase or a variant thereof, which can catalyze indole-3-acetic acid (IAA) to be a potent cytotoxin, thereby effecting cell killing.

In some embodiments, the safety switch may be iCasp9. Cysteine protease 9 is a component of the intrinsic mitochondrial apoptotic pathway that is activated under physiological conditions by the release of cytochrome C by the damaged mitochondria. The activated cysteine protease 9 then activates cysteine protease 3, which cysteine protease 3 triggers a terminal effector molecule leading to apoptosis. iCasp9 may be generated by fusing truncated cysteine protease 9 (without its physiological dimerization domain or cysteine protease activation domain) to FK506 binding protein (FKBP) FKBP12-F36V via a peptide linker. iCasp9 has a low dimer-dependent basal activity and can be stably expressed in host cells (e.g., human T cells) without compromising its phenotype, function, or antigen specificity. However, iCasp9 may undergo induced dimerization and activation of downstream cysteine protease molecules in the presence of chemical dimerization inducers (CIDs) such as Li Midu west (rimiducid) (AP 1903), AP20187, and rapamycin, resulting in apoptosis of the iCasp9 expressing cells. See, for example, PCT application publication No. WO2011/146862; stasi et al, n.engl.j.med.365;18 (2011); tey et al, biol. Blood Marrow Transplant13:913-924 (2007). In particular, the rapamycin inducible cysteine protease 9 variant is designated rapaCasp. See Stavrou et al, mal. Ther.26 (5): 1266-1276 (2018). Thus, iCasp9 can be used as a safety switch to achieve controlled killing of host cells.

In some embodiments, the safety switch may be a membrane-expressed protein that allows cell depletion following administration of antibodies specific for this protein. Such safety switches may include, for example, one or more transgenes encoding CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, or RQR8 for surface expression thereof. These proteins may have surface epitopes that can be targeted by specific antibodies. In some embodiments, the safety switch comprises CCR4, which can be recognized by an anti-CCR 4 antibody. Non-limiting examples of suitable anti-CCR 4 antibodies include Mo Geli bead mab and biological analogs thereof. In some embodiments, the safety switch comprises CD16 or CD30, which can be recognized by an anti-CD 16 or anti-CD 30 antibody. Non-limiting examples of such anti-CD 16 or anti-CD 30 antibodies include AFM13 and biological analogs thereof. In some embodiments, the safety switch comprises CD19, which can be recognized by an anti-CD 19 antibody. Non-limiting examples of such anti-CD 19 antibodies include MOR208 and biological analogs thereof. In some embodiments, the safety switch comprises CD20, which can be recognized by an anti-CD 20 antibody. Non-limiting examples of such anti-CD 20 antibodies include rituximab, oxcarbatuzumab, rituximab-RIIb, and biological analogs thereof. Thus, cells expressing the safety switch are CD20 positive and killing can be targeted by administration of an anti-CD 20 antibody as described above. In some embodiments, the safety switch comprises EGFR, which can be recognized by anti-EGFR antibodies. Non-limiting examples of such anti-EGFR antibodies include tobraziumab, RO5083945 (GA 201), cetuximab, and biological analogs thereof. In some embodiments, the safety switch comprises GD2, which can be recognized by an anti-GD 2 antibody. Non-limiting examples of such anti-GD 2 antibodies include Hul4.18K322A, hul4.18-IL2, hu3F8, rituximab, c.60C3-Rllc, and biological analogs thereof.

In some embodiments, the safety switch may be an exogenously administered agent that recognizes one or more tolerogenic factors on the surface of the engineered cell. In some embodiments, the exogenously administered agent is an antibody, e.g., an anti-CD 47 antibody, directed against or specific for the tolerogenic agent. By recognizing and blocking tolerogenic factors on engineered cells, exogenously administered antibodies can block the immunosuppressive function of tolerogenic factors, thereby re-sensitizing the immune system to the engineered cells. For example, for an engineered cell that overexpresses CD47, an exogenously administered anti-CD 47 antibody can be administered to a subject, resulting in masking of CD47 on the engineered cell and triggering an immune response to the engineered cell.

In some embodiments, the method further comprises introducing into the cell an expression vector comprising an inducible suicide switch.

In some embodiments, the engineered cells are derived from source cells that already contain one or more desired modifications. In some embodiments, in view of the teachings provided herein, one of ordinary skill in the art will readily understand how to evaluate which modifications are needed to achieve a desired final form of an engineered cell, and not all target component level decreases or increases are achieved via activity engineering. In some embodiments, the modification of the engineered cells may be in any order, and not necessarily in the order listed in the descriptive language provided herein.

In some embodiments, provided herein is a method of producing an engineered cell, comprising: (a) Reducing or eliminating expression of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules) in a cell; (b) reducing expression of CD142 in the cell; and (c) increasing expression of the tolerogenic factors in the cell. In some embodiments, the one or more tolerogenic factors are selected from the group consisting of DUX4, B2M-HLA-E, CD, CD52, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc receptor, IL15-RF and H2-M3. In some embodiments, the one or more tolerogenic factors is CD47. In some embodiments, the methods comprise reducing or eliminating expression of one or more MHC class I molecules and one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules). In some embodiments, decreasing or increasing expression comprises one or more modifications to the cell using a guide nuclease (e.g., CRISPR/Cas system). In some embodiments, the method further comprises introducing into the cell an expression vector comprising an inducible suicide switch. In some embodiments, the method further comprises increasing expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD55 in the cell.

In some embodiments, provided herein is a method of producing an engineered cell, comprising: (a) Increasing expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200, and MFGE8 in a cell, and (b) decreasing expression of CD142 in a cell. In some embodiments, decreasing or increasing expression comprises one or more modifications to the cell using a guide nuclease (e.g., CRISPR/Cas system). In some embodiments, the method further comprises introducing into the cell an expression vector comprising an inducible suicide switch. In some embodiments, the method further comprises increasing expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD55 in the cell.

Once altered, the presence of expression of any of the molecules described herein can be determined using known techniques, such as western blotting, ELISA assays, FACS assays, and the like.

A. Reduced target gene expression

1. Target gene

MHC class I and/or MHC class II molecules

In some embodiments, an engineered cell is provided that comprises a modification (e.g., genetic modification) of one or more target polynucleotide or protein sequences (also interchangeably referred to as target genes) that regulate (e.g., reduce or eliminate) expression of one or more MHC class I molecules, one or more MHC class II molecules, or one or more MHC class I molecules and one or more MHC class II molecules. In some embodiments, the cell to be modified or engineered is an unmodified cell or a non-engineered cell into which one or more modifications have not been previously introduced. In some embodiments, the gene editing system is used to modify one or more target polynucleotide sequences that modulate (e.g., reduce or eliminate) expression of one or more MHC class I molecules, one or more MHC class II molecules, or one or more MHC class I molecules and one or more MHC class II molecules. In certain embodiments, the genome of the cell has been altered to reduce or delete components required or involved in promoting HLA expression (such as expression of one or more MHC class I molecules and/or one or more MHC class II molecules on the cell surface). For example, in some embodiments, expression of β -2-microglobulin (B2M) (a component of one or more MHC class I molecules) in a cell is reduced or eliminated, thereby reducing or eliminating protein expression (e.g., cell surface expression) of one or more MHC class I molecules of the engineered cell.

In some embodiments, decreasing expression of one or more MHC class I molecules or MHC class II molecules decreases expression ：B2M、TAP I、NLRC5、CIITA、HLa-a、HLA-B、HLA-C、HLA-DP、HLA-DM、HLA-DOA、HLA-DOB、HLA-DQ、HLA-DR、RFX5、RFXANK、RFXAP、NFY-A、NFY-B and/or NFY-C of one or more of the following.

In some embodiments, any of the modifications in the engineered cells that regulate (e.g., reduce or eliminate) expression of one or more target polynucleotides or proteins in the engineered cells can be combined with one or more modifications to overexpress the polynucleotide described in section ii.b (e.g., tolerogenic factors such as CD 47).

In some embodiments, reducing expression of one or more MHC class I molecules and/or one or more MHC class II molecules may be achieved, for example, by one or more of the following: (1) Direct targeting of polymorphic HLA alleles (HLA-A, HLA-B, HLA-C) and one or more MHC class II genes; (2) Removal of B2M, which will reduce surface transport of all one or more MHC class I molecules; and/or (3) deletion of one or more MHC enhancer components critical to HLA expression, such as LRC5, RFX-5, RFXANK, RFXAP, IRFl, NF-Y (including NFY-A, NFY-B, NFY-C) and CIITA.

In certain embodiments, HLA expression is disrupted. In some embodiments, HLA expression is interfered with by: targeting individual HLA (e.g., knockout of HLA-a, HLA-B, and/or HLA-C), targeting transcriptional modulators of HLA expression (e.g., knockout of expression of NLRC5, CIITA, RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, and/or IRF-1), blocking surface transport of one or more MHC class I molecules (e.g., knockout of expression of B2M and/or TAP 1), and/or targeting with HLA-razors (see, e.g., WO 2016183041).

Human Leukocyte Antigen (HLA) complex is synonymous with human MHC. In some embodiments, the engineered cells disclosed herein are human cells. In certain aspects, the engineered cells disclosed herein do not express one or more human leukocyte antigens (e.g., HLA-A, HLa-B, and/or HLa-C) corresponding to one or more MHC class I molecules and/or one or more MHC class II molecules, and are therefore characterized as being hypoimmunogenic. For example, in certain aspects, the engineered cells disclosed herein have been modified such that the cells (including any stem cells or differentiated stem cells prepared therefrom) do not express or exhibit reduced expression of one or more of the following MHC class I molecules: HLA-A, HLA-B and HLA-C. In some embodiments, one or more of HLA-A, HLA-B, and HLA-C may be "knocked out" of the cell. Cells with knockouts of HLA-A genes, HLA-B genes, and/or HLA-C genes may exhibit reduced or eliminated expression of each knocked-out gene.

In certain embodiments, expression of one or more MHC class I molecules and/or one or more MHC class II molecules is modulated by targeting and deleting a contiguous stretch of genomic DNA, thereby reducing or eliminating expression of a target gene selected from the group consisting of B2M, CIITA and NLRC 5.

In some embodiments, the engineered cells provided comprise modifications that modulate one or more target polynucleotide sequences of one or more MHC class I molecules. Exemplary methods for reducing expression of one or more MHC class I molecules are described in the following sections. In some embodiments, the target polynucleotide sequence is one or both of B2M and NLRC 5. In some embodiments, the cell comprises a genetic editing modification (e.g., an indel) to the B2M gene. In some embodiments, the cell comprises a genetic editing modification (e.g., an indel) to the NLRC5 gene. In some embodiments, the cells comprise genetic editing modifications (e.g., indels) to B2M and CIITA genes.

In some embodiments, the modification that reduces expression of one or more MHC class I molecules is a modification that reduces expression of B2M. In some embodiments, the modification that reduces B2M expression reduces B2M mRNA expression. In some embodiments, the decrease in mRNA expression of B2M is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification. In some embodiments, mRNA expression of B2M is reduced by more than about 5%, such as by more than about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, mRNA expression of B2M is reduced by up to about 100%, such as by up to any of about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less. In some embodiments, mRNA expression of B2M is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, mRNA expression of B2M is eliminated (e.g., 0% expression of B2M mRNA). In some embodiments, modifications that reduce B2M mRNA expression eliminate B2M gene activity.

In some embodiments, the modification that reduces B2M expression reduces B2M protein expression. In some embodiments, the reduced protein expression of B2M is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification. In some embodiments, protein expression of B2M is reduced by more than about 5%, such as by more than about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, protein expression of B2M is reduced by up to about 100%, such as by up to any of about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less. In some embodiments, the protein expression of B2M is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, protein expression of B2M is eliminated (e.g., there is no detectable expression of B2M protein). In some embodiments, modifications that reduce B2M protein expression eliminate B2M gene activity.

In some embodiments, the modification that reduces B2M expression comprises inactivation or disruption of the B2M gene. In some embodiments, the modification that reduces B2M expression comprises inactivation or disruption of one allele of the B2M gene. In some embodiments, the modification that reduces B2M expression comprises inactivation or disruption of both alleles of the B2M gene.

In some embodiments, the modification comprises inactivation or disruption of one or more B2M coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption of all B2M coding sequences in the cell. In some embodiments, the modification comprises an indel in the B2M gene. In some embodiments, the modification is a frameshift mutation of genomic DNA of the B2M gene. In some embodiments, the modification is a deletion of genomic DNA of the B2M gene. In some embodiments, the modification is a deletion of a stretch of contiguous genomic DNA of the B2M gene. In some embodiments, the B2M gene is knocked out.

In some embodiments, the engineered cells provided comprise modifications that modulate one or more target polynucleotide sequences of one or more MHC class II molecules. Exemplary methods for reducing expression of one or more MHC class II molecules are described in the following sections. In some embodiments, the cell comprises a genetic editing modification to the CIITA gene.

In some embodiments, the modification that reduces expression of one or more MHC class II molecules is a modification that reduces expression of CIITA. In some embodiments, the modification that reduces CIITA expression reduces CIITA MRNA expression. In some embodiments, the decrease in mRNA expression of CIITA is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification. In some embodiments, the mRNA expression of CIITA is reduced by more than about 5%, such as by more than about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the mRNA expression of CIITA is reduced by up to about 100%, such as by up to any of about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less. In some embodiments, the mRNA expression of CIITA is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, mRNA expression of CIITA is eliminated (e.g., expression of 0% CIITA MRNA). In some embodiments, the modification that reduces CIITA MRNA expression eliminates CIITA gene activity.

In some embodiments, the modification that reduces CIITA expression reduces CIITA protein expression. In some embodiments, the reduced protein expression of CIITA is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification. In some embodiments, the protein expression of CIITA is reduced by more than about 5%, such as by more than about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the protein expression of CIITA is reduced by up to about 100%, such as by up to any of about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less. In some embodiments, the protein expression of CIITA is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, protein expression of CIITA is eliminated (e.g., expression of 0% CIITA protein). In some embodiments, modifications that reduce expression of the CIITA protein eliminate CIITA gene activity.

In some embodiments, the modification that reduces CIITA expression comprises inactivation or disruption of the CIITA gene. In some embodiments, the modification that reduces CIITA expression comprises inactivation or disruption of one allele of the CIITA gene. In some embodiments, the modification that reduces CIITA expression comprises inactivation or disruption of both alleles of the CIITA gene.

In some embodiments, the modification comprises inactivation or disruption of one or more B2M coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption of all B2M coding sequences in the cell. In some embodiments, the modification comprises an indel in the B2M gene. In some embodiments, the modification is a frameshift mutation of genomic DNA of the B2M gene. In some embodiments, the modification is a deletion of genomic DNA of the B2M gene. In some embodiments, the modification is a deletion of a stretch of contiguous genomic DNA of the B2M gene. In some embodiments, the CIITA gene is knocked out.

In some embodiments, the engineered cells provided comprise modifications that modulate one or more target polynucleotide sequences of one or more MHC class I molecules and one or more MHC class II molecules. Exemplary methods for reducing expression of one or more MHC class I molecules and one or more MHC class II molecules are described in the following sections. In some embodiments, the cell comprises genetic editing modifications to B2M and NLRC5 genes. In some embodiments, the cells comprise genetic editing modifications to the CIITA and NLRC5 genes. In particular embodiments, the cells comprise genetic editing modifications to the B2M, CIITA and NLRC5 genes.

B.CD142

In certain aspects, the techniques disclosed herein modulate (e.g., reduce or eliminate) expression of CD142, CD142 also being referred to as tissue factor, factor III, and F3. In some embodiments, modulation is performed using a CRISPR/Cas system.

In some embodiments, the target polynucleotide sequence is CD142 or a variant of CD 142. In some embodiments, the target polynucleotide sequence is a homolog of CD 142. In some embodiments, the target polynucleotide sequence is an ortholog of CD 142.

In some embodiments, the cells outlined herein comprise modifications that target the CD142 gene. In some embodiments, the modification to target the CD142 gene by the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the CD142 gene. Methods useful for identifying a CD 142-targeting gRNA sequence are described below.

Assays for testing whether the CD142 gene has been inactivated are known and described herein. In one embodiment, the modification of the CD142 gene and the reduction in CD142 expression by PCR can be determined by FACS analysis. In another embodiment, CD142 protein expression is detected using western blotting of cell lysates probed with antibodies to CD142 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating modifications.

Available genomic, polynucleotide and polypeptide information about human CD142 is provided, for example, in GeneCard identifiers GC01M094530, HGNC No.3541, NCBI Gene ID 2152, NCBI RefSeq No. nm_001178096.1, nm_001993.4, np_001171567.1 and np_001984.1, uniProt No. p13726, and the like.

2. Methods for reducing expression

In some embodiments, the cells provided herein are modified (e.g., genetically modified) to reduce expression of the one or more target polynucleotides or proteins. In some embodiments, the cell engineered with one or more modifications to reduce (e.g., eliminate) expression of the polynucleotide or protein is any source cell as described herein. In some embodiments, the source cell is any of the cells described in section ii.c. In certain embodiments, a cell disclosed herein (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell such as a beta islet cell or a liver cell, or a primary cell) comprises one or more modifications to reduce expression of one or more target polynucleotides. Non-limiting examples of one or more target polynucleotides include any of those described above (such as CD 142), and one or more of CIITA, B2M, NLRC, HLA-A, HLA-B, HLA-C, LRC5, RFX-ANK, RFX5, RFX-AP, NFY-A, NFY-B, NFY-C, IRF1, and TAP 1. In some embodiments, modifications that reduce the expression of one or more target polynucleotides are combined with one or more modifications that increase the expression of a desired transgene (such as any of the transgenes described in section ii.b). In some embodiments, the modification results in an immune-immune engineered cell or a low immunogenicity cell. Such cells exhibit reduced immune activation upon implantation into a recipient subject by modulating (e.g., reducing or deleting) expression of one or more target polynucleotides. In some embodiments, the cells are considered to be hypoimmunogenic, e.g., in a recipient subject or patient after administration.

Any method for reducing expression of a target polynucleotide may be used. In some embodiments, the modification results in permanent elimination or reduction of expression of the target polynucleotide. For example, in some embodiments, a target polynucleotide or gene is disrupted by introducing a DNA break in the target polynucleotide (such as by using a targeting endonuclease). In other embodiments, the modification results in a transient decrease in expression of the target polynucleotide. For example, in some embodiments, an inhibitory nucleic acid complementary to the target polynucleotide is used to selectively inhibit or suppress expression of the gene (e.g., using antisense technology, such as by RNA interference (RNAi), short interfering RNA (siRNA), short hairpin (shRNA), and/or ribozyme).

In some embodiments, the target polynucleotide sequence is a genomic sequence. In some embodiments, the target polynucleotide sequence is a human genomic sequence. In some embodiments, the target polynucleotide sequence is a mammalian genomic sequence. In some embodiments, the target polynucleotide sequence is a vertebrate genomic sequence.

In some embodiments, gene disruption is typically performed in a targeted manner by inducing one or more double strand breaks and/or one or more single strand breaks in the gene. In some embodiments, the double-or single-strand break is produced by a nuclease (e.g., an endonuclease, such as a gene-targeted nuclease). In some embodiments, the targeting nuclease is selected from the group consisting of Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases such as CRISPR-associated nucleases (Cas), which are specifically designed for the sequence of a gene or a portion thereof. In some embodiments, the targeted nuclease generates a double-stranded or single-stranded break, which is then repaired by error-prone non-homologous end joining (NHEJ), or in some cases, by precise Homology Directed Repair (HDR) using a template. In some embodiments, the targeted nuclease generates a DNA Double Strand Break (DSB). In some embodiments, the process of creating and repairing breaks is generally error-prone and results in insertions and deletions of DNA bases from NHEJ repair (indels). In some embodiments, the modification may induce deletion, insertion, or mutation of the nucleotide sequence of the target gene. In some cases, the modification may result in a frame shift mutation, which may result in a premature stop codon. In the case of nuclease-mediated gene editing, targeted editing occurs on both alleles of a gene, resulting in double allele disruption or editing of the gene. In some embodiments, the gene editing targets all alleles of a gene. In some embodiments, modification using a targeting nuclease (such as using a CRISPR/Cas system) results in a complete knockout of the gene.

In some embodiments, a nuclease (such as a rare-cutting endonuclease) is introduced into the cell containing the target polynucleotide sequence. The nuclease may be introduced into the cell in the form of a nucleic acid encoding the nuclease. The process of introducing the nucleic acid into the cell may be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid mediated transfection, electroporation and transduction or infection with viral vectors. In some embodiments, the nucleic acid introduced into the cell is DNA. In some embodiments, the nuclease is introduced into the cell in the form of a protein. For example, in the case of a CRISPR/Cas system, ribonucleoprotein (RNP) can be introduced into cells.

In some embodiments, the modification is performed using a CRISPR/Cas system. Any CRISPR/Cas system capable of altering a target polynucleotide sequence in a cell can be used. Such CRISPR-Cas systems can employ a variety of Cas proteins (Haft et al, PLoS Comput biol.2005;1 (6) e 60). Molecular mechanisms of such Cas proteins that allow CRISPR/Cas systems to alter target polynucleotide sequences in cells include RNA-binding proteins, endonucleases and exonucleases, helicases and polymerases. In some embodiments, the CRISPR/Cas system is a type I CRISPR system. In some embodiments, the CRISPR/Cas system is a type II CRISPR system. In some embodiments, the CRISPR/Cas system is a V-type CRISPR system.

CRISPR/Cas systems include targeting systems that can be used to alter any target polynucleotide sequence in a cell. In some embodiments, a CRISPR/Cas system provided herein includes a Cas protein and one or more (such as at least one to two) ribonucleic acids (e.g., guide RNAs (grnas)) capable of directing and hybridizing the Cas protein to a target motif of a target polynucleotide sequence.

In some embodiments, the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprise conservative amino acid substitutions. In some cases, the substitution and/or modification may prevent or reduce proteolytic degradation and/or extend the half-life of the polypeptide in the cell. In some embodiments, the Cas protein may comprise peptide bond substitutions (e.g., urea, thiourea, carbamate, sulfonylurea, etc.). In some embodiments, the Cas protein may comprise naturally occurring amino acids. In some embodiments, the Cas protein may comprise a surrogate amino acid (e.g., D-amino acid, β -amino acid, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein may comprise modifications to include moieties (e.g., pegylation, glycosylation, lipidation, acetylation, capping, etc.).

In some embodiments, the Cas protein comprises a core Cas protein. Exemplary Cas core proteins include, but are not limited to, cas1, cas2, cas3, cas4, cas5, cas6, cas7, cas8, and Cas9. In some embodiments, the Cas protein comprises a Cas protein of the e.coli subtype (also referred to as CASS 2). Exemplary Cas proteins of e.coli subtypes include, but are not limited to, cse1, cse2, cse3, cse4, and Cas5e. In some embodiments, the Cas protein comprises Cas protein of subtype Ypest (also referred to as CASS 3). Exemplary Cas proteins of subtype Ypest include, but are not limited to Csy1, csy2, csy3, and Csy4. In some embodiments, the Cas protein comprises Cas protein of subtype Nmeni (also referred to as CASS 4). Exemplary Cas proteins of subtype Nmeni include, but are not limited to Csn1 and Csn2. In some embodiments, the Cas protein comprises Cas protein of subtype Dvulg (also referred to as CASS 1). Exemplary Cas proteins of subtype Dvulg include Csd1, csd2, and Cas5d. In some embodiments, the Cas protein comprises Cas protein of subtype Tneap (also referred to as CASS 7). Exemplary Cas proteins of subtype Tneap include, but are not limited to Cst1, cst2, cas5t. In some embodiments, the Cas protein comprises Cas protein of subtype Hmari. Exemplary Cas proteins of subtype Hmari include, but are not limited to Csh1, csh2, and Cas5h. In some embodiments, the Cas protein comprises Cas protein of subtype Apern (also referred to as CASS 5). Exemplary Cas proteins of subtype Apern include, but are not limited to Csa1, csa2, csa3, csa4, csa5, and Cas5a. In some embodiments, the Cas protein comprises Cas protein of subtype Mtube (also referred to as CASS 6). Exemplary Cas proteins of subtype Mtube include, but are not limited to Csm1, csm2, csm3, csm4, and Csm5. In some embodiments, the Cas protein comprises a RAMP module Cas protein. Exemplary RAMP module Cas proteins include, but are not limited to, cmr1, cmr2, cmr3, cmr4, cmr5, and Cmr6. See, e.g., klompe et al, nature 571,219-225 (2019); strecker et al, science 365,48-53 (2019).

In some embodiments, methods for genetically modifying a cell to knock out, knock down, or otherwise modify one or more genes include the use of site-directed nucleases, including, for example, zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, transposases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems.

ZFNs are fusion proteins comprising a series of site-specific DNA binding domains adapted from zinc finger transcription factors attached to the endonuclease domain of bacterial fokl restriction enzymes. ZFNs can have one or more (e.g., 1,2, 3, 4,5, 6,7, 8, 9, 10, or more) DNA binding domains or zinc finger domains. See, for example, carroll et al Genetics Society of America (2011) 188:773-782; kim et al Proc.Natl.Acad.Sci.USA (1996) 93:1156-1160. Each zinc finger domain is a small protein structural motif stabilised by one or more zinc ions and typically recognizes a 3 to 4bp DNA sequence. Thus, the tandem domain can potentially bind to an extended nucleotide sequence unique in the cell genome.

Various zinc fingers of known specificity may be combined to produce multi-finger polypeptides that recognize about 6, 9, 12, 15 or 18bp sequences. Various selection and modular assembly techniques can be used to generate zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast single hybridization systems, bacterial single and double hybridization systems, and mammalian cells. The zinc fingers can be engineered to bind to a predetermined nucleic acid sequence. Criteria for engineering zinc fingers to bind to predetermined nucleic acid sequences are known in the art. See, for example, sera et al, biochemistry (2002) 41:7074-7081; liu et al, bioinformation (2008) 24:1850-1857.

ZFNs containing fokl nuclease domains or other dimeric nuclease domains are used as dimers. Thus, a pair of ZFNs is required to target non-palindromic DNA sites. Two separate ZFNs must bind opposite strands of DNA by properly spaced nucleases. See Bitinaite et al, proc.Natl. Acad. Sci. USA (1998) 95:10570-10575. To cleave a designated site in the genome, a pair of ZFNs is designed to recognize two sequences flanking the site, one on the forward strand and the other on the reverse strand. When ZFNs bind on either side of the site, the nuclease domain dimerizes and cleaves DNA at the site, generating a DSB with a 5' overhang. The HDR can then be used to introduce specific mutations by means of a repair template containing the desired mutation flanked by homology arms. Repair templates are typically exogenous double-stranded DNA vectors that are introduced into cells. See Miller et al, nat. Biotechnol. (2011) 29:143-148; hockemeyer et al, nat.Biotechnol. (2011) 29:731-734.

TALENs are another example of artificial nucleases that can be used to edit a target gene. TALENs are derived from a DNA binding domain called TALE repeat sequence, which typically comprises a tandem array of 10 to 30 repeats that bind and recognize an extended DNA sequence. Each repeat is 33 to 35 amino acids in length, with two adjacent amino acids (referred to as repeated variable double residues or RVDs) conferring specificity to one of the four DNA base pairs. Thus, there is a one-to-one correspondence between the repeated sequences and the base pairs in the target DNA sequence.

TALENs are artificially created by fusing one or more TALE DNA binding domains (e.g., 1,2, 3,4, 5, 6, 7, 8, 9, 10 or more) to a nuclease domain (e.g., a fokl endonuclease domain). See Zhang, nature biotech (2011) 29:149-153. For use in TALENs, several mutations have been made to fokl; for example, these improve cleavage specificity or activity. See Cermak et al, nucleic acids res (2011) 39:e82; miller et al, nature Biotech (2011) 29:143-148; hockemeyer et al, nature Biotech. (2011) 29:731-734; wood et al, science (2011) 333:307; doyon et al, nature Methods (2010) 8:74-79; szczepek et al, nature Biotech (2007) 25:786-793; guo et al, J.mol.biol. (2010) 200:96. The fokl domain acts as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with the correct orientation and spacing. The number of amino acid residues between the TALE DNA binding domain and the fokl nuclease domain, and the number of bases between two separate TALEN binding sites, appear to be important parameters for achieving high levels of activity. Miller et al Nature Biotech (2011) 29:143-148.

By combining an engineered TALE repeat sequence with a nuclease domain, a site-specific nuclease can be produced that is specific for any desired DNA sequence. Like ZFNs, TALENs can be introduced into cells to generate DSBs at desired target sites in the genome, and thus can be used to knock out genes or knock-in mutations in a similar HDR-mediated pathway. See Boch, nature Biotech. (2011) 29:135-136; boch et al Science (2009) 326:1509-1512; moscou et al, science (2009) 326:3501.

Meganucleases are enzymes in the endonuclease family, characterized by their ability to recognize and cleave large DNA sequences (14 to 40 base pairs). Meganucleases are divided into families based on structural motifs of meganucleases that affect nuclease activity and/or DNA recognition. The most widely and famous meganucleases are proteins in the LAGLIDADG family, the names of which originate from conserved amino acid sequences. See Chevalier et al, nucleic Acids Res. (2001) 29 (18): 3757-3774. In another aspect, GIY-YIG family members have a GIY-YIG module that is 70-100 residues in length and includes four or five conserved sequence motifs with four invariant residues, two of which are required for activity. See Van Roey et al, nature Structure. Biol. (2002) 9:806-811.His-Cys family meganucleases are characterized by a series of highly conserved histidines and cysteines in a region covering hundreds of amino acid residues. See Chevalier et al, nucleic Acids Res. (2001) 29 (18): 3757-3774. Members of the NHN family are defined by motifs containing two pairs of conserved histidines surrounded by asparagine residues. See Chevalier et al, nucleic Acids Res. (2001) 29 (18): 3757-3774.

Because of the high specificity requirements, the chance of identifying the native meganuclease of a particular target DNA sequence is low, various methods (including mutagenesis and high throughput screening methods) have been used to create meganuclease variants that recognize unique sequences. Strategies for engineering meganucleases with altered DNA binding specificity (e.g., to bind a predetermined nucleic acid sequence) are known in the art. See, e.g., chevalier et al, mol.cell. (2002) 10:895-905; epinat et al, nucleic Acids Res (2003) 31:2952-2962; silva et al, J mol. Biol. (2006) 361:744-754; seligman et al Nucleic Acids Res (2002) 30:3870-3879; sussman et al, J Mol Biol (2004) 342:31-41; doyon et al, J Am Chem Soc (2006) 128:2477-2484; chen et al, protein ENG DES SEL (2009) 22:249-256; arnould et al, J Mol biol. (2006) 355:443-458; smith et al, nucleic Acids Res. (2006) 363 (2): 283-294.

Like ZFNs and TALENs, meganucleases can produce DSBs in genomic DNA, which can produce frameshift mutations if improperly repaired (e.g., via NHEJ), resulting in reduced expression of the target gene in the cell. Alternatively, foreign DNA may be introduced into the cell along with the meganuclease. Depending on the sequence of the foreign DNA and the chromosomal sequence, this process can be used to modify the target gene. See Silva et al, current GENE THERAPY (2011) 11:11-27.

Transposases are enzymes that bind to the ends of a transposon and catalyze its movement to another part of the genome by a cut and paste mechanism or replicative transposition mechanism. By linking the transposase to other systems (such as the CRISPER/Cas system), new gene editing tools can be developed to achieve site-specific insertion or manipulation of genomic DNA. There are two known methods of DNA integration using transposons, using catalytically inactive Cas effector proteins and Tn 7-like transposons. Transposase-dependent DNA integration does not trigger DSBs in the genome, which may ensure safer and more specific DNA integration.

CRISPR systems were originally found in prokaryotes (e.g., bacteria and archaebacteria) as a system that was involved in defending against invading phages and plasmids to provide an adaptive immunity. It has now been adapted and used as a popular gene editing tool in research and clinical applications.

CRISPR/Cas systems typically comprise at least two components: one or more guide RNAs (grnas) and a Cas protein. Cas protein is a nuclease that introduces DSBs into the target site. There are two main classes of CRISPR-Cas systems: class 1 systems use complexes of multiple Cas proteins to degrade nucleic acids; class 2 systems use a single large Cas protein to achieve the same purpose. Class 1 is divided into I, III and type IV; class 2 is divided into types II, V and VI. Different Cas proteins suitable for gene editing applications include, but are not limited to Cas3、Cas4、Cas5、Cas8a、Cas8b、Cas8c、Cas9、Cas10、Cas12、Cas12a(Cpf1)、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12f(C2c10)、Cas12g、Cas12h、Cas12i、Cas12k(C2c5)、Cas13、Cas13a(C2c2)、Cas13b、Cas13c、Cas13d、C2c4、C2c8、C2c9、Cmr5、Cse1、Cse2、Csf1、Csm2、Csn2、Csx10、Csx11、Csy1、Csy2、Csy3 and Mad7. The most widely used Cas9 is a type II Cas protein and is described herein as illustrative. These Cas proteins may be derived from different source species. For example, cas9 may be derived from streptococcus pyogenes(s) or staphylococcus aureus (s.aureus).

In the original microbial genome, the type II CRISPR system incorporates sequences from the invasive DNA between CRISPR repeats encoded as an array within the host genome. Transcripts from the CRISPR repeat array are processed into CRISPR RNA (crrnas), each with a variable sequence transcribed from the invaded DNA (referred to as a "protospacer" sequence), and a portion of the CRISPR repeat. Each crRNA hybridizes to a second trans-activating CRISPR RNA (tracrRNA), and both RNAs form a complex with Cas9 nuclease. The protospacer-encoding portion of crRNA directs Cas9 complexes to cleave complementary target DNA sequences, provided that they are adjacent to a short sequence known as a "protospacer adjacent motif" (PAM).

Since discovery, CRISPR systems have been adapted to induce sequence specific DSBs and targeted genome editing in a wide range of cells and organisms, from bacteria to eukaryotic cells (including human cells). In the use of gene editing applications, artificially designed synthetic gRNAs have replaced the original crRNA-tracrRNA complex. For example, the gRNA may be a single guide RNA (sgRNA) composed of crRNA, tetracyclic and tracrRNA. crrnas typically contain complementary regions (also referred to as spacers, typically about 20 nucleotides in length) that are designed by the user to recognize the target DNA of interest. the tracrRNA sequence comprises a scaffold region for Cas nuclease binding. The crRNA sequence and the tracrRNA sequence are joined by four loops, each having a short repeat sequence for hybridization to each other, thus generating a chimeric sgRNA. The genomic target of the Cas nuclease can be altered by simply altering the spacer or complementary region sequences present in the gRNA. The complementary region will direct the Cas nuclease to the target DNA site by standard RNA-DNA complementary base pairing rules.

In order for Cas nuclease to function, PAM must be present immediately downstream of the target sequence in genomic DNA. The recognition of PAM by Cas proteins is believed to disrupt the stability of adjacent genomic sequences, allowing for gRNA interrogation sequences and resulting gRNA-DNA pairing when a matching sequence is present. The specific sequence of PAM varies depending on the kind of Cas gene. For example, the most commonly used Cas9 nucleases derived from streptococcus pyogenes recognize the PAM sequence of 5'-NGG-3', or recognize the PAM sequence of 5'-NAG-3' with lower efficiency, where "N" can be any nucleotide. Other Cas nuclease variants with alternative PAMs have also been characterized and successfully used for genome editing, which variants are summarized in table 1a below.

Table 1a exemplary Cas nuclease variants and PAM sequences thereof

R=a or G; y=c or T; w=a or T; v=a or C or G; n=any base

In some embodiments, cas nucleases can comprise one or more mutations to alter their activity, specificity, recognition, and/or other features. For example, a Cas nuclease may have one or more mutations that alter its fidelity to mitigate off-target effects (e.g., eSpCas of SpCas9, spCas9-HF1, HYPASPCAS9, heFSpCas, and evoSpCas9 high-fidelity variants). For another example, the Cas nuclease may have one or more mutations that alter its PAM specificity.

In some embodiments, the Cas protein comprises any one of the Cas proteins described herein or a functional portion thereof. As used herein, a "functional moiety" refers to a portion of a peptide that retains its ability to complex with at least one ribonucleic acid (e.g., a guide RNA (gRNA)) and cleave a target polynucleotide sequence. In some embodiments, the functional moiety comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional moiety comprises a combination of operably linked Cas12a (also referred to as Cpf 1) protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of a RuvC-like domain. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of an HNH nuclease domain. In some embodiments, the functional portion of the Cas12a protein comprises a functional portion of a RuvC-like domain.

In some embodiments, suitable Cas proteins include, but are not limited to, cas0, cas12a (i.e., cpf 1), cas12b, cas12i, casX, and Mad7.

In some embodiments, the exogenous Cas protein may be introduced into the cell in the form of a polypeptide. In certain embodiments, the Cas protein may be conjugated or fused to a cell penetrating polypeptide or a cell penetrating peptide. As used herein, "cell penetrating polypeptide" and "cell penetrating peptide" refer to a polypeptide or peptide, respectively, that facilitates uptake of a molecule into a cell. The cell penetrating polypeptide may contain a detectable label.

In certain embodiments, the Cas protein may be conjugated or fused to a charged protein (e.g., that carries a positive charge, a negative charge, or an overall neutral charge). Such linkages may be covalent. In some embodiments, the Cas protein may be fused to a superpositive GFP to significantly increase the ability of the Cas protein to penetrate cells (Cronican et al ACS Chem biol.2010;5 (8): 747-52). In certain embodiments, the Cas protein may be fused to a Protein Transduction Domain (PTD) to facilitate its entry into a cell. Exemplary PTDs include Tat, oligoarginine, and penetrating peptides. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a cell penetrating peptide. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a PTD. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a tat domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to an oligoarginine domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a penetrating peptide domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a superpositive GFP. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a cell penetrating peptide. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a PTD. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a tat domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to an oligoarginine domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a penetrating peptide domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a superpositive GFP.

In some embodiments, the Cas protein may be introduced into the cell containing the target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein. The process of introducing the nucleic acid into the cell may be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid mediated transfection, electroporation and transduction or infection with viral vectors. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises a modified DNA as described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises a modified mRNA (e.g., a synthetic modified mRNA) as described herein.

In the embodiments provided, the CRISPR/Cas system generally comprises two components: one or more guide RNAs (grnas) and a Cas protein. In some embodiments, the Cas protein is complexed with one or more, such as one to two, ribonucleic acids (e.g., guide RNAs (grnas)). In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., a synthetic modified mRNA) as described herein.

In some embodiments, the gRNA is a short synthetic RNA consisting of a scaffold sequence for Cas binding and a user designed spacer or complementary portion designated as crRNA. The cRNA consists of a crRNA targeting sequence (hereinafter also referred to as gRNA targeting sequence; typically about 20 nucleotides in length) and a crRNA repeat region (e.g., GUUUUAGAGCUA; SEQ ID NO: 19) defining the genomic target to be modified. The genomic target of the Cas protein can be altered by simply altering the complementary partial sequence present in the gRNA (e.g., the gRNA targeting sequence). In some embodiments, the scaffold sequence for Cas binding consists of a tracrRNA sequence (e.g., UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA AGUGGCACCGAGUCGGUGCUUU; SEQ ID NO: 20) that hybridizes to the crRNA through its anti-repeat sequence. crRNA the complex between the tracrRNA recruits Cas nuclease (e.g., cas 9) and cleaves upstream of the Protospacer Adjacent Motif (PAM). In order for the Cas protein to function, there must be PAM immediately downstream of the target sequence in the genomic DNA. The recognition of PAM by Cas proteins is believed to disrupt the stability of adjacent genomic sequences, allowing for gRNA interrogation sequences and resulting gRNA-DNA pairing when a matching sequence is present. The specific sequence of PAM varies depending on the kind of Cas gene. For example, the most commonly used Cas9 nucleases derived from streptococcus pyogenes recognize the PAM sequence of NGG. Other Cas9 variants and other nucleases with alternative PAMs have also been characterized and successfully used for genome editing. Thus, CRISPR/Cas systems can be used to create targeted DSBs at specific genomic loci that are complementary to grnas designed for the target loci. The crRNA and tracrRNA can be joined together with a loop sequence (e.g., four loops; GAAA) to generate the gRNA as chimeric single guide RNA (sgRNA; hsu et al 2013). The sgrnas may be generated for DNA-based expression or by chemical synthesis.

In some embodiments, the complementary partial sequence of the gRNA (e.g., the gRNA targeting sequence) will vary depending on the target site of interest. In some embodiments, the gRNA comprises a complementary portion specific for the gene sequences listed in table 1. In some embodiments, the gRNA-targeted genomic locus is within 4000bp, 3500bp, 3000bp, 2500bp, 2000bp, 1500bp, 1000bp, or 500bp of any of the loci described.

The methods disclosed herein contemplate the use of any ribonucleic acid capable of directing and hybridizing a Cas protein to a target motif of a target polynucleotide sequence. In some embodiments, at least one ribonucleic acid comprises

In some embodiments, the Cas protein is complexed with one to two ribonucleic acids (e.g., guide RNAs (grnas)). In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., a synthetic modified mRNA) as described herein.

The methods disclosed herein contemplate the use of any ribonucleic acid capable of directing and hybridizing a Cas protein to a target motif of a target polynucleotide sequence. In some embodiments, the at least one ribonucleic acid comprises a tracrRNA. In some embodiments, the at least one ribonucleic acid comprises CRISPR RNA (crRNA). In some embodiments, the single ribonucleic acid comprises a guide RNA that directs and hybridizes to a target motif of a target polynucleotide sequence in the cell to the Cas protein. In some embodiments, the at least one ribonucleic acid comprises a guide RNA that directs and hybridizes to a target motif of a target polynucleotide sequence in the cell to which the Cas protein is directed. In some embodiments, one or both ribonucleic acids comprise a guide RNA that directs and hybridizes to a target motif of a target polynucleotide sequence in a cell to which the Cas protein is directed. As will be appreciated by those of skill in the art, ribonucleic acids provided herein can be selected to hybridize to a variety of different target motifs, depending on the particular CRISPR/Cas system and sequence of target polynucleotide employed. One or two ribonucleic acids may also be selected to minimize hybridization to nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, one to two ribonucleic acids hybridize to a target motif containing at least two mismatches when compared to all other genomic nucleotide sequences in a cell. In some embodiments, one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared to all other genomic nucleotide sequences in a cell. In some embodiments, one or both ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleotide motif recognized by a Cas protein. In some embodiments, each of the one to two ribonucleic acids is designed to hybridize to a target motif immediately adjacent to a deoxyribonucleotide motif recognized by a Cas protein that flanks a mutant allele located between the target motifs.

In some embodiments, each of the one to two ribonucleic acids comprises a guide RNA that directs and hybridizes to a target motif of a target polynucleotide sequence in a cell to which the Cas protein is directed.

In some embodiments, one or both ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to sequences on the same strand of the target polynucleotide sequence. In some embodiments, one or both ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to sequences on opposite strands of the target polynucleotide sequence. In some embodiments, one or both ribonucleic acids (e.g., guide RNAs) are not complementary to and/or hybridize to sequences on opposite strands of the target polynucleotide sequence. In some embodiments, one or both ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to overlapping target motifs of a target polynucleotide sequence. In some embodiments, one or both ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to a complementing target motif of a target polynucleotide sequence.

In some embodiments, the nucleic acid encoding the Cas protein and the nucleic acid encoding at least one to two ribonucleic acids are introduced into the cell via viral transduction (e.g., lentiviral transduction). In some embodiments, the Cas protein is complexed with 1-2 ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., a synthetic modified mRNA) as described herein.

Exemplary gRNA targeting sequences that can be used for CRISPR/Cas-based targeting of the genes described herein are provided in table 1. Sequences can be found in WO2016183041 filed 5/9 in 2016, the disclosure of which including tables, appendices and sequence listing are incorporated herein by reference in their entirety.

TABLE 1 exemplary gRNA targeting sequences useful for targeting genes

Additional exemplary Cas9 guide RNA sequences that can be used for CRISPR/Cas-based targeting of the genes described herein are provided in table 1 c.

Table 2A additional exemplary Cas9 guide RNA sequences useful for targeting genes

In some embodiments, it is within the level of the skilled artisan to identify novel loci and/or gRNA targeting sequences for genetic disruption methods to reduce or eliminate expression of the genes. For example, for CRISPR/Cas systems, when an existing gRNA targeting sequence for a particular locus (e.g., within a target gene (e.g., listed in table 1)) is known, the "inch worm (inch worming)" approach can be used to identify additional loci to target an insertion transgene by scanning flanking regions flanking the locus for PAM sequences that typically occur about once every 100 base pairs (bp) in the genome. PAM sequences will depend on the particular Cas nuclease used, as different nucleases typically have different corresponding PAM sequences. Flanking regions on both sides of the locus may be about 500 to 4000bp long, for example about 500bp, about 1000bp, about 1500bp, about 2000bp, about 2500bp, about 3000bp, about 3500bp or about 4000bp long. When PAM sequences are identified within the search, new guides can be designed based on the sequence of this locus for use in genetic disruption methods. Although the CRISPR/Cas system is described as illustrative, any of the described gene editing methods can be used for this method of identifying new loci, including those using ZFNs, TALENs, meganucleases and transposases.

In some embodiments, the cells described herein are prepared using a transcription activator-like effector nuclease (TALEN) method. "TALE nuclease" (TALEN) means a fusion protein consisting of a nucleic acid binding domain typically derived from a transcription activator-like effector (TALE) and a nuclease catalytic domain that cleaves a nucleic acid target sequence. The catalytic domain in some embodiments is a nuclease domain, such as a domain having endonuclease (e.g., I-TevI, colE7, nucA, and Fok-I) activity. In a particular embodiment, the TALE domain can be fused to meganucleases such as I-CreI and I-OnuI or functional variants thereof. In some embodiments, the nuclease is a monomeric TALE nuclease. Monomeric TALE nucleases are TALE nucleases that do not require dimerization for specific recognition and cleavage, such as the fusion of an engineered TAL repeat with the catalytic domain of I-TevI described in WO 2012138927. A transcription activator-like effector (TALE) is a protein from the bacterial species Xanthomonas (Xanthomonas) comprising multiple repeat sequences, each comprising a diradical (RVD) in positions 12 and 13 specific for each nucleotide base of a nucleic acid targeting sequence. Binding domains with similar modular base-by-base nucleic acid binding properties (MBBBD) can also be derived from novel modular proteins recently discovered by applicants in different bacterial species. The novel modular proteins have the advantage of exhibiting more sequence variability than TAL repeats. In some embodiments, the RVD associated with identifying a different nucleotide is HD for identifying C; NG for identifying T; NI for identifying a; NN for identifying G or a; NS for identifying A, C, G or T; HG for identifying T; IG for identifying T; NK for identifying G; HA for identifying C; ND for identifying C; HI for identifying C; HN for identifying G; NA for identifying G; SN for identifying G or a; and YG for identifying T; TL for identifying a; VT for identifying a or G; and SW for identifying a. In another embodiment, the critical amino acids 12 and 13 may be mutated to other amino acid residues in order to modulate their specificity for nucleotides A, T, C and G, in particular to enhance such specificity. TALEN kits are commercially available.

In some embodiments, the cells are manipulated using Zinc Finger Nucleases (ZFNs). A "zinc finger binding protein" is a protein or polypeptide that binds DNA, RNA, and/or protein in a sequence-specific manner, for example, due to stabilization of the protein structure by coordination of zinc ions. The term zinc finger binding protein is commonly abbreviated as zinc finger protein or ZFP. The individual DNA binding domains are commonly referred to as "fingers". ZFP has at least one finger, typically two, three or six fingers. Each finger binds two to four DNA base pairs, typically three or four DNA base pairs. ZFP binds to a nucleic acid sequence called a target site or target segment. Each finger typically comprises a zinc chelating DNA binding subdomain of about 30 amino acids. Studies have shown that such single zinc fingers consist of an alpha helix containing two invariant histidine residues coordinated to zinc and two cysteine residues at a single beta turn (see, e.g., berg and Shi, science271:1081-1085 (1996)).

In some embodiments, homing endonucleases are used to prepare the cells described herein. Such homing endonucleases are well known in the art (Stoddard 2005). Homing endonucleases recognize DNA target sequences and generate single-or double-strand breaks. Homing endonucleases are highly specific, recognizing DNA target sites ranging in length from 12 to 45 base pairs (bp), typically ranging in length from 14bp to 40bp. The homing endonuclease may for example correspond to a LAGLIDADG endonuclease, an HNH endonuclease or a GIY-YIG endonuclease. In some embodiments, the homing endonuclease can be an I-CreI variant.

In some embodiments, the cells described herein are prepared using meganucleases. Meganucleases are, by definition, sequence-specific endonucleases recognizing large sequences (chemalier, b.s. And b.l.stoddard, nucleic Acids res.,2001,29,3757-3774). They can cleave unique sites in living cells, thereby enhancing gene targeting in the vicinity of the cleavage site 1000-fold or more (Puchta et al, nucleic Acids Res.,1993,21,5034-5040; rouet al, mol.cell.biol.,1994,14,8096-8106; choulika et al, mol.cell.biol.,1995,15,1968-1973; puchta et al, proc.Natl.Acad.Sci. USA,1996,93,5055-5060; sargent et al, mol.cell.biol.,1997,17,267-77; donoho et al, mol.cell.biol.,1998,18,4070-4078; elliott et al, mol.cell.biol.,1998,18,93-101; cohen-Tannoudji et al, mol.cell.biol.,1998,18,1444-1448).

In some embodiments, RNA silencing or RNA interference (RNAi) is used to knock down (e.g., reduce, eliminate, or inhibit) expression of a polypeptide, such as a tolerogenic factor, to produce a cell provided herein. Useful RNAi methods include methods utilizing synthetic RNAi molecules, short interfering RNAs (siRNAs), PIWI interacting NRAs (piRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), and other transient knockdown methods recognized by those skilled in the art. Reagents for RNAi, including sequence specificity shRNA, siRNA, miRNA and the like, are commercially available. For example, a target polynucleotide (such as any of those described above, e.g., CIITA, B2M, or NLRC 5) in a cell can be knocked down by RNA interference by introducing into the cell an inhibitory nucleic acid (such as an siRNA) that is complementary to the target motif of the target polynucleotide. In some embodiments, a target polynucleotide, e.g., any of the target polynucleotides described above (such as any of the above, e.g., CIITA, B2M, or NLRC 5) in a cell can be knocked down by transduction of shRNA-expressing virus into the cell. In some embodiments, RNA interference is employed to reduce or inhibit expression of at least one selected from the group consisting of CIITA, B2M and NLRC 5.

3. Exemplary target polynucleotides and methods for reducing expression

MHC class I molecules

In certain embodiments, the modification reduces or eliminates (such as knocks out) expression of one or more MHC class I molecules (e.g., one or more MHC class I genes encoding one or more MHC class I molecules) by targeting the helper strand B2M. In some embodiments, the modification is performed using a CRISPR/Cas system. By reducing or eliminating (such as knockout) expression of B2M, surface transport of one or more MHC class I molecules is blocked, and such cells exhibit immune tolerance when implanted into a recipient subject. In some embodiments, the cells are considered to be hypoimmunogenic, e.g., in a recipient subject or patient after administration.

In some embodiments, a target polynucleotide sequence provided herein is a variant of B2M. In some embodiments, the target polynucleotide sequence is a homolog of B2M. In some embodiments, the target polynucleotide sequence is an ortholog of B2M.

In some embodiments, reduced or eliminated B2M expression reduces or eliminates expression of one or more of the following MHC class I molecules: HLA-A, HLA-B and HLA-C.

In some embodiments, the engineered cell comprises a modification that targets the B2M gene. In some embodiments, the modification of the targeted B2M gene is performed by using a targeting nuclease system comprising a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the B2M gene. In some embodiments, at least one guide ribonucleic acid sequence (e.g., a gRNA targeting sequence) for specifically targeting the B2M gene is selected from the group consisting of appendix 2 of WO2016/183041 or SEQ ID NOs 81240-85644 of table 15, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, an exogenous nucleic acid or transgene encoding a polypeptide as disclosed herein (e.g., chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the B2M gene. Exemplary transgenes for targeted insertion at the B2M locus include any transgene as described in section ii.b.

Assays to test whether the B2M gene has been inactivated are known and described herein. In one embodiment, modification of the B2M gene and reduction of HLA-I expression by PCR can be determined by flow cytometry (such as by FACS analysis). In another embodiment, B2M protein expression is detected using western blotting of cell lysates probed with antibodies to B2M protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating modifications.

In some embodiments, the reduction in expression or function of one MHC class I molecule (HLA when the cell is derived from a human cell) in the engineered cell can be measured using techniques known in the art, for example FACS techniques using labeled antibodies that bind to HLA complexes (e.g., using commercially available HLA-a, B, C antibodies that bind to the alpha chain of a human major histocompatibility HLA class I antigen). In addition, cells can be tested to confirm that HLA I complexes are not expressed on the cell surface. This can be determined by FACS analysis using antibodies against one or more HLA cell surface components as discussed above. In addition to reducing HLA I (or MHC class I), the sensitivity of the engineered cells provided herein to macrophage phagocytosis and NK cell killing is also reduced. Methods for determining the low immunogenicity phenotype of an engineered cell are further described below.

MHC class II molecules

In certain aspects, the modification reduces or eliminates (such as knocks out) expression of one or more MHC class II genes by targeting class II transactivator (CIITA) expression. In some embodiments, the modification is performed using a CRISPR/Cas system. CIITA is a member of the LR or Nucleotide Binding Domain (NBD) Leucine Rich Repeat (LRR) protein family and regulates transcription of one or more MHC class II genes by association with MHC enhancers. By reducing or eliminating (such as knockout) expression of CIITA, expression of one or more MHC class II molecules is reduced, thereby also reducing surface expression. In some cases, such cells exhibit immune tolerance when implanted into a recipient subject. In some embodiments, the cells are considered to be hypoimmunogenic, e.g., in a recipient subject or patient after administration.

In some embodiments, the target polynucleotide sequence is a variant of CIITA. In some embodiments, the target polynucleotide sequence is a homolog of CIITA. In some embodiments, the target polynucleotide sequence is an ortholog of CIITA.

In some embodiments, the reduced or eliminated expression of CIITA reduces or eliminates expression of one or more of the following MHC class II molecules: HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ and HLA-DR.

In some embodiments, the engineered cell comprises a modification that targets the CIITA gene. In some embodiments, the modification of the targeted CIITA gene is performed by a targeting nuclease system comprising a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene. In some embodiments, at least one guide ribonucleic acid sequence (e.g., a gRNA targeting sequence) for specifically targeting the CIITA gene is selected from the group consisting of appendix 1 of WO2016183041 or SEQ ID NO:5184-36352 of table 12, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, an exogenous nucleic acid or transgene encoding a polypeptide as disclosed herein (e.g., chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the CIITA gene. Exemplary transgenes for targeted insertion at the B2M locus include any transgene as described in section ii.b.

Assays for testing whether the CIITA gene has been inactivated are known and described herein. In one embodiment, modification of the CIITA gene and reduction of HLA-II expression by PCR can be determined by flow cytometry (such as by FACS analysis). In another embodiment, CIITA protein expression is detected using western blotting of cell lysates probed with antibodies to CIITA proteins. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating modifications.

In some embodiments, the decrease in expression or function of one or more MHC class II molecules (HLA II when the cell is derived from a human cell) in the engineered cell can be measured using techniques known in the art, such as western blotting using antibodies to the protein, FACS techniques, RT-PCR techniques, and the like. In some embodiments, the engineered cells may be tested to confirm that HLA II complexes are not expressed on the cell surface. Methods of assessing surface expression include methods known in the art (see, e.g., figure 21 of WO 2018132783) and are typically performed using western blot or FACS analysis based on commercial antibodies that bind to human HLA class II HLA-DR, DP and most DQ antigens. In addition to reducing one or more HLA class II molecules (or one or more MHC class II molecules), the sensitivity of the engineered cells provided herein to macrophage phagocytosis and NK cell killing is also reduced. Methods for determining the low immunogenicity phenotype of an engineered cell are further described below.

C.CD142

In certain aspects, the modification reduces or eliminates (such as knocks out) expression of CD 142. In some embodiments, the modification is performed using a CRISPR/Cas system. CD142, also known as tissue factor (F3), is a membrane-bound protein that initiates blood clotting by forming a complex with circulating factor VII or factor VIIa. CD142 (TF) VIIa complex activates factor IX or X by specific limited proteolysis. CD142 (TF) plays a role in normal hemostasis by initiating cell surface assembly and transmission of the thrombin cascade. By reducing or eliminating (such as knockout) the expression of CD142, the expression of one or more MHC class II molecules is reduced, thereby also reducing surface expression. In some cases, such cells exhibit immune tolerance when implanted into a recipient subject. In some embodiments, the cells are considered to be hypoimmunogenic, e.g., in a recipient subject or patient after administration.

In some embodiments, the target polynucleotide sequence is a variant of CD 142. In some embodiments, the target polynucleotide sequence is a homolog of CD 142. In some embodiments, the target polynucleotide sequence is an ortholog of CD 142.

In some embodiments, the engineered cell comprises a modification targeting the CD142 gene. In some embodiments, the modification of the targeted CD142 gene is performed by a targeting nuclease system comprising a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the CD142 gene. In some embodiments, the target polynucleotide sequence is CD142 or a variant of CD 142. In some embodiments, the target polynucleotide sequence is a homolog of CD 142. In some embodiments, the target polynucleotide sequence is an ortholog of CD 142.

In some embodiments, an exogenous nucleic acid or transgene encoding a polypeptide as disclosed herein (e.g., chimeric antigen receptor, CD46, CD59, CD55, or CD47, or another tolerogenic factor disclosed herein) is inserted at the CD142 gene. Exemplary transgenes for targeted insertion at the CD142 locus include any transgene as described in section ii.b.

In some embodiments, the reduction in CD142 expression or function in the engineered cells can be measured using techniques known in the art, such as western blotting using antibodies to proteins, FACS techniques, RT-PCR techniques, and the like. In some embodiments, the engineered cells may be tested to confirm that CD142 is not expressed on the cell surface. Methods of assessing surface expression include methods known in the art (see, e.g., figure 21 of WO 2018132783) and are typically performed using western blot or FACS analysis based on commercial antibodies that bind to human CD 142. In addition to the reduction of CD142, the sensitivity of the engineered cells provided herein to IBMIR is also reduced. Methods for determining the low immunogenicity phenotype of an engineered cell are further described below.

In some embodiments, the modification that reduces CD142 expression reduces CD142mRNA expression. In some embodiments, the decrease in mRNA expression of CD142 is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification. In some embodiments, mRNA expression of CD142 is reduced by more than about 5%, such as by more than about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, mRNA expression of CD142 is reduced by up to about 100%, such as by up to any of about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less. In some embodiments, mRNA expression of CD142 is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, mRNA expression of CD142 is eliminated (e.g., 0% expression of CD142 mRNA). In some embodiments, modifications that reduce CD142mRNA expression eliminate CD142 gene activity.

In some embodiments, the modification that reduces CD142 expression reduces CD142 protein expression. In some embodiments, the reduced protein expression of CD142 is relative to an unmodified or wild-type cell of the same cell type that does not comprise the modification. In some embodiments, the protein expression of CD142 is reduced by more than about 5%, such as by more than about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, protein expression of CD142 is reduced by up to about 100%, such as by up to any of about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less. In some embodiments, the protein expression of CD142 is reduced by any of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, protein expression of CD142 is eliminated (e.g., 0% expression of CD142 protein). In some embodiments, modifications that reduce CD142 protein expression eliminate CD142 gene activity.

In some embodiments, the modification that reduces CD142 expression comprises inactivation or disruption of the CD142 gene. In some embodiments, the modification that reduces CD142 expression comprises inactivation or disruption of one allele of the CD142 gene. In some embodiments, the modification that reduces CD142 expression comprises inactivation or disruption of both alleles of the CD142 gene.

In some embodiments, the modification comprises inactivation or disruption of one or more CD142 coding sequences in the cell. In some embodiments, the modification comprises inactivation or disruption of all CD142 coding sequences in the cell. In some embodiments, the modification comprises an indel in the CD142 gene. In some embodiments, the modification is a frameshift mutation of genomic DNA of the CD142 gene. In some embodiments, the modification is a deletion of genomic DNA of the CD142 gene. In some embodiments, the modification is a deletion of a stretch of contiguous genomic DNA of the CD142 gene.

Exemplary guide sequences for targeting CD142 are known, for example:

B. overexpression of polynucleotides

In some embodiments, the engineered cells provided herein are genetically modified or engineered, such as by introducing one or more modifications into the cells to overexpress a desired polynucleotide in the cells. In some embodiments, the cell to be modified or engineered is an unmodified cell or a non-engineered cell into which one or more modifications have not been previously introduced. In some embodiments, the engineered cells provided herein are genetically modified to comprise one or more exogenous polynucleotides encoding an exogenous protein (also used interchangeably with the term "transgene"). As described, in some embodiments, the cells are modified to increase the expression of certain genes, tolerogenic (e.g., immune) factors that affect immune recognition and tolerance in the recipient. In some embodiments, an engineered cell (such as a T cell or NK cell) is provided that also expresses a Chimeric Antigen Receptor (CAR). One or more polynucleotides (e.g., exogenous polynucleotides) may be expressed (e.g., overexpressed) in an engineered cell along with one or more genetic modifications to reduce expression of a target polynucleotide described in section i.a above, such as one or more MHC class I and/or one or more MHC class II molecules or CD 142. In some embodiments, the engineered cells provided do not trigger or activate an immune response upon administration to a recipient subject.

In some embodiments, the engineered cell comprises 1,2,3, 4, 5, 6, 7, 8, 9, 10, or more different over-expressed polynucleotides. In some embodiments, the engineered cell comprises 1,2,3, 4, 5, 6, 7, 8, 9, 10, or more different over-expressed polynucleotides. In some embodiments, the overexpressed polynucleotide is an exogenous polynucleotide. In some embodiments, the engineered cell comprises 1,2,3, 4, 5, 6, 7, 8, 9, 10, or more different exogenous polynucleotides. In some embodiments, the engineered cell comprises 1,2,3, 4, 5, 6, 7, 8, 9, 10, or more different exogenous polynucleotides. In some embodiments, the over-expressed polynucleotide is an exogenous polynucleotide that is expressed free in the cell. In some embodiments, the overexpressed polynucleotide is an exogenous polynucleotide inserted or integrated into one or more genomic loci of the engineered cell.

In some embodiments, fusion proteins comprising a DNA targeting domain and a transcriptional activator are used to increase expression of a polynucleotide, i.e., over-expression of a polynucleotide. Targeting methods for increasing expression using transactivator domains are known to the skilled artisan.

In some embodiments, the engineered cells contain one or more exogenous polynucleotides, wherein the one or more exogenous polynucleotides are inserted or integrated into the genomic locus of the cell by a non-targeted insertion method (such as by transduction using a lentiviral vector). In some embodiments, one or more exogenous polynucleotides are inserted or integrated into the genome of the cell by a targeted insertion method, such as by using Homology Directed Repair (HDR). The exogenous polynucleotide can be inserted into the genomic locus of the engineered cell by HDR using any suitable method, including the gene editing methods described herein (e.g., CRISPR/Cas system). In some embodiments, one or more exogenous polynucleotides are inserted into one or more genomic loci, such as any of the genomic loci described herein (e.g., table 2). In some embodiments, the exogenous polynucleotide is inserted into the same genomic locus. In some embodiments, the exogenous polynucleotide is inserted into a different genomic locus. In some embodiments, two or more exogenous polynucleotides are inserted into the same genomic locus, such as any of the genomic loci described herein (e.g., table 2). In some embodiments, two or more exogenous polynucleotides are inserted into different genomic loci, such as the two or more genomic loci described herein (e.g., table 2).

Exemplary polynucleotides or overexpression and methods for overexpressing the same are described in the following subsections.

1. Complement inhibitors

In some embodiments, expression of one or more complement inhibitors is increased in a cell. In some embodiments, the one or more complement inhibitors are one or more membrane-bound complement inhibitors. In some embodiments, the at least one exogenous polynucleotide comprises a polynucleotide encoding a complement inhibitor. In some embodiments, the one or more complement inhibitors is CD46, CD59, CD55, or any combination thereof. For example, in some embodiments, at least one exogenous polynucleotide is a polynucleotide encoding one or more complement inhibitors (such as CD 46). In some embodiments, the one or more complement inhibitors are CD46 and CD59, or CD46, CD59, and CD55. In some embodiments, the expression of CD46 and CD59 or CD46, CD59 and CD55 protects the cell or population thereof from complement dependent cytotoxicity, including in the presence of antibodies to cell surface antigens expressed by the cell.

In some embodiments, the present disclosure provides a cell or population thereof that has been modified to express one or more complement inhibitors, such as CD46, CD59, CD55, or any combination thereof. In some embodiments, the one or more complement inhibitors are CD46 and CD59. In some embodiments, the one or more complement inhibitors are CD46, CD59, and CD55. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express one or more complement inhibitors. In some embodiments, the engineered cells express one or more exogenous complement inhibitors, such as exogenous CD46 and CD59 or CD46, CD59, and CD55. In some cases, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD46 polypeptide. In some cases, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD59 polypeptide. In some cases, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD55 polypeptide. In some embodiments, the expression vector comprises nucleotide sequences encoding any combination of two or more complement inhibitors. In some embodiments, the expression vector comprises nucleotide sequences encoding CD46 and CD59. In some embodiments, the expression vector comprises nucleotide sequences encoding CD46, CD59, and CD55.

D.CD46

In some embodiments, the engineered cells contain an over-expressed polynucleotide encoding CD46 (such as human CD 46). In some embodiments, the engineered cells contain an exogenous polynucleotide encoding CD46 (such as human CD 46). In some embodiments, CD46 is overexpressed in the cell. In some embodiments, the expression of CD46 in the engineered cell is increased compared to a similar reference or unmodified cell (including having any other modification), except that the reference or unmodified cell does not comprise an exogenous polynucleotide encoding CD 46. CD46 is a membrane bound complement inhibitor. It acts as a cofactor for complement factor I, a serine protease that protects autologous cells from complement-mediated damage by cleaving C3b and C4 b. Available genomic, polynucleotide and polypeptide information about human CD46 is provided, for example, in GeneCard identifiers GC01P207752, HGNC No.6953, NCBI Gene ID 4179, uniprot No. P15529, and NCBI Ref Seq No.NM_002389.4、NM_153826.3、NM_172350.2、NM_172351.2、NM_172352.2NP_758860.1、NM_172353.2、NM_172359.2、NM_172361.2、NP_002380.3、NP_722548.1、NP_758860.1、NP_758861.1、NP_758862.1、NP_758863.1、NP_758869.1 and NP 758871.1.

In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD46 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to an amino acid sequence as listed in NCBI Ref.Sequence No.NP_002380.3、NP_722548.1、NP_758860.1、NP_758861.1、NP_758862.1、NP_758863.1、NP_758869.1 and np_ 758871.1. In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD46 polypeptide having the amino acid sequences as listed in NCBI Ref.Sequence No.NP_002380.3、NP_722548.1、NP_758860.1、NP_758861.1、NP_758862.1、NP_758863.1、NP_758869.1 and np_ 758871.1. In some embodiments, the cell comprises an over-expressed nucleotide sequence of CD46 that has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the sequences listed in NCBI Ref.No.NM_002389.4、NM_153826.3、NM_172350.2、NM_172351.2、NM_172352.2、NP_758860.1、NM_172353.2、NM_172359.2 and nm_ 172361.2. In some embodiments, the cell comprises an over-expressed nucleotide sequence of CD46 as listed in NCBI ref.sequence nos. nm_001777.3 and nm_002389.4, nm_153826.3, nm_172350.2, nm_172351.2, nm_172352.2np_758868.1, nm_172353.2, nm_172359.2, and nm_ 172361.2.

In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding CD46 polypeptides having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequences as listed in NCBI Ref.Sequence No.NP_002380.3、NP_722548.1、NP_758860.1、NP_758861.1、NP_758862.1、NP_758863.1、NP_758869.1 and np_ 758871.1. In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding CD46 polypeptides having the amino acid sequences as listed in NCBI Ref.Sequence No.NP_002380.3、NP_722548.1、NP_758860.1、NP_758861.1、NP_758862.1、NP_758863.1、NP_758869.1 and np_ 758871.1. In some embodiments, the cell comprises an exogenous nucleotide sequence of CD46 that has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the sequences listed in NCBI Ref.No.NM_002389.4、NM_153826.3、NM_172350.2、NM_172351.2、NM_172352.2NP_758860.1、NM_172353.2、NM_172359.2 and nm_ 172361.2. In some embodiments, the cell comprises an exogenous nucleotide sequence such as CD46 listed in NCBI ref.sequence nos. nm_001777.3 and nm_002389.4, nm_153826.3, nm_172350.2, nm_172351.2, nm_172352.2np_758856.1, nm_172353.2, nm_172359.2, and nm_ 172361.2.

In some embodiments, the cells comprise an over-expressed CD46 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to an amino acid sequence as set forth in NCBI ref. Sequence No. np_722548.1, np_758860.1, np_758861.1, np_758862.1, np_758863.1, np_758869.1 and np_ 758871.1. In some embodiments, the cell comprises an exogenous CD46 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to an amino acid sequence as set forth in NCBI ref. Sequence No. np_722548.1, np_758860.1, np_758861.1, np_758862.1, np_758863.1, np_758869.1, and np_ 758871.1. In some embodiments, the cells outlined herein comprise over-expressed CD46 polypeptides having the amino acid sequences as listed in NCBI ref.sequence No. np_722548.1, np_758860.1, np_758861.1, np_758862.1, np_758863.1, np_758869.1, and np_ 758871.1. In some embodiments, the cells outlined herein comprise exogenous CD46 polypeptides having the amino acid sequences as listed in NCBI ref.sequence No. np_722548.1, np_758860.1, np_758861.1, np_758862.1, np_758863.1, np_758869.1, and np_ 758871.1.

In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD46 polypeptide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 4. In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding a CD46 polypeptide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 4. In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence that encodes a CD46 polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 4. In some embodiments, the cells outlined herein comprise an exogenous nucleotide sequence that encodes a CD46 polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 4.

In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding a CD46 polypeptide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 3. In some embodiments, the cells outlined herein comprise an exogenous nucleotide sequence that encodes a CD46 polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 3. In some embodiments, the exogenous nucleotide sequence encoding a CD46 polypeptide is operably linked to a sequence encoding a heterologous signal peptide.

In some embodiments, all or a functional portion of CD46 may be linked to other components, such as a signal peptide, a leader sequence, a secretion signal, a marker (e.g., a reporter), or any combination thereof. In some embodiments, the nucleic acid sequence encoding the signal peptide of CD46 is replaced with a nucleic acid sequence encoding a signal peptide from a heterologous protein. The heterologous protein may be, for example, CD8 alpha, CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF receptor (GM-CSFRa), or an immunoglobulin (e.g., igE or IgK). In some embodiments, the signal peptide is a signal peptide from an immunoglobulin (such as an IgG heavy chain or an IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2) or CD 33), serum albumin (e.g., HSA or albumin), a human azurin preproprotein signal sequence, luciferase, trypsinogen (e.g., chymotrypsinogen or trypsinogen), or other signal peptide capable of efficiently expressing a protein by or on a cell.

In certain embodiments, the exogenous polynucleotide encoding CD46 is operably linked to a promoter.

In some embodiments, the polynucleotide encoding CD46 is inserted into any one of the loci depicted in table 2. In some cases, the polynucleotide encoding CD46 is inserted into a safe harbor locus, such as but not limited to a locus selected from AAVS1, CCR5, CLYBL, ROSA26, SHS 231. In particular embodiments, the polynucleotide encoding CD46 is inserted into the CCR5 locus, the PPP1R12C (also known as AAVS 1) locus, or the CLYBL locus. In some embodiments, the polynucleotide encoding CD46 is inserted into the B2M locus, the CIITA locus, or the CD142 locus. In some embodiments, the engineered cell is a T cell and the polynucleotide encoding CD46 is inserted into the TRAC locus or the TRBC locus. In some embodiments, insertion of the polynucleotide encoding CD46 into the genomic locus of the cell is facilitated using a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein).

In some embodiments, CD46 protein expression is detected using western blotting of cell lysates that are probed with antibodies to CD46 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous CD46 mRNA.

E.CD59

In some embodiments, the engineered cells contain an over-expressed polynucleotide encoding CD59 (such as human CD 59). In some embodiments, the engineered cells contain an exogenous polynucleotide encoding CD59 (such as human CD 59). In some embodiments, CD59 is overexpressed in the cell. In some embodiments, the expression of CD59 in the engineered cell is increased compared to a similar reference or unmodified cell (including having any other modification), except that the reference or unmodified cell does not comprise an exogenous polynucleotide encoding CD 59. CD59 is a membrane-bound complement inhibitor. More specifically, CD59 is an inhibitor of complement Membrane Attack Complex (MAC) activity. CD59 works by binding to the C8 and/or C9 complement of the assembled MAC, thereby preventing incorporation of multiple copies of C9 required to fully form the permeation aperture. Available genomic, polynucleotide and polypeptide information about human CD59 is provided, for example, in GeneCard identifiers GC11M033704, HGNC No.1689, NCBI Gene ID 966, uniprot No. p13987, and NCBI RefSeq No.NP_000602.1、NM_000611.5、NP_001120695.1、NM_001127223.1、NP_001120697.1、NM_001127225.1、NP_001120698.1、NM_001127226.1、NP_001120699.1、NM_001127227.1、NP_976074.1、NM_203329.2、NP_976075.1、NM_203330.2、NP_976076.1 and nm_203331.2.

In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD59 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to an amino acid sequence as listed in NCBI Ref.Sequence No.NP_000602.1、NP_001120695.1、NP_001120697.1、NP_001120698.1、NP_001120699.1、NP_976074.1、NP_976075.1 and np_ 976076.1. In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD59 polypeptide having the amino acid sequences as listed in NCBI Ref.Seq uence No.NP_000602.1、NP_001120695.1、NP_001120697.1、NP_001120698.1、NP_001120699.1、NP_976074.1、NP_976075.1 and np_ 976076.1. In some embodiments, the cell comprises an over-expressed nucleotide sequence of CD59 that has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the sequences listed in NCBI Ref.No.NM_000611.5、NM_001127223.1、NM_001127225.1、NM_001127226.1、NM_001127227.1、NM_203329.2、NM_203330.2 and nm_ 203331.2. In some embodiments, the cell comprises an over-expressed nucleotide sequence of CD59 as listed in NCBI Ref.Seq uence No.NM_000611.5、NM_001127223.1、NM_001127225.1、NM_001127226.1、NM_001127227.1、NM_203329.2、NM_203330.2 and nm_ 203331.2.

In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD59 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to an amino acid sequence as listed in NCBI Ref.Sequence No.NP_000602.1、NP_001120695.1、NP_001120697.1、NP_001120698.1、NP_001120699.1、NP_976074.1、NP_976075.1 and np_ 976076.1. In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding CD59 polypeptides having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequences as listed in NCBI Ref.Seque nce No.NP_000602.1、NP_001120695.1、NP_001120697.1、NP_001120698.1、NP_001120699.1、NP_976074.1、NP_976075.1 and np_ 976076.1. In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD59 polypeptide having the amino acid sequences as listed in NCBI Ref.Sequenc e No.NP_000602.1、NP_001120695.1、NP_001120697.1、NP_001120698.1、NP_001120699.1、NP_976074.1、NP_976075.1 and np_ 976076.1. In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding CD59 polypeptides having the amino acid sequences as listed in NCBI Ref.SequenceNo.NP_000602.1、NP_001120695.1、NP_001120697.1、NP_001120698.1、NP_001120699.1、NP_976074.1、NP_976075.1 and np_ 976076.1. In some embodiments, the cell comprises an over-expressed nucleotide sequence of CD59 that has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the sequences listed in NCBI Ref.No.NM_000611.5、NM_001127223.1、NM_001127225.1、NM_001127226.1、NM_001127227.1、NM_203329.2、NM_203330.2 and nm_ 203331.2. In some embodiments, the cell comprises an exogenous nucleotide sequence of CD59 that has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the sequences listed in NCBI Ref.No.NNM_000611.5、NM_001127223.1、NM_001127225.1、NM_001127226.1、NM_001127227.1、NM_203329.2、NM_203330.2 and nm_ 203331.2. In some embodiments, the cell comprises an over-expressed nucleotide sequence of CD59 as listed in NCBI Ref.Sequence No.NM_000611.5、NM_001127223.1、NM_001127225.1、NM_001127226.1、NM_001127227.1、NM_203329.2、NM_203330.2 and nm_ 203331.2. In some embodiments, the cell comprises an exogenous nucleotide sequence for CD59 as set forth in NCBI Ref.Sequence No.NM_000611.5、NM_001127223.1、NM_001127225.1、NM_001127226.1、NM_001127227.1、NM_203329.2、NM_203330.2 and nm_ 203331.2.

In some embodiments, the cell comprises an over-expressed CD59 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to an amino acid sequence as set forth in NCBI Ref.Sequence No.NP_000602.1、NP_001120695.1、NP_001120697.1、NP_001120698.1、NP_001120699.1、NP_976074.1、NP_976075.1 and np_ 976076.1. In some embodiments, the cell comprises an exogenous CD59 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to an amino acid sequence as set forth in NCBI Ref.Sequence No.NP_000602.1、NP_001120695.1、NP_001120697.1、NP_001120698.1、NP_001120699.1、NP_976074.1、NP_976075.1 and np_ 976076.1. In some embodiments, the cells outlined herein comprise an over-expressed CD59 polypeptide having the amino acid sequences as listed in NCBI Ref.Sequence No.NP_000602.1、NP_001120695.1、NP_001120697.1、NP_001120698.1、NP_001120699.1、NP_976074.1、NP_976075.1 and np_ 976076.1. In some embodiments, the cells outlined herein comprise exogenous CD59 polypeptides having the amino acid sequences as listed in NCBI Ref.Sequence No.NP_000602.1、NP_001120695.1、NP_001120697.1、NP_001120698.1、NP_001120699.1、NP_976074.1、NP_976075.1 and np_ 976076.1.

In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD59 polypeptide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 6. In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding a CD59 polypeptide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 6. In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence that encodes a CD59 polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 6. In some embodiments, the cells outlined herein comprise an exogenous nucleotide sequence that encodes a CD59 polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 6.

In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD59 polypeptide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding a CD59 polypeptide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence that encodes a CD59 polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the cells outlined herein comprise an exogenous nucleotide sequence that encodes a CD59 polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, the exogenous nucleotide sequence encoding a CD59 polypeptide is operably linked to a sequence encoding a heterologous signal peptide.

In some embodiments, all or a functional portion of CD59 may be linked to other components, such as a signal peptide, a leader sequence, a secretion signal, a marker (e.g., a reporter), or any combination thereof. In some embodiments, the nucleic acid sequence encoding the signal peptide of CD59 is replaced with a nucleic acid sequence encoding a signal peptide from a heterologous protein. The heterologous protein may be, for example, CD8 alpha, CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF receptor (GM-CSFRa), or an immunoglobulin (e.g., igE or IgK). In some embodiments, the signal peptide is a signal peptide from an immunoglobulin (such as an IgG heavy chain or an IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2) or CD 33), serum albumin (e.g., HSA or albumin), a human azurin preproprotein signal sequence, luciferase, trypsinogen (e.g., chymotrypsinogen or trypsinogen), or other signal peptide capable of efficiently expressing a protein by or on a cell.

In certain embodiments, the exogenous polynucleotide encoding CD59 is operably linked to a promoter.

In some embodiments, a polynucleotide encoding CD59 is inserted into any one of the loci depicted in table 2. In some cases, the polynucleotide encoding CD59 is inserted into a safe harbor locus, such as, but not limited to, a locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS 231. In particular embodiments, the polynucleotide encoding CD59 is inserted into the CCR5 locus, the PPP1R12C (also known as AAVS 1) locus, or the CLYBL locus. In some embodiments, the polynucleotide encoding CD59 is inserted into the B2M locus, the CIITA locus, or the CD142 locus. In some embodiments, the engineered cell is a T cell and the polynucleotide encoding CD59 is inserted into the TRAC locus or the TRBC locus. In some embodiments, insertion of the polynucleotide encoding CD59 into the genomic locus of the cell is facilitated using a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein).

In some embodiments, CD59 protein expression is detected using western blotting of cell lysates that are probed with antibodies to CD59 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous CD59 mRNA.

F.CD55

In some embodiments, the engineered cells contain an over-expressed polynucleotide encoding CD55 (such as human CD 55). In some embodiments, the engineered cells contain an exogenous polynucleotide encoding CD55 (such as human CD 55). In some embodiments, CD55 is overexpressed in the cell. In some embodiments, the expression of CD55 in the engineered cell is increased compared to a similar reference or unmodified cell (including having any other modification), except that the reference or unmodified cell does not comprise an exogenous polynucleotide encoding CD 55. CD55 is a membrane bound complement inhibitor. In some embodiments, the interaction of CD55 with cell-associated C4B and C3B polypeptides interferes with their ability to catalyze the conversion of C2 and factor B to enzymatically active C2a and Bb, thereby preventing the formation of C4B2a and C3bBb (the amplified invertase of the complement cascade). In some embodiments, CD55 inhibits complement activation by disrupting the stabilization of C3 and C5 convertases and preventing the formation of C3 and C5 convertases. Available genomic, polynucleotide and polypeptide information about human CD55 (also known as complement decay acceleration factor) is provided, for example, in GeneCard identifiers GC01P207321, HGNC No.2665, NCBI Gene ID 1604, uniprot No. P08174, as well as NCBI RefSeq Nos.NM_000574.4、NM_001114752.2、NM_001300903.1、NM_001300904.1、NP_000565.1、NP_001108224.1、NP_001287832.1 and np_001287833.1.

In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD55 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequences as listed in NCBI ref. Sequence No. np_000565.1, np_001108224.1, np_001287832.1, and np_ 001287833.1. In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD55 polypeptide having the amino acid sequences as listed in NCBI ref. Sequence No. np_000565.1, np_001108224.1, np_001287832.1, and np_ 001287833.1. In some embodiments, the cell comprises an over-expressed nucleotide sequence of CD55 that has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the sequences listed in NCBI ref.no. nm_001777.3 and nm_ 198793.2. In some embodiments, the cell comprises an over-expressed nucleotide sequence of CD55 as listed in NCBI ref.sequence No. nm_000574.4, nm_001114752.2, nm_001300903.1, and nm_ 001300904.1.

In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding CD55 polypeptides having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to amino acid sequences as listed in NCBI ref. Sequence No. np_000565.1, np_001108224.1, np_001287832.1, and np_ 001287833.1. In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding CD55 polypeptides having the amino acid sequences as listed in NCBI ref. Sequence No. np_000565.1, np_001108224.1, np_001287832.1, and np_ 001287833.1. In some embodiments, the cell comprises an exogenous nucleotide sequence of CD55 that has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to a sequence listed in NCBI ref.no. nm_001777.3 and nm_ 198793.2. In some embodiments, the cell comprises an exogenous nucleotide sequence of CD55 as set forth in NCBI ref.sequence No. nm_000574.4, nm_001114752.2, nm_001300903.1, and nm_ 001300904.1.

In some embodiments, the cells comprise an over-expressed CD55 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to an amino acid sequence as set forth in NCBI ref.sequence No. np_000565.1, np_001108224.1, np_001287832.1, and np_ 001287833.1. In some embodiments, the cell comprises an exogenous CD55 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to an amino acid sequence as set forth in NCBI ref.sequence No. np_000565.1, np_001108224.1, np_001287832.1, and np_ 001287833.1. In some embodiments, the cells outlined herein comprise an over-expressed CD55 polypeptide having the amino acid sequences as listed in NCBI ref.sequence No. np_000565.1, np_001108224.1, np_001287832.1 and np_ 001287833.1. In some embodiments, the cells outlined herein comprise exogenous CD55 polypeptides having the amino acid sequences as listed in NCBI ref.sequence No. np_000565.1, np_001108224.1, np_001287832.1, and np_ 001287833.1.

In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD55 polypeptide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 9. In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding a CD55 polypeptide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 9. In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence that encodes a CD55 polypeptide comprising the amino acid sequence set forth in SEQ ID NO 9. In some embodiments, the cells outlined herein comprise an exogenous nucleotide sequence that encodes a CD55 polypeptide comprising the amino acid sequence set forth in SEQ ID NO 9.

In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence encoding a CD55 polypeptide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 8. In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding a CD55 polypeptide having at least 85% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 8. In some embodiments, the cells outlined herein comprise an over-expressed nucleotide sequence that encodes a CD55 polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 8. In some embodiments, the cells outlined herein comprise an exogenous nucleotide sequence that encodes a CD55 polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 8. In some embodiments, the exogenous nucleotide sequence encoding a CD59 polypeptide is operably linked to a sequence encoding a heterologous signal peptide.

In some embodiments, all or a functional portion of CD55 may be linked to other components, such as a signal peptide, a leader sequence, a secretion signal, a marker (e.g., a reporter), or any combination thereof. In some embodiments, the nucleic acid sequence encoding the signal peptide of CD55 is replaced with a nucleic acid sequence encoding a signal peptide from a heterologous protein. The heterologous protein may be, for example, CD8 alpha, CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF receptor (GM-CSFRa), or an immunoglobulin (e.g., igE or IgK). In some embodiments, the signal peptide is a signal peptide from an immunoglobulin (such as an IgG heavy chain or an IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2) or CD 33), serum albumin (e.g., HSA or albumin), a human azurin preproprotein signal sequence, luciferase, trypsinogen (e.g., chymotrypsinogen or trypsinogen), or other signal peptide capable of efficiently expressing a protein by or on a cell.

In certain embodiments, the exogenous polynucleotide encoding CD55 is operably linked to a promoter.

In some embodiments, the polynucleotide encoding CD55 is inserted into any one of the loci depicted in table 2. In some cases, the polynucleotide encoding CD55 is inserted into a safe harbor locus, such as, but not limited to, a locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS 231. In particular embodiments, the polynucleotide encoding CD55 is inserted into the CCR5 locus, the PPP1R12C (also known as AAVS 1) locus, or the CLYBL locus. In some embodiments, the polynucleotide encoding CD55 is inserted into the B2M locus, the CIITA locus, or the CD142 locus. In some embodiments, the engineered cell is a T cell and the polynucleotide encoding CD55 is inserted into the TRAC locus or the TRBC locus. In some embodiments, insertion of the polynucleotide encoding CD55 into the genomic locus of the cell is facilitated using a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein).

In some embodiments, CD55 protein expression is detected using western blotting of cell lysates that are probed with antibodies to CD55 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous CD55 mRNA.

G. Combination of complement inhibitors

In some embodiments, the cell comprises increased expression of two or more complement inhibitors selected from any combination of the group consisting of CD46, CD59, and CD 55.

In some embodiments, the engineered cells contain an over-expressed polynucleotide encoding CD46 (such as any of the polynucleotides described above) and an over-expressed polynucleotide encoding CD59 (such as any of the polynucleotides described above).

In some embodiments, the engineered cells contain an exogenous polynucleotide encoding CD46 (such as any of the polynucleotides described above) and an exogenous polynucleotide encoding CD59 (such as any of the polynucleotides described above).

In some embodiments, the engineered cells (comprising one or more modifications that increase CD46 and CD59 expression) comprise increased CD46 and CD59 expression relative to cells that do not comprise modifications (e.g., relative to endogenous expression of CD46 and CD 59). In some embodiments, the engineered cells comprise increased expression of CD46 and CD59 between 1.5-fold and 2-fold, between 2-fold and 3-fold, between 3-fold and 4-fold, between 4-fold and 5-fold, between 5-fold and 10-fold, between 10-fold and 15-fold, between 15-fold and 20-fold, between 20-fold and 40-fold, between 40-fold and 60-fold, between 60-fold and 80-fold, between 80-fold and 100-fold, or between 100-fold and 200-fold compared to cells without modification (e.g., compared to endogenous expression of CD46 and CD 59). In some embodiments, the unmodified cell does not have endogenous expression of CD46 and CD59 or has no detectable expression of CD46 and CD 59. In some embodiments, the fold increase in expression is greater than 200 fold as compared to a cell lacking the modification.

In some embodiments, the engineered cells (comprising one or more modifications that increase the expression of CD46 and CD 59) comprise between 2-fold and 200-fold, between 2-fold and 100-fold, between 2-fold and 50-fold, or between 2-fold and 20-fold increased expression of CD46 and CD59 compared to cells without the modification (e.g., compared to endogenous expression of CD46 and CD 59). In some embodiments, the engineered cells (comprising one or more modifications that increase the expression of CD46 and CD 59) comprise between 5-fold and 200-fold, between 5-fold and 100-fold, between 5-fold and 50-fold, or between 5-fold and 20-fold increased expression of CD46 and CD59 compared to cells without the modification (e.g., compared to endogenous expression of CD46 and CD 59).

In some embodiments, the engineered cells (comprising one or more modifications that increase CD46 and CD59 expression) comprise increased CD46 and CD59 expression relative to cells that do not comprise modifications (e.g., relative to endogenous expression of CD46 and CD 59). In some embodiments, the engineered cells comprise increased expression of CD46 and CD59 of at least or about 2-fold, at least or about 4-fold, at least or about 6-fold, at least or about 10-fold, at least or about 15-fold, at least or about 20-fold, at least or about 30-fold, at least or about 50-fold, at least or about 60-fold, at least or about 70-fold, at least or about 80-fold, at least or about 100-fold, or any value in between any of the foregoing, as compared to cells without modification (e.g., as compared to endogenous expression of CD46 and CD 59).

In some embodiments, the engineered cells (comprising one or more modifications that increase CD46 and CD59 expression) comprise increased CD46 and CD59 expression relative to cells that do not comprise modifications (e.g., relative to endogenous expression of CD46 and CD 59). In some embodiments, the engineered cells comprise increased expression of CD46 and CD59 at or about 2-fold, at or about 4-fold, at or about 6-fold, at or about 10-fold, at or about 15-fold, at or about 20-fold, at or about 30-fold, at or about 50-fold, at or about 60-fold, at or about 70-fold, at or about 80-fold, at or about 100-fold, or any value in between, as compared to cells without modification (e.g., as compared to endogenous expression of CD46 and CD 59).

In some embodiments, the cells comprise one or more transgenes encoding CD46 and CD 59. In some embodiments, the transgene is a monocistronic or polycistronic vector, as described in section ii.b.4 below. In some embodiments, CD46 and CD59 consist of the same polycistronic vector, optionally in combination with one or more tolerogenic factors (such as CD 47). In some embodiments, CD46 and CD59 consist of different transgenes, optionally in combination with one or more tolerogenic factors (such as CD 47).

In some embodiments, the engineered cells contain an over-expressed polynucleotide encoding CD46 (such as any of the polynucleotides described above), an over-expressed polynucleotide encoding CD59 (such as any of the polynucleotides described above), and an over-expressed polynucleotide encoding CD55 (such as any of the polynucleotides described above).

In some embodiments, the engineered cells contain an exogenous polynucleotide encoding CD46 (such as any of the polynucleotides described above), an exogenous polynucleotide encoding CD59 (such as any of the polynucleotides described above), and an exogenous polynucleotide encoding CD55 (such as any of the polynucleotides described above).

In some embodiments, the engineered cells (comprising one or more modifications that increase CD46, CD59, and CD55 expression) comprise increased expression of CD46, CD59, and CD55 relative to cells that do not comprise modifications (e.g., relative to endogenous expression of CD46, CD59, and CD 55). In some embodiments, the engineered cells comprise increased expression of CD46, CD59, and CD55 between 1.5-fold and 2-fold, between 2-fold and 3-fold, between 3-fold and 4-fold, between 4-fold and 5-fold, between 5-fold and 10-fold, between 10-fold and 15-fold, between 15-fold and 20-fold, between 20-fold and 40-fold, between 40-fold and 60-fold, between 60-fold and 80-fold, between 80-fold and 100-fold, or between 100-fold and 200-fold compared to cells without modification (e.g., compared to endogenous expression of CD46, CD59, and CD 55). In some embodiments, the unmodified cells do not have endogenous expression of CD46, CD59, and CD55 or do not have detectable expression of CD46, CD59, and CD 55. In some embodiments, the fold increase in expression is greater than 200 fold as compared to a cell lacking the modification.

In some embodiments, the engineered cells (comprising one or more modifications that increase the expression of CD46, CD59, and CD 55) comprise an increase in the expression of CD46, CD59, and CD55 of between 2-fold and 200-fold, between 2-fold and 100-fold, between 2-fold and 50-fold, or between 2-fold and 20-fold compared to cells without the modification (e.g., compared to endogenous expression of CD46, CD59, and CD 55). In some embodiments, the engineered cells (comprising one or more modifications that increase the expression of CD46, CD59, and CD 55) comprise between 5-fold and 200-fold, between 5-fold and 100-fold, between 5-fold and 50-fold, or between 5-fold and 20-fold increased expression of CD46, CD59, and CD55 as compared to cells without the modifications (e.g., as compared to endogenous expression of CD46, CD59, and CD 55).

In some embodiments, the engineered cells (comprising one or more modifications that increase the expression of CD46, CD59, and CD 55) comprise increased expression of CD46, CD59, and CD55 relative to cells that do not comprise modifications (e.g., relative to endogenous expression of CD46 and CD 59). In some embodiments, the engineered cells comprise increased expression of CD46, CD59, and CD55 of at least or about 2-fold, at least or about 4-fold, at least or about 6-fold, at least or about 10-fold, at least or about 15-fold, at least or about 20-fold, at least or about 30-fold, at least or about 50-fold, at least or about 60-fold, at least or about 70-fold, at least or about 80-fold, at least or about 100-fold, or any value in between any of the foregoing, as compared to cells without modification (e.g., as compared to endogenous expression of CD46, CD59, and CD 55).

In some embodiments, the engineered cells (comprising one or more modifications that increase CD46, CD59, and CD55 expression) comprise increased expression of CD46, CD59, and CD55 relative to cells that do not comprise modifications (e.g., relative to endogenous expression of CD46, CD59, and CD 55). In some embodiments, the engineered cells comprise increased expression of CD46, CD59, and CD55 at or about 2-fold, at or about 4-fold, at or about 6-fold, at or about 10-fold, at or about 15-fold, at or about 20-fold, at or about 30-fold, at or about 50-fold, at or about 60-fold, at or about 70-fold, at or about 80-fold, at or about 100-fold, or any value in between, as compared to cells without modification (e.g., as compared to endogenous expression of CD46, CD59, and CD 55).

In some embodiments, the cells comprise one or more transgenes encoding CD46, CD59, and CD 55. In some embodiments, the transgene is a monocistronic or polycistronic vector, as described in section ii.b.4 below. In some embodiments, CD46, CD59, and CD55 consist of the same polycistronic vector, optionally in combination with one or more tolerogenic factors (such as CD 47). In some embodiments, CD46, CD59, and CD55 consist of different transgenes, optionally in combination with one or more tolerogenic factors (such as CD 47).

2. Tolerogenic factors

In some embodiments, expression of the tolerogenic factors is over-expressed or increased in the cell. In some embodiments, the engineered cell comprises increased expression, i.e., overexpression, of at least one tolerogenic factor. In some embodiments, a tolerogenic factor is any factor that promotes or helps promote or induce tolerance of the immune system (e.g., the innate or adaptive immune system) to an engineered cell. In some embodiments, tolerogenic factors are DUX4, B2M-HLA-E, CD16, CD52, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc receptor, IL15-RF and H2-M3. In some embodiments, the tolerogenic factors are CD47, PD-L1, HLA-E or HLA-G, CCL, fasL, serpin 9, CD200 or Mfge8, or any combination thereof. In some embodiments, the cell comprises at least one exogenous polynucleotide comprising a polynucleotide encoding a tolerogenic factor. For example, in some embodiments, the at least one exogenous polynucleotide is a polynucleotide encoding CD 47. Provided herein are cells that do not trigger or activate an immune response upon administration to a recipient subject. As described above, in some embodiments, the cells are modified to increase expression of genes and tolerogenic (e.g., immune) factors that affect immune recognition and tolerance in the recipient.

In some embodiments, the disclosure provides a cell or population thereof that has been modified to express a tolerizing factor (e.g., an immunomodulatory polypeptide), such as CD47. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express a tolerogenic factor (e.g., an immunomodulatory polypeptide) such as CD47. In some embodiments, the engineered cells express an exogenous tolerogenic factor (e.g., an immunomodulatory polypeptide), such as exogenous CD47. In some cases, over-expressing the exogenous polynucleotide or increasing expression of the exogenous polynucleotide is achieved by introducing into a cell (e.g., a transduced cell) an expression vector comprising a nucleotide sequence encoding a human CD47 polypeptide. In some embodiments, the expression vector may be a viral vector (such as a lentiviral vector), or may be a non-viral vector. In some embodiments, the cell is engineered to contain one or more exogenous polynucleotides, wherein at least one exogenous polynucleotide comprises a polynucleotide encoding a tolerogenic factor. In some of any of the embodiments, the tolerogenic factors are DUX4, B2M-HLA-E, CD16, CD52, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc receptor, IL15-RF and H2-M3. In some embodiments, the tolerogenic factors are selected from CD47, PD-L1, HLA-E or HLA-G, CCL, fasL, serpin 9, CD200 or Mfge8, or any combination thereof (e.g., all of them). For example, in some embodiments, the at least one exogenous polynucleotide is a polynucleotide encoding CD47.

In some embodiments, the tolerogenic factor is CD47. In some embodiments, the engineered cells contain an exogenous polynucleotide encoding CD47 (such as human CD 47). In some embodiments, CD47 is overexpressed in the cell. In some embodiments, expression of CD47 is over-expressed or increased in an engineered cell as compared to a similar cell of the same cell type that is not engineered with the modification, such as a reference or unmodified cell (e.g., a cell that is not engineered with an exogenous polynucleotide encoding CD 47). CD47 is a leukocyte surface antigen and plays a role in cell adhesion and integrin regulation. It is usually expressed on the cell surface and signals circulating macrophages that they are not to phagocytic. Available genomic, polynucleotide and polypeptide information about human CD47 is provided, for example, in np_001768.1, np_942088.1, nm_001777.3 and nm_ 198793.2.

In some embodiments, the engineered cell comprises increased expression, i.e., overexpression, of at least one tolerogenic factor. In some embodiments, the cell comprises at least one exogenous polynucleotide comprising a polynucleotide encoding a tolerogenic factor. In some embodiments, tolerogenic factors include DUX4, B2M-HLA-E, CD16, CD52, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc receptor, IL15-RF, and H2-M3, or any combination thereof. For example, in some embodiments, at least one over-expressed (e.g., exogenous) polynucleotide is a polynucleotide encoding CD 47.

In some embodiments, the disclosure provides a cell or population thereof that has been modified to express a tolerizing factor (e.g., an immunomodulatory polypeptide), such as CD47. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express a tolerogenic factor (e.g., an immunomodulatory polypeptide) such as CD47. In some embodiments, the engineered cells express an exogenous tolerogenic factor (e.g., an immunomodulatory polypeptide), such as exogenous CD47. In some cases, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD47 polypeptide.

In some embodiments, the engineered cells contain an over-expressed polynucleotide encoding CD47 (such as human CD 47). In some embodiments, the engineered cells contain an exogenous polynucleotide encoding CD47 (such as human CD 47). In some embodiments, CD47 is overexpressed in the cell. In some embodiments, the expression of CD47 in the engineered cell is increased compared to a similar reference or unmodified cell (including having any other modification), except that the reference or unmodified cell does not comprise an exogenous polynucleotide encoding CD 47. CD47 is a leukocyte surface antigen and plays a role in cell adhesion and integrin regulation. It is usually expressed on the cell surface and signals circulating macrophages that they are not to phagocytic. Available genomic, polynucleotide and polypeptide information about human CD47 is provided, for example, in np_001768.1, np_942088.1, nm_001777.3 and nm_ 198793.2.

In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding CD47 polypeptides having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to amino acid sequences as listed in NCBI ref. Sequence nos. np_001768.1 and np_ 942088.1. In some embodiments, the cells outlined herein comprise exogenous nucleotide sequences encoding CD47 polypeptides having the amino acid sequences as listed in NCBI ref. Sequence No. np_001768.1 and np_ 942088.1. In some embodiments, the cell comprises an exogenous nucleotide sequence of CD47 that has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to a sequence listed in NCBI ref.no. nm_001777.3 and nm_ 198793.2. In some embodiments, the cell comprises an exogenous nucleotide sequence of CD47 as set forth in NCBI ref.sequence No. nm_001777.3 and nm_ 198793.2.

In some embodiments, the cells comprise exogenous CD47 polypeptides having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to amino acid sequences as listed in NCBI ref. Sequence nos. np_001768.1 and np_ 942088.1. In some embodiments, the cells outlined herein comprise exogenous CD47 polypeptides having the amino acid sequences as listed in NCBI ref.sequence No. np_001768.1 and np_ 942088.1.

In some embodiments, the cell comprises an over-expressed polynucleotide encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 1. In some embodiments, the cell comprises an exogenous polynucleotide encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in SEQ ID No. 1. In some embodiments, the cell comprises an over-expressed polynucleotide encoding a CD47 polypeptide having the amino acid sequence set forth in SEQ ID No. 1. In some embodiments, the cell comprises an exogenous polynucleotide encoding a CD47 polypeptide having the amino acid sequence set forth in SEQ ID NO. 1.

In some embodiments, the cell comprises an over-expressed CD47 polypeptide having at least 95% sequence identity (e.g., 95%,96%,97%,98%,99% or more) to the amino acid sequence set forth in SEQ ID No. 2. In some embodiments, the cell comprises an exogenous CD47 polypeptide having at least 95% sequence identity (e.g., 95%,96%,97%,98%,99% or more) to the amino acid sequence set forth in SEQ ID No. 2. In some embodiments, the cell comprises an over-expressed CD47 polypeptide having the amino acid sequence set forth in SEQ ID NO. 2. In some embodiments, the cell comprises an exogenous CD47 polypeptide having the amino acid sequence set forth in SEQ ID NO. 2. In some embodiments, the exogenous nucleotide sequence encoding a CD59 polypeptide is operably linked to a sequence encoding a heterologous signal peptide. In some embodiments, the exogenous polynucleotide encoding CD47 is integrated into the cell genome by targeted or non-targeted insertion methods (such as described further below). In some embodiments, targeted insertion is by homology-dependent insertion into a target locus, such as by insertion into any one of the loci depicted in table 2 (e.g., B2M gene, CIITA gene, TRAC gene, TRBC gene). In some embodiments, targeted insertion is by homologous independent insertion, such as by insertion into a safe harbor locus. In some cases, the polynucleotide encoding CD47 is inserted into a safe harbor locus, such as, but not limited to, a locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS 231. In particular embodiments, the polynucleotide encoding CD47 is inserted into the CCR5 locus, the PPP1R12C (also known as AAVS 1) locus, or the CLYBL locus.

In some embodiments, all or a functional portion of CD47 may be linked to other components, such as a signal peptide, a leader sequence, a secretion signal, a marker (e.g., a reporter), or any combination thereof. In some embodiments, the nucleic acid sequence encoding the signal peptide of CD47 is replaced with a nucleic acid sequence encoding a signal peptide from a heterologous protein. The heterologous protein may be, for example, CD8 alpha, CD28, tissue plasminogen activator (tPA), growth hormone, granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF receptor (GM-CSFRa), or an immunoglobulin (e.g., igE or IgK). In some embodiments, the signal peptide is a signal peptide from an immunoglobulin (such as an IgG heavy chain or an IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2) or CD 33), serum albumin (e.g., HSA or albumin), a human azurin preproprotein signal sequence, luciferase, trypsinogen (e.g., chymotrypsinogen or trypsinogen), or other signal peptide capable of efficiently expressing a protein by or on a cell.

In certain embodiments, the exogenous polynucleotide encoding CD47 is operably linked to a promoter.

In some embodiments, an exogenous polynucleotide encoding CD47 is inserted into any one of the loci depicted in table 2. In some cases, an exogenous polynucleotide encoding CD47 is inserted into a safe harbor locus, such as but not limited to a locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS 231. In particular embodiments, an exogenous polynucleotide encoding CD47 is inserted into the CCR5 locus, the PPP1R12C (also known as AAVS 1) locus, or the CLYBL locus. In some embodiments, an exogenous polynucleotide encoding CD47 is inserted into the B2M locus, the CIITA locus, or the CD142 locus. In some embodiments, the engineered cell is a T cell and the exogenous polynucleotide encoding CD47 is inserted into the TRAC locus or the TRBC locus. In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding CD47 into a genomic locus of a cell.

In some embodiments, CD47 protein expression is detected using western blotting of cell lysates that are probed with antibodies to CD47 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous CD47 mRNA.

In some embodiments, the engineered cells contain an exogenous polynucleotide encoding CD200 (such as human CD 200). In some embodiments, CD200 is overexpressed in the cell. In some embodiments, the expression of CD200 in the engineered cell is increased compared to a similar reference or unmodified cell (including having any other modification), except that the reference or unmodified cell does not comprise an exogenous polynucleotide encoding CD 200. Available genomic, polynucleotide and polypeptide information about human CD200 is provided, for example, in GeneCard identifiers GC03P112332, HGNC No.7203, NCBI Gene ID 4345, uniprot No. P41217, and NCBI RefSeq No.NP_001004196.2、NM_001004196.3、NP_001305757.1、NM_001318828.1、NP_005935.4、NM_005944.6、XP_005247539.1 and xm_005247482.2. In certain embodiments, the polynucleotide encoding CD200 is operably linked to a promoter.

In some embodiments, a polynucleotide encoding CD200 is inserted into any one of the loci depicted in table 2. In some cases, the polynucleotide encoding CD200 is inserted into a safe harbor locus, such as, but not limited to, a locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS 231. In particular embodiments, the polynucleotide encoding CD200 is inserted into the CCR5 locus, the PPP1R12C (also known as AAVS 1) locus, or the CLYBL locus. In some embodiments, the polynucleotide encoding CD200 is inserted into the B2M locus, the CIITA locus, or the CD142 locus. In some embodiments, the engineered cell is a T cell and the polynucleotide encoding CD200 is inserted into the TRAC locus or the TRBC locus. In some embodiments, insertion of the polynucleotide encoding CD200 into the genomic locus of a cell is facilitated using a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein).

In some embodiments, CD200 protein expression is detected using western blotting of cell lysates that are probed with antibodies to CD200 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous CD200 mRNA.

In some embodiments, the engineered cells contain an exogenous polynucleotide encoding HLA-E (such as human HLA-E). In some embodiments, HLA-E is overexpressed in the cell. In some embodiments, the expression of HLA-E in the engineered cell is increased compared to a similar reference or unmodified cell (including having any other modification), except that the reference or unmodified cell does not comprise an exogenous polynucleotide encoding HLA-E. Available genomic, polynucleotide and polypeptide information about human HLA-E is provided, for example, in GeneCard identifiers GC06P047281, HGNC No.4962, NCBI Gene ID 3133, uniprot No. P13747, NCBI RefSeq No. np_005507.3 and nm_005516.5. In certain embodiments, the polynucleotide encoding HLA-E is operably linked to a promoter.

In some embodiments, a polynucleotide encoding HLA-E is inserted into any one of the loci depicted in table 2. In some cases, a polynucleotide encoding HLA-E is inserted into a safe harbor locus, such as but not limited to a locus selected from AAVS1, CCR5, CLYBL, ROSA26, SHS 231. In particular embodiments, a polynucleotide encoding HLA-E is inserted into the CCR5 locus, PPP1R12C (also known as AAVS 1) locus, or CLYBL locus. In some embodiments, a polynucleotide encoding HLA-E is inserted into the B2M locus, CIITA locus, or CD142 locus. In some embodiments, the engineered cell is a T cell and the polynucleotide encoding HLA-E is inserted into the TRAC locus or TRBC locus. In some embodiments, insertion of the HLA-E encoding polynucleotide into the genomic locus of the cell is facilitated using a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein).

In some embodiments, HLA-E protein expression is detected using Western blotting of cell lysates probed with antibodies to HLA-E proteins. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous HLA-E mRNA.

In some embodiments, the engineered cells contain an exogenous polynucleotide encoding HLA-G (such as human HLA-G). In some embodiments, HLA-G is overexpressed in the cell. In some embodiments, the expression of HLA-G in the engineered cell is increased compared to a similar reference or unmodified cell (including having any other modification), except that the reference or unmodified cell does not comprise an exogenous polynucleotide encoding HLA-G. Available genomic, polynucleotide and polypeptide information about human HLA-G is provided, for example, in GeneCard identifiers GC06P047256, HGNC No.4964, NCBI Gene ID 3135, uniprot No. P17693, and NCBI RefSeq No. np_002118.1 and nm_002127.5. In certain embodiments, the polynucleotide encoding HLA-G is operably linked to a promoter.

In some embodiments, a polynucleotide encoding HLA-G is inserted into any one of the loci depicted in table 2. In some cases, a polynucleotide encoding HLA-G is inserted into a safe harbor locus, such as, but not limited to, a locus selected from AAVS1, CCR5, CLYBL, ROSA26, SHS 231. In particular embodiments, a polynucleotide encoding HLA-G is inserted into the CCR5 locus, PPP1R12C (also known as AAVS 1) locus, or CLYBL locus. In some embodiments, a polynucleotide encoding HLA-G is inserted into the B2M locus, CIITA locus, or CD142 locus. In some embodiments, the engineered cell is a T cell and the polynucleotide encoding HLA-G is inserted into the TRAC locus or the TRBC locus. In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding HLA-G into a genomic locus of a cell.

In some embodiments, HLA-G protein expression is detected using Western blotting of cell lysates probed with antibodies to HLA-G proteins. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous HLA-G mRNA.

In some embodiments, the engineered cell contains an exogenous polynucleotide encoding PD-L1 (such as human PD-L1). In some embodiments, PD-L1 is overexpressed in the cell. In some embodiments, the expression of PD-L1 in the engineered cell is increased compared to a similar reference or unmodified cell (including having any other modification), except that the reference or unmodified cell does not comprise an exogenous polynucleotide encoding PD-L1. Available genomic, polynucleotide and polypeptide information about human PD-L1 or CD274 is provided, for example, in GeneCard identifiers GC09P005450, HGNC No.17635, NCBI Gene ID 29126, uniprot No. q9nzq7, and NCBI RefSeq nos. np_001254635.1, nm_001267706.1, np_054862.1 and nm_014143.3. In certain embodiments, the polynucleotide encoding PD-L1 is operably linked to a promoter.

In some embodiments, a polynucleotide encoding PD-L1 is inserted into any one of the loci depicted in table 2. In some cases, the polynucleotide encoding PD-L1 is inserted into a safe harbor locus, such as, but not limited to, a locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS 231. In particular embodiments, the polynucleotide encoding PD-L1 is inserted into the CCR5 locus, PPP1R12C (also known as AAVS 1) locus, or CLYBL locus. In some embodiments, the polynucleotide encoding PD-L1 is inserted into the B2M locus, CIITA locus, or CD142 locus. In some embodiments, the engineered cell is a T cell and the polynucleotide encoding PD-L1 is inserted into the TRAC locus or TRBC locus. In some embodiments, insertion of a polynucleotide encoding PD-L1 into a genomic locus of a cell is facilitated using a suitable gene editing system (e.g., a CRISPR/Cas system or any of the gene editing systems described herein).

In some embodiments, PD-L1 protein expression is detected using western blotting of cell lysates probed with antibodies to PD-L1 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous PD-L1 mRNA.

In some embodiments, the engineered cells contain an exogenous polynucleotide encoding FasL (such as human FasL). In some embodiments, fasL is overexpressed in the cell. In some embodiments, the expression of FasL in the engineered cell is increased compared to a similar reference or unmodified cell (including having any other modification), except that the reference or unmodified cell does not comprise an exogenous polynucleotide encoding FasL. Available genomic, polynucleotide and polypeptide information about human Fas ligand (which is also referred to as FasL, FASLG, CD178, TNFSF6, etc.) is provided, for example, in GeneCard identifiers GC01P172628, HGNC No.11936, NCBI Gene ID 356, uniprot No. P48023, and NCBI Refseq No. NP-000630.1, NM-000639.2, NP-001289675.1, and NM-001302746.1. In certain embodiments, a polynucleotide encoding Fas-L is operably linked to a promoter.

In some embodiments, a polynucleotide encoding Fas-L is inserted into any one of the loci depicted in Table 2. In some cases, a polynucleotide encoding Fas-L is inserted into a safe harbor locus, such as, but not limited to, a locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS 231. In particular embodiments, a polynucleotide encoding Fas-L is inserted into the CCR5 locus, PPP1R12C (also known as AAVS 1) locus, or CLYBL locus. In some embodiments, a polynucleotide encoding Fas-L is inserted into the B2M locus, CIITA locus, or CD142 locus. In some embodiments, the engineered cell is a T cell and the polynucleotide encoding Fas-L is inserted into the TRAC locus or TRBC locus. In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding Fas-L into a genomic locus of a cell.

In some embodiments, western blotting of cell lysates detected with antibodies to Fas-L protein is used to detect Fas-L protein expression. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous Fas-LmRNA.

In some embodiments, the engineered cell contains an exogenous polynucleotide encoding CCL21 (such as human CCL 21). In some embodiments, CCL21 is overexpressed in a cell. In some embodiments, the expression of CCL21 in an engineered cell is increased compared to a similar reference or unmodified cell (including having any other modification), except that the reference or unmodified cell does not comprise an exogenous polynucleotide encoding CCL 21. Available genomic, polynucleotide and polypeptide information about human CCL21 is provided, for example, in GeneCard identifiers GC09M034709, HGNC No.10620, NCBI Gene ID 6366, uniprot No. o00585, NCBI RefSeq No. np_002980.1 and nm_002989.3. In certain embodiments, a polynucleotide encoding CCL21 is operably linked to a promoter.

In some embodiments, a polynucleotide encoding CCL21 is inserted into any one of the loci depicted in table 2. In some cases, a polynucleotide encoding CCL21 is inserted into a safe harbor locus, such as, but not limited to, a locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS 231. In particular embodiments, a polynucleotide encoding CCL21 is inserted into the CCR5 locus, PPP1R12C (also known as AAVS 1) locus, or CLYBL locus. In some embodiments, a polynucleotide encoding CCL21 is inserted into the B2M locus, CIITA locus, or CD142 locus. In some embodiments, the engineered cell is a T cell and the polynucleotide encoding CCL21 is inserted into a TRAC locus or a TRBC locus. In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding CCL21 into a genomic locus of a cell.

In some embodiments, CCL21 protein expression is detected using western blotting of cell lysates probed with antibodies to CCL21 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous CCL21 mRNA.

In some embodiments, the engineered cell contains an exogenous polynucleotide encoding CCL22 (such as human CCL 22). In some embodiments, CCL22 is overexpressed in a cell. In some embodiments, the expression of CCL22 in an engineered cell is increased compared to a similar reference or unmodified cell (including having any other modification), except that the reference or unmodified cell does not comprise an exogenous polynucleotide encoding CCL 22. Available genomic, polynucleotide and polypeptide information about human CCL22 is provided, for example, in GeneCard identifiers GC16P057359, HGNC No.10621, NCBI Gene ID 6367, uniprot No. o00626, NCBI RefSeq No. np_002981.2, nm_002990.4, xp_016879020.1 and xm_017023531.1. In certain embodiments, a polynucleotide encoding CCL22 is operably linked to a promoter.

In some embodiments, a polynucleotide encoding CCL22 is inserted into any one of the loci depicted in table 2. In some cases, a polynucleotide encoding CCL22 is inserted into a safe harbor locus, such as, but not limited to, a locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS 231. In particular embodiments, a polynucleotide encoding CCL22 is inserted into the CCR5 locus, PPP1R12C (also known as AAVS 1) locus, or CLYBL locus. In some embodiments, the polynucleotide encoding CCL22 is inserted into the B2M locus, CIITA locus, or CD142 locus. In some embodiments, the engineered cell is a T cell and the polynucleotide encoding CCL22 is inserted into a TRAC locus or a TRBC locus. In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding CCL22 into a genomic locus of a cell.

In some embodiments, CCL22 protein expression is detected using western blotting of cell lysates probed with antibodies to CCL22 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous CCL22 mRNA.

In some embodiments, the engineered cell contains an exogenous polynucleotide encoding Mfge (such as human Mfge 8). In some embodiments Mfge8 is overexpressed in the cell. In some embodiments, the expression of Mfge in the engineered cell is increased compared to a similar reference or unmodified cell (including having any other modifications), except that the reference or unmodified cell does not comprise the exogenous polynucleotide encoding Mfge. Available genomic, polynucleotide and polypeptide information about human Mfge is provided, for example, in GeneCard identifier GC15M088898, hgncno.7036, NCBI Gene ID 4240, uniprot No. q08431, and NCBI RefSeq No.NP_001108086.1、NM_001114614.2、NP_001297248.1、NM_001310319.1、NP_001297249.1、NM_001310320.1、NP_001297250.1、NM_001310321.1、NP_005919.2 and nm_005928.3. In certain embodiments, the polynucleotide encoding Mfge8 is operably linked to a promoter.

In some embodiments, the polynucleotide encoding Mfge8 is inserted into any one of the loci depicted in table 2. In some cases, the polynucleotide encoding Mfge is inserted into a safe harbor locus, such as, but not limited to, a locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS 231. In particular embodiments, the polynucleotide encoding Mfge8 is inserted into the CCR5 locus, the PPP1R12C (also known as AAVS 1) locus, or the CLYBL locus. In some embodiments, the polynucleotide encoding Mfge8 is inserted into the B2M locus, CIITA locus, CD142 locus. In some embodiments, the engineered cell is a T cell and the polynucleotide encoding Mfge is inserted into the TRAC locus or the TRBC locus. In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein) is used to facilitate insertion of the polynucleotide encoding Mfge into a genomic locus of a cell.

In some embodiments, the Mfge protein expression is detected using western blotting of cell lysates probed with antibodies to Mfge protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous Mfge mRNA.

In some embodiments, the engineered cell contains an exogenous polynucleotide encoding SerpinB (such as human SerpinB 9). In some embodiments SerpinB9 is overexpressed in the cell. In some embodiments, the expression of SerpinB in the engineered cell is increased compared to a similar reference or unmodified cell (including having any other modifications), except that the reference or unmodified cell does not comprise the exogenous polynucleotide encoding SerpinB 9. Available genomic, polynucleotide and polypeptide information about human SerpinB is provided, for example, in GeneCard identifiers GC06M002887, HGNC No.8955, NCBI Gene ID 5272, uniprot No. p50453, and NCBI RefSeq No. np_004146.1, nm_004155.5, xp_005249241.1, and xm_005249184.4. In certain embodiments, the polynucleotide encoding SerpinB9 is operably linked to a promoter.

In some embodiments, the polynucleotide encoding SerpinB9 is inserted into any one of the loci depicted in table 2. In some cases, the polynucleotide encoding SerpinB is inserted into a safe harbor locus, such as, but not limited to, a locus selected from AAVS1, CCR5, CLYBL, ROSA26, and SHS 231. In particular embodiments, the polynucleotide encoding SerpinB9 is inserted into the CCR5 locus, the PPP1R12C (also known as AAVS 1) locus, or the CLYBL locus. In some embodiments, the polynucleotide encoding SerpinB9 is inserted into the B2M locus, the CIITA locus, or the CD142 locus. In some embodiments, the engineered cell is a T cell and the polynucleotide encoding SerpinB is inserted into the TRAC locus or the TRBC locus. In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein) is used to facilitate insertion of the polynucleotide encoding SerpinB into a genomic locus of a cell.

In some embodiments, the SerpinB protein expression is detected using western blotting of cell lysates that are probed with antibodies to SerpinB protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous SerpinB mRNA.

3. Chimeric antigen receptor

In some embodiments, the engineered cells provided are further modified to express a Chimeric Antigen Receptor (CAR). In some embodiments, provided cells contain genetic modification of one or more target polynucleotide sequences that regulate expression of one or more MHC class I molecules, one or more MHC class II molecules, or one or more MHC class I molecules and one or more MHC class II molecules, regulate immediate blood-mediated inflammatory responses, overexpress tolerogenic factors (e.g., CD 47) as described herein, and express a CAR. In some embodiments, the cell is one in which: B2M reduction or elimination (e.g., knockout), CIITA reduction or elimination (e.g., knockout), CD142 reduction or elimination, CD47 overexpression, and CAR expression. In some embodiments, the cells are B2M ^-/-、CIITA^-/-、CD142^-/-, CD47tg, car+. In some embodiments, the cell (e.g., T cell) may also be a cell in which TRAC is reduced or eliminated (e.g., knocked out). In some embodiments, the cell is B2 ^-/-、CIITA^-/-、CD142^-/-、CD47tg、TRAC^-/- car+.

In some embodiments, the polynucleotide encoding the CAR is introduced into a cell. In some embodiments, the cell is a T cell, such as a primary T cell or a T cell differentiated from a pluripotent cell (e.g., iPSC). In some embodiments, the cell is a Natural Killer (NK) cell, such as a primary NK cell or an NK cell differentiated from a pluripotent cell (e.g., iPSC).

In some embodiments, the CAR is selected from the group consisting of a first generation CAR, a second generation CAR, a third generation CAR, and a fourth generation CAR. In some embodiments, the CAR is or comprises a first generation CAR comprising an antigen binding domain, a transmembrane domain, and at least one signaling domain (e.g., one, two, or three signaling domains). In some embodiments, the CAR comprises a second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains. In some embodiments, the CAR comprises a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, the fourth generation CAR comprises an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces cytokine gene expression upon successful signaling of the CAR. In some embodiments, the antigen binding domain is or comprises an antibody, antibody fragment, scFv, or Fab.

In some embodiments, any of the cells described herein comprise a nucleic acid encoding a CAR or a first generation CAR. In some embodiments, the first generation CAR comprises one antigen binding domain, one transmembrane domain, and one signaling domain. In some embodiments, the signaling domain mediates downstream signaling during T cell activation.

In some embodiments, any of the cells described herein comprise a nucleic acid encoding a CAR or a second generation CAR. In some embodiments, the second generation CAR comprises one antigen binding domain, one transmembrane domain, and two signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a co-stimulatory domain. In some embodiments, the costimulatory domain enhances cytokine production, CAR-T cell proliferation, and/or CAR-T cell persistence during T cell activation.

In some embodiments, any of the cells described herein comprise a nucleic acid encoding a CAR or a third generation CAR. In some embodiments, the third generation CAR comprises one antigen binding domain, one transmembrane domain, and at least three signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a co-stimulatory domain. In some embodiments, the costimulatory domain enhances cytokine production, CAR-T cell proliferation, and or CAR-T cell persistence during T cell activation. In some embodiments, the third generation CAR comprises at least two co-stimulatory domains. In some embodiments, the at least two co-stimulatory domains are different.

In some embodiments, any of the cells described herein comprise a nucleic acid encoding a CAR or a fourth generation CAR. In some embodiments, the fourth generation CAR comprises one antigen binding domain, one transmembrane domain, and at least two, three, or four signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a co-stimulatory domain. In some embodiments, the costimulatory domain enhances cytokine production, CAR-T cell proliferation, and or CAR-T cell persistence during T cell activation.

In some embodiments, an engineered cell provided herein (e.g., a primary or iPSC-derived T cell or a primary or iPSC-derived NK cell) comprises a polynucleotide encoding a CAR, wherein the polynucleotide is inserted into a genomic locus. In some embodiments, the polynucleotide is inserted into a safe harbor locus (such as, but not limited to, AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD 142), MICA, MICB, LRP1 (also known as CD 91), HMGB1, ABO, RHD, FUT1, or KDM5D locus). In some embodiments, the polynucleotide is inserted into the B2M, CIITA, TRAC, TRB, PD1 or CTLA4 gene. The CAR can be inserted into the genomic locus of the low-immunogenicity cell using any suitable method, including the gene editing methods described herein (e.g., CRISPR/Cas system).

In some embodiments, the first, second, third, or fourth generation CAR further comprises a domain that induces cytokine gene expression upon successful signaling of the CAR. In some embodiments, the cytokine gene is endogenous or exogenous to a target cell comprising a CAR comprising a domain that induces expression of the cytokine gene upon successful signaling of the CAR. In some embodiments, the cytokine gene encodes a proinflammatory cytokine. In some embodiments, the cytokine gene encodes IL-1, IL-2, IL-9, IL-12, IL-18, TNF or IFN-gamma or a functional fragment thereof. In some embodiments, the domain that induces cytokine gene expression upon successful signaling of the CAR is or comprises a transcription factor or a functional domain or fragment thereof. In some embodiments, the domain that induces cytokine gene expression upon successful signaling of the CAR is or comprises a transcription factor or a functional domain or fragment thereof. In some embodiments, the transcription factor or functional domain or fragment thereof is or comprises a Nuclear Factor (NFAT), NF-kB, or functional domain or fragment thereof of an activated T cell. See, e.g., zhang. C. Et al, ENGINEERING CAR-T cells. Biomarker research.5:22 (2017); WO 2016126608; sha, H.et al CHIMAERIC ANTIGEN receiver T-CELL THERAPY for tumour immunotherapy.bioscience Reports 2017, 1 month 27, 37 (1).

The skilled artisan is familiar with the CAR and the different components and configurations of the CAR. Any known CAR may be used in conjunction with the provided embodiments. In addition to the CARs described herein, various CARs and nucleotide sequences encoding the same are known in the art and will be suitable for use in engineered cells as described herein. See, for example, WO2013040557; WO2012079000; WO2016030414; smith T et al, nature nanotechnology.2017.DOI 10.1038/NNANO.2017.57, the disclosure of which is incorporated herein by reference. Exemplary properties and components of CA R are described in the following subsections.

H. antigen binding domains

In some embodiments, the CAR Antigen Binding Domain (ABD) is or comprises an antibody, or antigen binding portion thereof. In some embodiments, the CAR antigen binding domain is or comprises an scFv or Fab.

In some embodiments, the antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, the cell surface antigen is characteristic of (e.g., expressed by) a particular or specific cell type. In some embodiments, the cell surface antigen is characteristic of more than one type of cell.

In some embodiments, the antigen may be an antigen expressed on tumor cells alone or preferentially on tumor cells, or an antigen characteristic of an autoimmune or inflammatory disease. In some embodiments, the Antigen Binding Domain (ABD) targets an antigen characteristic of a tumor cell. For example, the antigen binding domain targets an antigen expressed by a tumor cell or cancer cell. In some embodiments, ABD binds a tumor associated antigen. In some embodiments, the tumor cell-characteristic antigen (e.g., an antigen associated with a tumor cell or cancer cell) or tumor-associated antigen is selected from the group consisting of a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-associated receptor, a receptor-like tyrosine phosphatase, a receptor serine/threonine kinase, a receptor guanylate cyclase, a histidine kinase-associated receptor.

In some embodiments, the target antigen is an antigen including, but not limited to: epidermal Growth Factor Receptor (EGFR) (including ErbB1/EGFR, erbB2/HER2, erbB3/HER3 and ErbB4/HER 4), fibroblast Growth Factor Receptor (FGFR) (including FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF18 and FGF 21), vascular Endothelial Growth Factor Receptor (VEGFR) (including VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF), vascular endothelial growth factor receptor (FGF), RET receptors and Eph receptor families (including EphA1, ephA2, ephA3, ephA4, ephA5, ephA6, ephA7, ephA8, ephA9, ephA10, ephB1, ephB2, ephB3, ephB4 and EphB6)、CXCR1、CXCR2、CXCR3、CXCR4、CXCR6、CCR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR8、CFTR、CIC-1、CIC-2、CIC-4、CIC-5、CIC-7、CIC-Ka、CIC-Kb、 macular proteins (Bestrophins), TMEM16A, GABA receptor, glycine receptor, ABC transporter, NAV1.1, NAV 1.2), NAV1.3, NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9, sphingosine-1-phosphate receptor (S1P 1R), NMDA channel, transmembrane protein, multiple transmembrane protein, T cell receptor motif; T cell alpha chain; t cell beta chain; t cell gamma chain; T cell delta chain 、CCR7、CD3、CD4、CD5、CD7、CD8、CD11b、CD11c、CD16、CD19、CD20、CD21、CD22、CD25、CD28、CD34、CD35、CD40、CD45RA、CD45RO、CD52、CD56、CD62L、CD68、CD80、CD95、CD117、CD127、CD133、CD137(4-1BB)、CD163、F4/80、IL-4Ra、Sca-1、CTLA-4、GITR、GARP、LAP、 granzyme B, LFA-1, transferrin receptor, NKp46, perforin, CD4+, th1, th2, th17, th40, th22, th9, tfh, canonical Treg, foxP3+, tr1, th3, treg17, T _RE G, CDCP, NT5E, epCAM, CEA, gpA33, mucin, TAG-72, carbonic anhydrase IX, PSMA, folate binding proteins, gangliosides (e.g., ,CD2、CD3、GM2)、Lewis-γ²、VEGF、VEGFR 1/2/3、αVβ3、α5β1、ErbB1/EGFR、ErbB1/HER2、ErB3、c-MET、IGF1R、EphA3、TRAIL-R1、TRAIL-R2、RANKL、FAP、 tenascin 、PDL-1、BAFF、HDAC、ABL、FLT3、KIT、MET、RET、IL-1β、ALK、RANKL、mTOR、CTLA-4、IL-6、IL-6R、JAK3、BRAF、PTCH、Smoothened、PIGF、ANPEP、TIMP1、PLAUR、PTPRJ、LTBR or ANTXR1, folate receptor alpha (FRa), ERBB2 (Her 2/neu), ephA2, IL-13Ra2, epidermal Growth Factor Receptor (EGFR), gangliosides, Mesothelin 、TSHR、CD19、CD123、CD22、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、MUC16(CA125)、L1CAM、LeY、MSLN、IL13Rα1、L1-CAM、Tn Ag、 Prostate Specific Membrane Antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B H3, KIT, interleukin-11 receptor a (IL-11 Ra), PSCA, PRSS21, VEGFR2, lewis Y, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, MUC1, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-1 receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLACl, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, asparaginase (legumain), HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, major histocompatibility complex class I related gene protein (MR 1), urokinase-type plasminogen activator receptor (uPAR), fos-related antigen 1, p53 mutant, Prostate specific protein (prostein), survivin, telomerase, PCTA-1/galectin 8, melanA/MART1, ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, rhoC, TRP-2, CYPIB I, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, enterocarboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST, EMR2, LY75, GPC3, FCRL5, IGLL1, neoantigen 、CD133、CD15、CD184、CD24、CD56、CD26、CD29、CD44、HLa-a、HLA-B、HLA-C、(HLa-a,B,C)CD49f、CD151、CD340、CD200、tkrA、trkB or trkC or antigenic fragments or antigenic portions thereof.

In some embodiments, exemplary target antigens include, but are not limited to, CDS, CD19, CD20, CD22, CD23, CD30, CD70, κ, λ, and B Cell Maturation Agent (BCMA) (associated with leukemia); CS1/SLAMF7, CD38, CD138, GPRC5D, TACI, and BCMA (associated with myeloma); GD2, HER2, EGFR, EGFRvlll, B H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, fra, IL-13 ra, mesothelin, MUC1, MUC16, and ROR1 (associated with solid tumors).

In some embodiments, the CAR is a CD19 CAR. In some embodiments, the extracellular binding domain of a CD19 CAR comprises an antibody that specifically binds CD19 (e.g., human CD 19). In some embodiments, the extracellular binding domain of the CD19 CAR comprises a scFv antibody fragment derived from a FMC63 monoclonal antibody (FMC 63) comprising a heavy chain variable region (VH) and a light chain variable region (VL) of FMC63 linked by a linker peptide. In some embodiments, the linker peptide is a "Whitlow" linker peptide. FMC63 and derived scFv have been described in Nicholson et al, mal.lmmun.34 (16-17): 1157-1165 (1997) and PCT application publication No. WO2018/213337A 1, the entire contents of each of which are incorporated herein by reference.

In some embodiments, the extracellular binding domain of CD19 CAR comprises an antibody derived from one of the CD 19-specific antibodies, including, for example, SJ25C1 (Bejcek et al, cancer Res.55:2346-2351 (1995)), HD37 (Pezutto et al, J.lmmunol.138 (9): 2793-2799 (1987)), 4G7 (Meeker et al, hybrid 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al, 70:418-427 (1987)), B4 HB12B (Kansas and Tedder, J.lmmunol.147:4094-4102 (1991)), yazawa et al, proc.Natl. Acad.Sci.USA 102:15178-15183 (2005)), herbst.exp.335:213-222 (2010), B4:418-427 (1987), B4:9 (1987), B4 HB12B (Kansas and 37-37.147.147), and CD.381 (1999-299).

In some embodiments, the CAR is a CD22 CAR. CD22 is a transmembrane protein that is found predominantly on the surface of mature B cells and acts as an inhibitory receptor for B Cell Receptor (BCR) signaling. CD22 is expressed in 60-70% of B cell lymphomas and leukemias (e.g., chronic B cell leukemia, hairy cell leukemia, acute Lymphoblastic Leukemia (ALL) and Burkitt's lymphoma) and is absent on the cell surface or stem cells at the early stages of B cell development. In some embodiments, the CD22 CAR comprises an extracellular binding domain, a transmembrane domain, an intracellular signaling domain, and/or an intracellular co-stimulatory domain that specifically binds CD 22. In some embodiments, the extracellular binding domain of the CD22 CAR comprises a scFv antibody fragment derived from an m971 monoclonal antibody (m 971) comprising a heavy chain variable region (VH) and a light chain variable region (VL) of m971 linked by a linker. In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv antibody fragment derived from m971-L7, which is an affinity matured variant of m971 with significantly increased CD22 binding affinity (from about 2nM to less than 50 pM) compared to the parent antibody m 971. In some embodiments, the scFv antibody fragment derived from m971-L7 comprises a VH and a VL of m971-L7 linked by a 3xG4S linker. In some embodiments, the extracellular binding domain of the CD22 CAR comprises immunotoxin HA22 or BL22. Immunotoxins BL22 and HA22 are therapeutic agents comprising scFv specific for CD22 fused to a bacterial toxin, and thus can bind to the surface of and kill cancer cells expressing CD 22. BL22 comprises dsFv, RFB4 of an anti-CD 22 antibody fused to a 38-kDa truncated form of Pseudomonas exotoxin A (Bang et al Clin. Cancer Res.,11:1545-50 (2005)). HA22 (CAT 8015, mositumomab (moxetumomab pasudotox)) is a mutated, higher affinity form of BL22 (Ho et al, j. Biol. Chem.,280 (1): 607-17 (2005)). Suitable sequences of the antigen binding domains of HA22 and BL22 specific for CD22 are disclosed, for example, in U.S. Pat. nos. 7,541,034;7,355,012; and 7,982,011, which are hereby incorporated by reference in their entirety.

In some embodiments, the CAR is BCMACAR. BCMA is a member of the Tumor Necrosis Family Receptor (TNFR) expressed on cells of the B cell lineage, with highest expression on terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. Recently, BCMA expression has been found to be associated with a variety of cancers such as multiple myeloma, hodgkin and non-hodgkin lymphomas, various leukemias and glioblastomas. In some embodiments BCMACAR comprises an extracellular binding domain, a transmembrane domain, an intracellular signaling domain, and/or an intracellular co-stimulatory domain that specifically binds BCMA. In some embodiments, the extracellular binding domain of BCMACAR comprises an antibody that specifically binds BCMA (e.g., human BCMA). CARs for BCMA have been described in PCT application publication nos. WO2016/014789, WO2016/014565, WO2013/154760 and WO 2015/128653. BCMA binding antibodies are also disclosed in PCT application publication nos. WO2015/166073 and WO 2014/068079. In some embodiments, the extracellular binding domain of BCMACAR comprises an scFv antibody fragment derived from a murine monoclonal antibody, as described in Carpenter et al, clin.cancer Res.19 (8): 2048-2060 (2013). In some embodiments, the scFv antibody fragment is a humanized form of a murine monoclonal antibody (Sommermeyer et al, leukemia31:2191-2199 (2017)). In some embodiments, the extracellular binding domain of BCMACAR comprises a single variable fragment of two heavy chains (VHH) that can bind to two epitopes of BCMA as described in Zhao et al, j.Hematol. Oneal.11 (1): 141 (2018). In some embodiments, the extracellular binding domain of BCMACAR comprises a fully human heavy chain variable domain (FHVH), as described in Lam et al, nat.Commun.11 (1): 283 (2020).

In some embodiments, the antigen binding domain targets an antigen characteristic of an autoimmune or inflammatory disorder. In some embodiments, ABD binds an antigen associated with an autoimmune or inflammatory disorder. In some cases, the antigen is expressed by a cell associated with an autoimmune or inflammatory disorder. In some embodiments, the autoimmune or inflammatory disorder is selected from chronic Graft Versus Host Disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, godpasture's disease (goodpasture), uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes mellitus, multiple sclerosis, collectinopathies, pemphigus vulgaris, graves' disease, autoimmune hemolytic anemia, hemophilia A, primary sjogren's syndrome, thrombotic thrombocytopenic purpura, neuromyelitis optica, eventuri's syndrome, igM-mediated neuropathy, cryoglobulinemia, dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired angioedema, chronic urticaria, anti-phospholipid demyelinating polyneuropathy and autoimmune thrombocytopenia or neutropenia or pure erythrocyte regeneration disorders, although illustrative non-limiting examples of alloimmune disorders include allogeneic allergic diseases (see e.g., blazar, 2015. 5. Can be treated with an allogeneic or alternative haemolytic therapy, such as that of human, 5-amp, adult human, or neonatal blood transfusion, hematopoietic tumor, or other conditions, hematopoietic disorders, and hematopoietic disorders, such as those obtained from a replacement therapy, or a genetic therapy of a neonatal or a J-type of a disease. In some cases, allosensitization refers to the occurrence of an immune response (such as a circulating antibody) against an MHC molecule (e.g., a human leukocyte antigen) that is considered by the immune system of the recipient subject or pregnant subject to be a non-self antigen. In some embodiments, the antigen characteristic of an autoimmune or inflammatory disorder is selected from the group consisting of a cell surface receptor, an ion channel linked receptor, an enzyme linked receptor, a G protein coupled receptor, a receptor tyrosine kinase, a tyrosine kinase-related receptor, a receptor-like tyrosine phosphatase, a receptor serine/threonine kinase, a receptor guanylate cyclase, or a histidine kinase-related receptor.

In some embodiments, the antigen binding domain of the CAR binds to a ligand expressed on B cells, plasma cells, or plasmablasts. In some embodiments, the antigen binding domain of the CAR binds to CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD, CD319, BCMA, CD28, TNF, interferon receptor, GM-CSF, ZAP-70, LFA-1, CD3 γ, CD5, or CD2. See US 2003/0077149; WO 2017/058753; WO 2017/058850, the contents of which are incorporated herein by reference. In some embodiments, the CAR is an anti-CD 19 CAR. In some embodiments, the CAR is anti-BCMACAR.

In some embodiments, the antigen binding domain targets an antigen characteristic of senescent cells, such as urokinase type plasminogen activator receptor (uPAR). In some embodiments, the ABD binds to an antigen associated with a senescent cell. In some cases, the antigen is expressed by senescent cells. In some embodiments, the CAR can be used to treat or prevent a disorder characterized by abnormal accumulation of senescent cells, such as liver and lung fibrosis, atherosclerosis, diabetes, and osteoarthritis.

In some embodiments, the antigen binding domain targets an antigen characteristic of an infectious disease. In some embodiments, ABD binds an antigen associated with an infectious disease. In some cases, the antigen is expressed by cells of the infectious disease. In some embodiments, wherein the infectious disease is selected from the group consisting of HIV, hepatitis B virus, hepatitis C virus, human herpesvirus type 8 (HHV-8, kaposi's sarcoma-associated herpesvirus (KSHV)), human T-lymphocyte virus-1 (HTLV-1), merck cell polyoma virus (MCV), simian Virus 40 (SV 40), epstein-Barr virus, CMV, human papilloma virus. In some embodiments, the infectious disease signature antigen is selected from the group consisting of a cell surface receptor, an ion channel linked receptor, an enzyme linked receptor, a G protein coupled receptor, a receptor tyrosine kinase, a tyrosine kinase related receptor, a receptor-like tyrosine phosphatase, a receptor serine/threonine kinase, a receptor guanylate cyclase, a histidine kinase related receptor, an HIV Env, gpl20, or a CD4 induced epitope on HIV-1 Env.

In any of these embodiments, the extracellular binding domain of the CAR can be codon optimized for expression in a host cell, or have a variant sequence to increase the function of the extracellular binding domain.

In some embodiments, the CAR has dual specificity for both target antigens. In some embodiments, the target antigen is a different target antigen. In some of any such embodiments, the two different target antigens are any two different antigens described above. In some embodiments, the extracellular binding domains are different and bind to two different antigens from: (i) CD19 and CD20, (ii) CD20 and L1-CAM, (iii) L1-CAM and GD2, (iv) EGFR and L1-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and ROR1. In some embodiments, each of the two different antigen binding domains is an scFv. In some embodiments, the C-terminus of one variable domain (VH or VL) of a first scFv is linked to the N-terminus of a second scFv (VL or VH, respectively) via a polypeptide linker. In some embodiments, the linker connects the N-terminus of VH to the C-terminus of VL or connects the C-terminus of VH to the N-terminus of VL. These scFv lack constant regions (Fc) present in the heavy and light chains of natural antibodies. scFv specific for at least two different antigens are arranged in tandem and linked to the costimulatory domain and the intracellular signaling domain via a transmembrane domain. In one embodiment, the extracellular spacer domain may be connected between the antigen specific binding region and the transmembrane domain.

In a further embodiment, each antigen-specific targeting region of the CAR comprises a bivalent (divalent or bivalent) single-chain variable fragment (di-scFv, bi-scFv). In a CAR comprising a di-scFV, two scFV specific for each antigen are linked together by producing a single peptide chain having two VH and two VL regions, thereby producing a CAR comprising at least two antigen-specific targeting regions in tandem scFv.(Xiong,Cheng-Yi;Natarajan,A;Shi,X B;Denardo,G L;Denardo,S J(2006)."Development of tumor targeting anti-MUC-1multimer:effects of di-scFv unpaired cysteine location on PEGylation and tumor binding".Protein Engineering Design and Selection 19(8):359-367;Kufer,Peter;Lutterbüse,Ralf;Baeuerle,Patrick A.(2004)."A revival of bispecific antibodies".Trends in Biotechnology 22(5):238-244). that will express two scFV specific for each of the two antigens. The resulting antigen-specific targeting region specific for at least two different antigens is linked via a transmembrane domain and a co-stimulatory domain and an intracellular signaling domain. In one embodiment, the extracellular spacer domain may be connected between the antigen specific binding domain and the transmembrane domain.

In another embodiment, each antigen-specific targeting region of the CAR comprises a diabody. In diabodies, scfvs are produced using a linker peptide that is too short to allow the two variable regions to fold together, driving scFv dimerization. Shorter linkers (one or two amino acids) lead to the formation of trimers, so-called triplex antibodies (triabodies) or trisomes (tribody). Four-chain antibodies (Tetrabody) may also be used.

In some embodiments, the cell is engineered to express more than one CAR, such as two different CARs, wherein each CAR has an antigen binding domain to a different target antigen. In some of any such embodiments, the two different target antigens are any two different antigens described above. In some embodiments, the extracellular binding domains are different and bind to two different antigens from: (i) CD19 and CD20, (ii) CD20 and L1-CAM, (iii) L1-CAM and GD2, (iv) EGFR and L1-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and ROR1.

In some embodiments, two different engineered cells are prepared, which contain the modifications provided, and each cell is engineered with a different CAR. In some embodiments, each of the two different CARs has an antigen binding domain to a different target antigen. In some of any such embodiments, the two different target antigens are any two different antigens described above. In some embodiments, the extracellular binding domains are different and bind to two different antigens from: (i) CD19 and CD20, (ii) CD20 and L1-CAM, (iii) L1-CAM and GD2, (iv) EGFR and L1-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and ROR1. In some embodiments, an engineered population of cells (e.g., low immunogenicity) expressing a first CAR against a first target antigen and an engineered population of cells (e.g., low immunogenicity) expressing a second CAR against a second target antigen are administered to a subject separately. In some embodiments, the first and second cell populations are administered sequentially in any order. For example, the population of cells expressing the second CAR is administered after the population of cells expressing the first CAR.

I. Spacer region

In some embodiments, the CAR further comprises one or more spacers, e.g., wherein the spacer is the first spacer between the antigen binding domain and the transmembrane domain. In some embodiments, the first spacer region comprises at least a portion of an immunoglobulin constant region or variant or modified form thereof. In some embodiments, the spacer is a second spacer between the transmembrane domain and the signaling domain. In some embodiments, the second spacer is an oligopeptide, for example, wherein the oligopeptide comprises glycine and serine residues, such as, but not limited to, glycine-serine duplex. In some embodiments, the CAR comprises two or more spacers, e.g., a spacer between the antigen binding domain and the transmembrane domain and a spacer between the transmembrane domain and the signaling domain.

J. Transmembrane domain

In some embodiments, the CAR transmembrane domain comprises at least the following transmembrane regions: the α, β or ζ chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional variants thereof. In some embodiments, the transmembrane domain comprises at least transmembrane region ：CD8α、CD8β、4-1BB/CD137、CD28、CD34、CD4、FcεRIγ、CD16、OX40/CD134、CD3ζ、CD3ε、CD3γ、CD3δ、TCRα、TCRβ、TCRζ、CD32、CD64、CD64、CD45、CD5、CD9、CD22、CD37、CD80、CD86、CD40、CD40L/CD154、VEGFR2、FAS and FGFR2B or a functional variant thereof.

K. Signaling domains

In some embodiments, a CAR described herein comprises one or at least one signaling domain ：B7-1/CD80;B7-2/CD86;B7-H1/PD-L1;B7-H2;B7-H3;B7-H4;B7-H6;B7-H7;BTLA/CD272;CD28;CTLA-4;Gi24/VISTA/B7-H5;ICOS/CD278;PD-1;PD-L2/B7-DC;PDCD6);4-1BB/TNFSF9/CD137;4-1BB ligand/TNFSF 9 selected from one or more of the following; BAFF/BLyS/TNFSF13B; BAFF R/TNFRSF13C; CD27/TNFRSF7; CD27 ligand/TNFSF 7; CD30/TNFRSF8; CD30 ligand/TNFSF 8; CD40/TNFRSF5; CD40/TNFSF5; CD40 ligand/TNFSF 5; DR3/TNFRSF25; GITR/TNFRSF18; GITR ligand/TNFSF 18; HVEM/TNFRSF14; LIGHT/TNFSF14; lymphotoxin-alpha/TNF-beta; OX40/TNFRSF4; OX40 ligands /TNFSF 4;RELT/TNFRSF19L;TACI/TNFRSF13B;TL1A/TNFSF15;TNF-α;TNF RII/TNFRSF1B);2B4/CD244/SLAMF4;BLAME/SLAMF8;CD2;CD2F-10/SLAMF9;CD48/SLAMF2;CD58/LFA-3;CD84/SL AMF5;CD229/SLAMF3;CRACC/SLAMF7;NTB-A/SLAMF6;SL AM/CD150);CD2;CD7;CD53;CD82/Kai-1;CD90/Thy1;CD96;CD160;CD200;CD300a/LMIR1;HLA I class; HLA-DR; ikaros; integrin alpha 4/CD49d; integrin alpha 4 beta 1; integrin α4β7/LPAM-1;LAG-3;TCL1A;TCL1B;CRTAM;DAP12;Dectin-1/CLEC7A;DPPI V/CD26;EphB6;TIM-1/KIM-1/HAVCR;TIM-4;TSLP;TSLP R; lymphocyte function-associated antigen 1 (LFA-1); NKG2C, CD zeta domain, immune receptor tyrosine based activation motif (ITAM), CD27, CD28, 4-1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, lymphocyte function associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, or a functional fragment thereof.

In some embodiments, at least one signaling domain comprises a cd3ζ domain or an immunoreceptor tyrosine based activation motif (ITAM) or functional variant thereof.

In some embodiments, the CAR comprises a signaling domain that is a co-stimulatory domain. In some embodiments, the CAR comprises a second co-stimulatory domain. In some embodiments, the CAR comprises at least two co-stimulatory domains. In some embodiments, the CAR comprises at least three co-stimulatory domains. In some embodiments, the CAR comprises a co-stimulatory domain selected from one or more of CD27, CD28, 4-1BB, CD134/OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B-H3, a ligand that specifically binds to CD 83. In some embodiments, if the CAR comprises two or more co-stimulatory domains, the two co-stimulatory domains are different. In some embodiments, if the CAR comprises two or more co-stimulatory domains, the two co-stimulatory domains are identical.

In other embodiments, at least one signaling domain comprises (i) a cd3ζ domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; and (ii) a CD28 domain, or a 4-1BB domain, or a functional variant thereof. In other embodiments, at least one signaling domain comprises (i) a cd3ζ domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof. In some embodiments, at least one signaling domain comprises (i) a cd3ζ domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain, or a CD134 domain, or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes.

In some embodiments, at least two signaling domains comprise a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or functional variant thereof. In other embodiments, at least two signaling domains comprise (i) a cd3ζ domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; and (ii) a CD28 domain, or a 4-1BB domain, or a functional variant thereof. In other embodiments, at least one signaling domain comprises (i) a cd3ζ domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof. In some embodiments, at least two signaling domains comprise (i) a cd3ζ domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain, or a CD134 domain, or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes.

In some embodiments, the at least three signaling domains comprise a cd3ζ domain or an immunoreceptor tyrosine based activation motif (ITAM) or functional variant thereof. In other embodiments, at least three signaling domains comprise (i) a cd3ζ domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; and (ii) a CD28 domain, or a 4-1BB domain, or a functional variant thereof. In other embodiments, at least three signaling domains comprise (i) a cd3ζ domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof. In some embodiments, the at least three signaling domains comprise (i) a cd3ζ domain, or an immunoreceptor tyrosine based activation motif (ITAM), or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain, or a CD134 domain, or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes.

In some embodiments, the CAR comprises a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain or a functional variant thereof.

In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof.

In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) A CD28 domain or a 4-1BB domain or a functional variant thereof, and/or (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof.

In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes.

L. exemplary CAR

In some embodiments, the CAR comprises an extracellular antigen binding domain (e.g., an antibody or antibody fragment, such as scFv) that binds an antigen (e.g., a tumor antigen), a spacer (e.g., comprising a hinge domain, such as any of the herein described), a transmembrane domain (e.g., any of the herein described), and an intracellular signaling domain (e.g., any intracellular signaling domain, such as a primary signaling domain or a co-stimulatory signaling domain described herein). In some embodiments, the intracellular signaling domain is or includes a primary cytoplasmic signaling domain. In some embodiments, the intracellular signaling domain additionally comprises an intracellular signaling domain of a costimulatory molecule (e.g., a costimulatory domain). Any such component may be any of the components described above.

Examples of exemplary components of the CAR are described in table 3. In aspects provided, the sequence of each component in the CAR can include any combination listed in table 3.

4. Methods for increasing expression (e.g., overexpression) of polynucleotides

In some embodiments, increased expression of the polynucleotide may be performed by any of a variety of techniques. For example, methods for regulating gene and factor (protein) expression include genome editing techniques and RNA or protein expression techniques, and the like. For all of these techniques, well-known recombinant techniques are used to generate recombinant nucleic acids as outlined herein. In some embodiments, the cell engineered with one or more modifications to overexpress or increase expression of a polynucleotide is any source cell as described herein. In some embodiments, the source cell is any of the cells described in section ii.c.

In some embodiments, gene expression is increased by increasing endogenous gene activity (e.g., increasing transcription of a foreign gene). In some cases, endogenous gene activity is increased by increasing activity of a promoter or enhancer operably linked to the endogenous gene. In some embodiments, increasing the activity of the promoter or enhancer comprises one or more modifications to the endogenous promoter or enhancer that increase the activity of the endogenous promoter or enhancer. In some cases, increasing the gene activity of an endogenous gene comprises modifying an endogenous promoter of the gene. In some embodiments, increasing the gene activity of the endogenous gene comprises introducing a heterologous promoter. In some embodiments, the heterologous promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter for HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter for HIV, moloney virus promoter, epstein Barr Virus (EBV) promoter, rous Sarcoma Virus (RSV) promoter, and UBC promoter.

M.DNA binding fusion proteins

In some embodiments, expression of a target gene (e.g., CD47 or another tolerogenic factor) is increased by expressing a fusion protein or protein complex comprising: (1) A site-specific binding domain specific for endogenous CD47 or other genes and (2) a transcriptional activator.

In some embodiments, the regulatory factor consists of a site-specific DNA binding nucleic acid molecule, such as a guide RNA (gRNA). In some embodiments, the methods are accomplished by site-specific DNA binding to a target protein, such as by a Zinc Finger Protein (ZFP) or a ZFP-containing fusion protein, also known as a Zinc Finger Nuclease (ZFN).

In some embodiments, the regulatory factor comprises a site-specific binding domain, such as using a DNA binding protein or DNA binding nucleic acid, that specifically binds or hybridizes to a gene of the targeted region. In some embodiments, the provided polynucleotides or polypeptides are coupled or complexed with a site-specific nuclease (such as a modified nuclease). For example, in some embodiments, administration is achieved using a fusion of a DNA targeting protein comprising a modified nuclease, such as using a meganuclease or RNA-guided nuclease, such as a clustered regularly interspaced short palindromic nucleic acid (CRISPR) -Cas system, such as a CRISPR-Cas9 system. In some embodiments, the nuclease is modified to lack nuclease activity. In some embodiments, the modified nuclease is dCAS9 that catalyzes death.

In some embodiments, the site-specific binding domain may be derived from a nuclease. For example, recognition sequences for homing endonucleases and meganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. See also U.S. patent No. 5,420,032; U.S. patent No. 6,833,252; belfort et al, (1997) Nucleic Acids Res.25:3379-3388; dujon et al, (1989) Gene 82:115-118; perler et al, (1994) Nucleic Acids Res.22,1125-1127; jasin (1996) Trends Genet.12:224-228; gimble et al, (1996) J.mol.biol.263:163-180; argast et al, (1998) J.mol. Biol.280:345-353 and NEW ENGLAND Biolabs catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind non-native target sites. See, e.g., chevalier et al, (2002) molecular cell 10:895-905; epinat et al, (2003) Nucleic Acids Res.31:2952-2962; ashworth et al, (2006) Nature 441:656-659; paques et al, (2007) Current GENE THERAPY 7:49-66; U.S. patent publication No. 2007/017128.

The zinc finger, TALE and CRISPR system binding domains can be "engineered" to bind to a predetermined nucleotide sequence, for example via engineering (changing one or more amino acids) of a recognition helix region of a naturally occurring zinc finger or TALE protein. The engineered DNA binding protein (zinc finger or TALE) is a non-naturally occurring protein. Reasonable design criteria include the application of substitution rules and computerized algorithms to process information in a database storing information of existing ZFP and/or TALE designs and binding data. See, for example, U.S. patent No. 6,140,081;6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO02/016536 and WO 03/016496 and U.S. publication No. 20110301073.

In some embodiments, the site-specific binding domain comprises one or more Zinc Finger Proteins (ZFPs) or domains thereof that bind DNA in a sequence-specific manner. ZFP or a domain thereof is a protein or domain within a larger protein that binds DNA in a sequence-specific manner by one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized by zinc ion coordination.

In ZFP, there is an artificial ZFP domain that targets a specific DNA sequence, typically 9-18 nucleotides in length, created by individual finger assembly. ZFPs include ZFPs in which the single finger domain is about 30 amino acids in length and contains an alpha helix containing two unchanged histidine residues coordinated to two cysteines of a single beta turn by zinc, and has two, three, four, five or six fingers. In general, the sequence specificity of ZFP can be altered by making amino acid substitutions at the four helix positions (-1, 2,3, and 6) on the zinc finger recognition helix. Thus, in some embodiments, ZFP or ZFP-containing molecules are non-naturally occurring, e.g., engineered to bind to a selected target site. See, for example, beerli et al (2002) Nature Biotechnol.20:135-141; pabo et al (2001) Ann.Rev.biochem.70:313-340; isalan et al (2001) Nature Biotechnol.19:656-660; segal et al (2001) curr.Opin.Biotechnol.12:632-637; choo et al (2000) curr.Opin. Structure. Biol.10:411-416; U.S. publication No. 6,453,242;6,534,261;6,599,692;6,503,717;6,689,558;7,030,215;6,794,136;7,067,317;7,262,054;7,070,934;7,361,635;7,253,273; and U.S. patent publication No. 2005/0064474;2007/0218528;2005/0267061, which is incorporated herein by reference in its entirety.

Many genetically engineered zinc fingers are commercially available. For example, sangamo Biosciences (Richmond, CA, USA) in concert with Sigma-Aldrich (St.Louis, MO, USA) developed a platform (CompoZr) for zinc finger construction that allowed researchers to bypass zinc finger construction and verification and provide specific targeted zinc fingers for thousands of proteins (Gaj et al Trends in Biotechnology,2013,31 (7), 397-405). In some embodiments, commercially available zinc fingers are used or custom designed.

In some embodiments, the site-specific binding domain comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as the domain in a transcription activator-like protein effector (TALE) protein, see, e.g., U.S. patent publication No. 20110301073, which is incorporated herein by reference in its entirety.

In some embodiments, the site-specific binding domain is derived from a CRISPR/Cas system. Generally, "CRISPR system" refers to transcripts and other elements involved in expressing or directing the activity of a CRISPR-associated ("Cas") gene, including sequences encoding Cas genes, tracr (trans-activated CRISPR) sequences (e.g., tracrRNA or active moiety tracrRNA), tracr mate sequences (covering "direct repeats" and partially direct repeats of tracrRNA processing in the context of an endogenous CRISPR system), guide sequences (also referred to as "spacers" or "targeting sequences" in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.

Generally, the guide sequence comprises a targeting domain comprising a polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and guide sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. In some examples, the targeting domain (e.g., targeting sequence) of the gRNA is complementary, e.g., at least 80%, 85%, 90%, 95%, 98%, or 99% complementary, e.g., fully complementary, to a target sequence on a target nucleic acid.

In some embodiments, the gRNA can be any gRNA as described herein. In certain embodiments, the gRNA has a targeting sequence complementary to a target site of: CD47, such as any one of SEQ ID NOs 200784-231885 (Table 29 of WO2016183041, appendix 22); HLA-E, such as any one of SEQ ID NOs 189859-193183 (Table 19 of WO2016183041, appendix 12); HLA-F, such as any one of SEQ ID NOs 688808-699754 (Table 45 of WO2016183041, appendix 38); HLA-G such as any one of SEQ ID NOs 188372-189858 (Table 18 of WO2016183041, appendix 11); or PD-L1 such as any of SEQ ID NO:193184-200783 (Table 21 of WO2016183041, appendix 14).

In some embodiments, the target site is upstream of the transcription initiation site of the target gene. In some embodiments, the target site is adjacent to the transcription initiation site of the gene. In some embodiments, the target site is adjacent to an RNA polymerase pause site downstream of the gene transcription initiation site.

In some embodiments, the targeting domain is configured to target a promoter region of a target gene to facilitate transcription initiation, binding of one or more transcription enhancers or activators, and/or RNA polymerase. One or more grnas may be used to target the promoter region of a gene. In some embodiments, one or more regions of a gene may be targeted. In certain aspects, the target site is located within 600 base pairs on either side of the Transcription Start Site (TSS) of the gene.

It is within the level of the skilled artisan to design or identify a gRNA sequence (i.e., a gRNA targeting sequence) that is or comprises the sequence of the targeted gene, including the sequence of the exonic sequences and regulatory regions, including promoters and activators. A whole genome gRNA database for CRISPR genome editing is publicly available that contains an exemplary single guide RNA (sgRNA) target sequence in a constitutive exon of a gene in the human genome or mouse genome (see, e.g., geneescript.com/gRNA-database.html; see also Sanjana et al (2014) Nat. Methods,11:783-4; www.e-crisp.org/E-CRISP/; crispr.mit.edu /). In some embodiments, the gRNA sequence is or comprises a targeting sequence that has minimal off-target binding to a non-target gene.

In some embodiments, the regulatory factor further comprises a functional domain, such as a transcriptional activator.

In some embodiments, the transcriptional activator is or contains one or more regulatory elements, such as one or more transcriptional control elements of a target gene, thereby recognizing the site-specific domain as provided above to drive expression of such gene. In some embodiments, the transcriptional activator drives expression of a target gene. In some cases, the transcriptional activator may be or contain all or a portion of a heterologous transactivation domain. For example, in some embodiments, the transcriptional activator is selected from the group consisting of a herpes simplex-derived transactivation domain, a Dnmt3a methyltransferase domain, p65, VP16, and VP64.

In some embodiments, the regulatory factor is a zinc finger transcription factor (ZF-TF). In some embodiments, the regulatory factor is VP64-p65-Rta (VPR).

In certain embodiments, the regulatory factor further comprises a transcriptional regulatory domain. Common domains include, for example, transcription factor domains (activators, inhibitors, co-activators, co-inhibitors), silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members, etc.); DNA repair enzyme and related factors and modifying factors thereof; DNA rearranging enzyme and related factors and modifying factors thereof; chromatin-related proteins and their modifiers (e.g., kinases, acetylases, and deacetylases); and DNA modifying enzymes (e.g., methyltransferases such as DNMT family members (e.g., DNMT1, DNMT3A, DNMT3B, DNMT L, etc., topoisomerase, helicase, ligase, kinase, phosphatase, polymerase, endonuclease) and related factors and modifying factors see, e.g., U.S. publication No. 2013/0253040, which is incorporated herein by reference in its entirety.

Suitable domains for achieving activation include the HSV VP 16 activation domain (see, e.g., hagmann et al, J.Virol.71,5952-5962 (1 97)) nuclear hormone receptor (see, e.g., torchia et al, curr.Opin.cell.biol.10:373-383 (1998)); the p65 subunit of nuclear factor κB (Bitko and Bank, J.Virol.72:5610-5618 (1998) and Doyle and Hunt, neuroreport 8:2937-2942 (1997)); Liu et al CANCER GENE Ther.5:3-28 (1998)) or artificial chimeric functional domains such as VP64 (Beerli et al, (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degradation determinants (Molinari et al, (1999) EMBO J.18, 6439-6447). Additional exemplary activation domains include Oct 1, oct-2A, spl, AP-2 and CTF1 (Seipel et al, EMBOJ.11,4961-4968 (1992) and p300, CBP, PCAF, SRC1 PvALF, atHD2A and ERF-2. See, for example, robyr et al, (2000) mol. Endocrinol.14:329-347; collingwood et al, (1999) J.mol.Endocrinol 23:255-275; leo et al, (2000) Gene245:1-11; manteuffel-Cymborowska (1999) Acta biochem. Pol.46:77-89; mcKenna et al, (1999) J.Steroid biochem. Mol. Biol.69:3-12; malik et al, (2000) Trends biochem. Sci.25:277-283; and Lemon et al, (1999) Curr.Opin.Genet.Dev.9:499-504. Additional exemplary activation domains include, but are not limited to, osGAI, HALF-1, cl, AP1, ARF-5, -6, -1 and-8, CPRF1, CPRF4, MYC-RP/GP and TRAB1, see, e.g., ogawa et al, (2000) Gene 245:21-29; Okanami et al, (1996) GENES CELLS 1:87-99; goff et al, (1991) Genes Dev.5:298-309; cho et al, (1999) Plant Mol Biol 40:419-429; ulmason et al, (1999) Proc.Natl.Acad.Sci.USA 96:5844-5849; sprenger-Haussels et al, (2000) Plant J.22:1-8; gong et al, (1999) Plant mol. Biol.41:33-44; And Hobo et al, (1999) Proc.Natl.Acad.Sci.USA 96:15,348-15,353.

Exemplary repressor domains that can be used to make genetic repressors include, but are not limited to, KRAB A/B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, DNMT family members (e.g., DNMT1, DNMT3A, DNMT3B, DNMT L, etc.), rb, and MeCP2. See, e.g., bird et al, (1999) Cell99:451-454; tyler et al, (1999) Cell 99:443-446; knoepfler et al, (1999) Cell 99:447-450; and Robertson et al, (2000) Nature Genet.25:338-342. Additional exemplary inhibitory domains include, but are not limited to, ROM2 and AtHD2A. See, e.g., chem et al, (1996) PLANT CELL 8:305-321; and Wu et al, (2000) Plant J.22:19-27.

In some cases, the domain is involved in epigenetic regulation of the chromosome. In some embodiments, the domain is a Histone Acetyltransferase (HAT), e.g., type a, nuclear localization, such as MYST family members MOZ, ybf 2/sam 3, MOF and Tip60, GNAT family members Gcn5 or pCAF, p300 family members CBP, p300 or Rttl (Bemdsen and Denu (2008) Curr Opin Struct Biol (6): 682-689). In other cases, the domain is a histone deacetylase (HD AC), such as class I (HDAC-l, 2, 3, and 8), class II (HDAC IIA (HDAC-4, 5, 7, and 9), HD AC IIB (HDAC 6 and 10)), class IV (HDAC-l 1), class III (also known as Sirtuin (SIRT); SIRT 1-7) (see Mottamal et al, (2015) Molecules (3): 3898-3941). Another domain used in some embodiments is a histone phosphorylase or kinase, examples of which include MSK1, MSK2, ATR, ATM, DNA-PK, bubl, vprBP, IKK-a, PKCpi, dik/Zip, JAK2, PKC5, WSTF, and CK2. In some embodiments, a methylation domain is used and may be selected from the group such as Ezh2、PRMT1/6、PRMT5/7、PRMT2/6、CARM1、set7/9、MLL、ALL-1、Suv 39h、G9a、SETDB1、Ezh2、Set2、Dotl、PRMT 1/6、PRMT 5/7、PR-Set7 and Suv4-20 h. Domains involved in hematoxylin and biotinylation (Lys 9, 13, 4, 18 and 12) may also be used in some embodiments (for reviews see Kousarides (2007) Cell 128:693-705).

Fusion molecules are constructed by cloning and biochemical conjugation methods well known to those skilled in the art. The fusion molecule comprises a DNA binding domain and a functional domain (e.g., a transcriptional activation or inhibition domain). The fusion molecule also optionally comprises a nuclear localization signal (e.g., a signal from the SV40 medium T antigen) and an epitope tag (such as, e.g., FLAG and hemagglutinin). The fusion proteins (and the nucleic acids encoding them) are designed such that the translational reading frame remains in the fusion component.

Fusions between the polypeptide component of a functional domain (or functional fragment thereof) on the one hand and a non-protein DNA binding domain (e.g., antibiotic, intercalator, minor groove binder, nucleic acid) on the other hand are constructed by biochemical conjugation methods known to those skilled in the art. See, e.g., PIERCE CHEMICAL Company (Rockford, ill.). Methods and compositions for performing fusion between minor groove binders and polypeptides have been described. Mapp et al, (2000) Proc.Natl.Acad.Sci.USA 97:3930-3935. Likewise, CRISPR/Cas TF and nucleases comprising an sgRNA nucleic acid component associated with a functional domain of a polypeptide component are also known to those of skill in the art and are described in detail herein.

N. exogenous polypeptides

In some embodiments, increased expression (i.e., overexpression) of a polynucleotide is mediated by introducing into a cell an exogenous polynucleotide encoding the polynucleotide to be overexpressed. In some embodiments, the exogenous polynucleotide is a recombinant nucleic acid. Well known recombinant techniques can be used to generate recombinant nucleic acids as outlined herein. In some embodiments, the exogenous polynucleotide encoding an exogenous polypeptide herein comprises a codon-optimized nucleic acid sequence.

In certain embodiments, a recombinant nucleic acid encoding an exogenous polypeptide (such as a tolerogenic factor or chimeric antigen receptor) may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences are generally suitable for the host cell and recipient subject to be treated. For a variety of host cells, a variety of types of suitable expression vectors and suitable regulatory sequences are known in the art. In general, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are also contemplated. The promoter may be a naturally occurring promoter or a hybrid promoter combining elements of more than one promoter. The expression construct may be present in the cell on an episome (such as a plasmid), or the expression construct may be inserted into a chromosome. In a specific embodiment, the expression vector comprises a selectable marker gene to allow selection of transformed host cells. Certain embodiments include expression vectors comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. Regulatory sequences as used herein include promoters, enhancers and other expression control elements. In certain embodiments, the expression vector is designed for selection of the host cell to be transformed, the particular variant polypeptide to be expressed, the copy number of the vector, the ability to control the copy number, and/or expression of any other protein encoded by the vector (such as an antibiotic marker).

In some embodiments, the exogenous polynucleotide is operably linked to a promoter for expression of the exogenous polynucleotide in the engineered cell. Examples of suitable mammalian promoters include, for example, promoters from the following genes: elongation factor 1 alpha (EF 1 alpha) promoter, hamster ubiquitin/S27 a promoter (WO 97/15664), simian cavitation virus 40 (SV 40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, long terminal repeat of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter (MMTV), moloney murine leukemia virus long terminal repeat and human Cytomegalovirus (CMV) early promoter. Examples of other heterologous mammalian promoters are actin, immunoglobulin or heat shock promoters. In additional embodiments, the promoters for mammalian host cells may be obtained from the genome of viruses such as polyomavirus, fowlpox virus (UK 2,211,504 disclosed in 7, 5, 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis b virus, and simian virus 40 (SV 40). In other embodiments, heterologous mammalian promoters are used. Examples include actin promoters, immunoglobulin promoters and heat shock promoters. The early and late promoters of SV40 are conveniently obtained as SV40 restriction fragments that also contain the SV40 viral origin of replication (Fiers et al, nature 273:113-120 (1978)). The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII restriction enzyme fragment (Greenaway et al, gene 18:355-360 (1982)). The foregoing references are incorporated by reference in their entirety.

In some embodiments, the expression vector is a bicistronic or polycistronic expression vector. A bicistronic or polycistronic expression vector may comprise (1) a plurality of promoters fused to each open reading frame; (2) insertion of splicing signals between genes; (3) expression of fusion of genes driven by a single promoter; and/or (4) insertion of proteolytic cleavage sites (self-cleaving peptides) between genes or insertion of Internal Ribosome Entry Sites (IRES) between genes.

In some embodiments, the expression vector or construct herein is a polycistronic construct. The terms "polycistronic construct" and "polycistronic vector" are used interchangeably herein and refer to a recombinant DNA construct to be transcribed into a single mRNA molecule, wherein the single mRNA molecule encodes two or more genes (e.g., two or more transgenes). If the polycistronic construct encodes two genes, it is referred to as a bicistronic construct; if the polycistronic construct encodes three genes, it is referred to as a tricistronic construct; if the polycistronic construct encodes four genes, it is called a tetracistronic construct, and so on.

In some embodiments, the vector or construct comprises two or more exogenous polynucleotides (e.g., transgenes) that are each separated by a polycistronic separation element. In some embodiments, the polycistronic isolation element is an IRES or a sequence encoding a cleavable peptide or ribosome jump element. In some embodiments, the polycistronic isolation element is an IRES, such as an Encephalomyocarditis (EMCV) virus IRES. In some embodiments, the polycistronic separating element is a cleavable peptide, such as a 2A peptide. Exemplary 2A peptides include P2A peptides, T2A peptides, E2A peptides, and F2A peptides. In some embodiments, the cleavable peptide is T2A. In some embodiments, two or more exogenous polynucleotides (e.g., a first exogenous polynucleotide and a second exogenous polynucleotide) are operably linked to a promoter. In some embodiments, the first exogenous polynucleotide and the second exogenous polynucleotide are each operably linked to a promoter. In some embodiments, the promoters are the same promoters. In some embodiments, the promoter is an EF1 promoter.

In some cases, an exogenous polynucleotide encoding an exogenous polypeptide (e.g., an exogenous polynucleotide encoding a tolerogenic factor or complement inhibitor described herein) encodes a cleavable peptide or ribosome-hopping element (such as T2A at the N-terminus or C-terminus of the exogenous polypeptide encoded by the polycistronic vector). In some embodiments, the inclusion of a cleavable peptide or ribosome jump element allows expression of two or more polypeptides from a single translation initiation site. In some embodiments, the cleavable peptide is T2A. In some embodiments, T2A is or comprises the amino acid sequence set forth in SEQ ID NO. 11. In some embodiments, T2A is or comprises the amino acid sequence set forth in SEQ ID NO. 12. In some embodiments, T2A is or comprises the amino acid sequence set forth in SEQ ID NO. 17. In some embodiments, T2A is or comprises the amino acid sequence set forth in SEQ ID NO. 18.

In some embodiments, the vector or construct comprises a single promoter that drives expression of one or more transcription units of the exogenous polynucleotide. In some embodiments, such vectors or constructs may be polycistronic (bicistronic or tricistronic, see, e.g., U.S. patent No.6,060,273). For example, in some embodiments, the transcription unit may be engineered to contain a bicistronic unit of IRES (internal ribosome entry site), which allows for the co-expression of gene products (e.g., one or more tolerogenic factors such as CD47 and/or one or more complement inhibitors such as CD46, CD59 and CD 55) from RNA transcribed from a single promoter. In some embodiments, the vectors or constructs provided herein are bicistronic, allowing the vectors or constructs to express two separate polypeptides. In some cases, the two separate polypeptides encoded by the vector or construct are tolerogenic factors (e.g., two factors selected from the group consisting of CD47, DUX4, CD24, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, IL-39, fasL, CCL21, CCL22, mfge8, and Serpinb). In some cases, the two separate polypeptides encoded by the vector or construct are CD46 and CD59. In some embodiments, the two separate polypeptides encoded by the vector or construct are tolerogenic factors (e.g., CD 47) and complement inhibitors selected from the group consisting of CD46, CD59, and CD55. In some embodiments, the vectors or constructs provided herein are tricistronic, allowing the vectors or constructs to express three separate polypeptides. In some cases, the three nucleic acid sequences of the tricistronic vector or construct are tolerogenic factors such as CD47, CD46, and CD59. In some cases, the three nucleic acid sequences of the tricistronic vector or construct are CD46, CD59, and CD55. In some embodiments, the three nucleic acid sequences of the tricistronic vector or construct are three tolerogenic factors selected from the group consisting of: CD47, DUX4, CD24, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, IL-39, fasL, CCL21, CCL22, mfge8 and Serpinb9. In some embodiments, the vectors or constructs provided herein are tetracistronic, allowing the vectors or constructs to express four separate polypeptides. In some cases, the four separate polypeptides of the tetracistronic vector or construct are CD47, CD46, CD59, and CD55. In some cases, the four individual polypeptides of the tetracistronic vector or construct are four tolerogenic factors selected from the group consisting of: CD47, DUX4, CD24, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, IL-39, fasL, CCL21, CCL22, mfge8 and Serpinb9.

In some embodiments, the cell comprises one or more vectors or constructs, wherein each vector or construct is a monocistronic or polycistronic construct as described above, and the monocistronic or polycistronic construct encodes one or more tolerogenic factors and/or complement inhibitors in any combination or order.

In some embodiments, a single promoter directs expression of RNA containing two, three, or four genes (e.g., encoding tolerogenic factors (e.g., CD 47) and/or one or more complement inhibitors selected from CD46, CD59, and CD 55) separated from each other by a sequence encoding a self-cleaving peptide (e.g., a 2A sequence) or a protease recognition site (e.g., furin) in a single Open Reading Frame (ORF). Thus, the ORF encodes a single polypeptide that is processed into separate proteins during translation (in the case of 2A) or post-translationally. In some cases, peptides such as T2A may cause ribosome skipping (ribosome skipping) to synthesize a peptide bond at the C-terminus of the 2A element, resulting in separation between the 2A sequence end and the next peptide downstream (see, e.g., de Felipe. Genetic VACCINES AND Ther.2:13 (2004) and deFelipe et al Traffic 5:616-626 (2004)). Many 2A elements are known in the art. Examples of 2A sequences that may be used in the methods and nucleic acids disclosed herein include, but are not limited to, 2A sequences from foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 16), equine rhinitis virus (E2A, e.g., SEQ ID NO: 15), leptopetalum album (thosea asigna) virus (T2A, e.g., SEQ ID NO:11, 12, 17, or 18), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO:13 or 14), as described in U.S. patent publication No. 20070116690.

Where the vector or construct (e.g., transgene) contains more than one nucleic acid sequence encoding a protein (e.g., a first exogenous polynucleotide encoding CD46 and a second exogenous polynucleotide encoding CD59, or a first exogenous polynucleotide encoding CD47, a second exogenous polynucleotide encoding CD56, and a third exogenous polynucleotide encoding CD 59), the vector or construct (e.g., transgene) may also contain a nucleic acid sequence encoding a peptide between the first and second exogenous polynucleotide sequences. In some cases, the nucleic acid sequence located between the first and second exogenous polynucleotides encodes a peptide that separates the translation products of the first and second exogenous polynucleotides during or after translation. In some embodiments, the peptide contains a self-cleaving peptide or a peptide that causes ribosome jump (ribosome jump element), such as a T2A peptide. In some embodiments, the inclusion of a cleavable peptide or ribosome jump element allows expression of two or more polypeptides from a single translation initiation site. In some embodiments, the peptide is a self-cleaving peptide, which is a T2A peptide. In some embodiments, T2A is or comprises the amino acid sequence set forth in SEQ ID NO. 11. In some embodiments, T2A is or comprises the amino acid sequence set forth in SEQ ID NO. 12. In some embodiments, T2A is or comprises the amino acid sequence set forth in SEQ ID NO. 17. In some embodiments, T2A is or comprises the amino acid sequence set forth in SEQ ID NO. 18.

The process of introducing the polynucleotides described herein into a cell may be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid mediated transfection, electroporation, fusogenic and transduction or infection with viral vectors. In some embodiments, the polynucleotide is introduced into the cell via viral transduction (e.g., lentiviral transduction) or otherwise delivered on a viral vector (e.g., fusogenic mediated delivery). Suitable techniques include calcium phosphate or lipid mediated transfection, electroporation, transposase mediated delivery, and transduction or infection with viral vectors. In some embodiments, the polynucleotide is introduced into the cell via viral transduction (e.g., lentiviral transduction) or otherwise delivered on a viral vector (e.g., fusogenic mediated delivery). In some embodiments, vectors that package polynucleotides encoding exogenous polynucleotides may be used to deliver the packaged polynucleotides to a cell or population of cells. These vectors may be of any type, including DNA vectors, RNA vectors, plasmids, viral vectors and particles. In some embodiments, the lipid nanoparticle may be used to deliver an exogenous polynucleotide to a cell. In some embodiments, viral vectors may be used to deliver exogenous polynucleotides to cells. Viral vector technology is well known and described in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York). Viruses that may be used as vectors include, but are not limited to, lentiviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes simplex virus vectors, retroviral vectors, oncolytic viruses, and the like. In some embodiments, the introduction of the exogenous polynucleotide into the cell may be specific (targeted) or non-specific (e.g., non-targeted). In some embodiments, the introduction of an exogenous polynucleotide into a cell may result in integration or insertion into the genome of the cell. In other embodiments, the introduced exogenous polynucleotide may be non-integrated or free in the cell. The skilled artisan is familiar with methods of introducing nucleic acid transgenes into cells, including any of the exemplary methods described herein, and can select an appropriate method.

1) Non-targeted delivery

In some embodiments, the exogenous polynucleotide is introduced into the cell (e.g., the source cell) by any of a variety of non-targeting methods. In some embodiments, the exogenous polynucleotide is inserted into a random genomic locus of the host cell. As known to those skilled in the art, viral vectors (including, for example, retroviral vectors and lentiviral vectors) are commonly used to deliver genetic material into a host cell and randomly insert foreign or exogenous genes into the host cell genome to promote stable expression and replication of the genes. In some embodiments, the non-targeted introduction of the exogenous polynucleotide into the cell is performed under conditions in which the exogenous polynucleotide is stably expressed in the cell. In some embodiments, the method for introducing a nucleic acid for stable expression in a cell involves any method that results in stable integration of the nucleic acid into the cell genome, such that if the cell into which it is integrated divides, it can propagate.

In some embodiments, the viral vector is a lentiviral vector. Lentiviral vectors are particularly useful means of successful viral transduction because they allow stable expression of genes contained in the nucleic acid transcripts delivered. Lentiviral vectors express reverse transcriptase and integrase, two enzymes required for stable expression of genes contained in the delivered nucleic acid transcripts. Reverse transcriptase converts RNA transcripts into DNA, while integrase inserts and integrates DNA into the genome of the target cell. Once the DNA is stably integrated into the genome, it will split with the host. The gene of interest contained in the integrated DNA may be expressed constitutively, or it may be inducible. As part of the host cell genome, it may undergo cellular regulation, including activation or inhibition, depending on many factors in the target cell.

Lentiviruses are a subgroup of the retroviral family of viruses, which are named because they require reverse transcription of the viral RNA genome into DNA prior to integration into the host genome. Thus, the most important property of lentiviral vectors/particles is their integration of genetic material into the genome of the target/host cell. Some examples of lentiviruses include human immunodeficiency virus: HIV-1 and HIV-2, simian Immunodeficiency Virus (SIV), feline Immunodeficiency Virus (FIV), bovine Immunodeficiency Virus (BIV), jem Brasenia virus (Jembrana Disease Virus) (JDV), equine Infectious Anemia Virus (EIAV), weissner-Medi virus, and Caprine Arthritis Encephalitis Virus (CAEV).

Typically, lentiviral particles that make up the gene delivery vehicle are themselves replication defective (also known as "self-inactivating"). Lentiviruses are able to infect dividing and non-dividing cells by an entry mechanism through the intact host nuclear envelope (Naldini L et al, curr. Opin. Bioiecknol,1998, 9:457-463). Recombinant lentiviral vectors/particles are created by multiple attenuation of HIV virulence genes, such as gene Env, vif, vpr, vpu, nef and Tat deletions, thereby rendering the vector biosafety. Correspondingly, lentiviral vectors derived from HIV-1/HIV-2, for example, can mediate efficient delivery, integration and long term expression of transgenes to non-dividing cells.

Lentiviral particles can be generated by co-expressing the viral packaging element and the vector genome itself in a producer cell (such as a human HEK293T cell). These elements are typically provided as three (in the second generation lentiviral system) or four separate plasmids (in the third generation lentiviral system). The producer cell is co-transfected with a plasmid encoding a lentiviral component, including the core (i.e., structural protein) and the enzyme component of the virus, and the envelope protein (referred to as the packaging system), as well as the plasmid encoding the genome comprising the foreign transgene to be transferred into the target cell, the vector itself (also referred to as the transfer vector). Generally, the plasmid or vector is contained in a producer cell line. The plasmid/vector is introduced into the producer cell line via transfection, transduction or infection. Methods of transfection, transduction or infection are well known to those skilled in the art. As non-limiting examples, packaging and transfer constructs can be introduced into a production cell line by calcium phosphate transfection, lipofection, or electroporation, typically together with a dominant selectable marker, such as neomycin (neo), dihydrofolate reductase (DHFR), glutamine synthetase, or Adenosine Deaminase (ADA), followed by selection and isolation of clones in the presence of an appropriate drug.

The producer cell produces recombinant viral particles containing the foreign gene, e.g., a polynucleotide encoding an exogenous polynucleotide. Recombinant viral particles are recovered from the culture medium and titrated by standard methods used by those skilled in the art. Recombinant lentiviral vectors may be used to infect target cells, such source cells including any of the cells described in section ii.c.

Cells that can be used to produce high titer lentiviral particles can include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al, mol Ther.2005, 11:452-459), freeStyle ^TM 293 expression systems (ThermoFisher, waltham, mass.) and other HEK 293T-based producer cell lines (e.g., stewart et al, hum Gene Ther. _2011,2,2. (3): 357-369; lee et al, biotechnol Bioeng,2012,10996): 1551-1560; throm et al blood 2009,113 (21): 5104-5110).

Additional elements provided in the lentiviral particle may include the retroviral LTR (long terminal repeat) at the 5 'or 3' end, the retroviral export element, optionally the lentiviral Reverse Response Element (RRE), the promoter or active portion thereof, and the Locus Control Region (LCR) or active portion thereof. Other elements include a central polypurine tract (cPPT) sequence that increases transduction efficiency in non-dividing cells, a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) that enhances expression of the transgene and increases titer.

Methods for generating recombinant lentiviral particles are known to the skilled artisan, for example, U.S. patent No.: 8,846,385;7,745,179;7,629,153;7,575,924;7,179,903; and 6,808,905. The lentiviral vector used may be selected from, but is not limited to pLVX、pLenti、pLenti6、pLJMl、FUGW、pWPXL、pWPI、pLenti CMV puro DEST、pLJMl-EGFP、pULTRA、pInducer2Q、pHIV-EGFP、pCW57.1、pTRPE、pELPS、pRRL and pLionII, and any known lentiviral vector may be used (see U.S. Pat. Nos. 9,260,725;9,068,199;9,023,646;8,900,858;8,748,169;8,709,799;8,420,104;8,329,462;8,076,106;6,013,516; and 5,994,136; international patent publication No. WO 2012079000).

In some embodiments, the exogenous polynucleotide is introduced into the cell under conditions of transient expression by the cell, such as by a method that results in free delivery of the exogenous polynucleotide.

In some embodiments, polynucleotides encoding exogenous polynucleotides may be packaged into recombinant adeno-associated virus (rAAV) vectors. Such vectors or viral particles may be designed to utilize any known serotype capsid or combination of serotype capsids. Serotype capsids may include capsids from any identified AAV serotype and variants thereof, such as AAV1, AAV2G9, AAV3, AAV4-4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAVrh10. In some embodiments, the AAV serotype may be or have the sequences as set forth in: U.S. publication No. US20030138772; pulicherla et al Molecular Therapy,2011,19 (6): 1070-1078; U.S. patent No.: 6,156,303;7,198,951; U.S. patent publication No.: US2015/0159173 and US2014/0359799; international patent publication No.: WO 1998/011024, WO2005/033321 and WO2014/14422.

AAV vectors include not only single stranded vectors, but also self-complementary AAV vectors (scAAV). scAAV vectors contain DNA that anneals together to form a double stranded vector genome. scAAV can be expressed rapidly in cells by skipping second strand synthesis. rAAV vectors can be prepared by standard methods in the art, such as by triple transfection in sf9 insect cells or in suspension cell cultures of human cells (such as HEK293 cells).

In some embodiments, non-viral based methods may be used. For example, in some aspects, a vector comprising a polynucleotide may be transferred to a cell by non-viral methods (by physical methods such as needle, electroporation, sonications (sonoporation), aqua-perforation (hyrdoporation); chemical carriers such as inorganic particles (e.g., calcium phosphate, silica, gold), and/or chemical methods). In some aspects, synthetic or natural biodegradable agents can be used for delivery, such as cationic lipids, lipid nanoemulsions, nanoparticles, peptide-based carriers, or polymer-based carriers.

2) Targeted delivery

The exogenous polynucleotide may be inserted into any suitable target genomic locus of the cell. In some embodiments, the exogenous polynucleotide is introduced into the cell by targeted integration into the target locus. In some embodiments, targeted integration may be achieved by gene editing using one or more nucleases and/or nicking enzymes and donor templates during a process involving homologous dependent or homologous independent recombination.

A number of gene editing methods can be used to insert exogenous polynucleotides into a particular genomic locus of choice, including, for example, homology directed repair (HOR), homology-mediated end ligation (HMEJ), homology Independent Targeted Integration (HITI), obligate ligation-gated recombination (ObliGaRe), or precise integration into a target chromosome (PITCh).

In some embodiments, the nuclease creates a specific double-strand break (DSB) at a desired location in the genome (e.g., a target site), and utilizes the endogenous mechanisms of the cell to repair the induced break. Nicking enzymes produce specific single-strand breaks at desired locations in the genome. In one non-limiting example, two nicking enzymes may be used to create two single strand breaks on opposite strands of the target DNA, thereby creating a blunt end or sticky end. Any suitable nuclease may be introduced into the cells to induce genome editing of the target DNA sequence, including, but not limited to, CRISPR-associated protein (Cas) nucleases, zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, other endonucleases or exonucleases, variants thereof, fragments thereof, and combinations thereof. In some embodiments, when a nuclease or nicking enzyme is introduced with a donor template containing an exogenous polynucleotide sequence (also referred to as a transgene) flanking a homologous sequence (e.g., homology arm) that is homologous to a sequence at or near an endogenous genomic target locus (e.g., safe harbor locus), the DNA damage repair pathway can result in integration of the transgene sequence at the target site in the cell. This can occur through a homology dependent process. In some embodiments, the donor template is a circular double stranded plasmid DNA, a single stranded donor oligonucleotide (ssODN), a linear double stranded Polymerase Chain Reaction (PCR) fragment, or a homologous sequence of the intact sister chromatid. Depending on the form of the donor template, homology-mediated gene insertion and replacement may be via specific DNA repair pathways, such as homology-directed repair (HDR), synthesis-dependent strand annealing (SDSA), microhomology-mediated end ligation (MMEJ), and homology-mediated end ligation (HMEJ) pathways.

For example, the DNA repair mechanism may be induced by nucleases after: (i) Two SSBs, one on each strand, to induce single-stranded overhang; (ii) DSBs present at the same cleavage site on both strands, thereby inducing blunt end fragmentation. After cleavage by one of these agents, the target locus with SSB or DSB undergoes one of two major pathways for DNA damage repair: (1) Error-prone non-homologous end joining (NHEJ), or (2) a high fidelity Homology Directed Repair (HDR) pathway. In some embodiments, introducing a donor template (e.g., circular plasmid DNA or a linear DNA fragment, such as ssODN) into a cell in which SSB or DSB is present can result in HDR and integration of the donor template into the target locus. Generally, in the absence of donor template, the NHEJ process rejoins the ends of the cleaved DNA strand, which typically results in nucleotide deletions and insertions at the cleavage site.

In some embodiments, site-directed insertion of an exogenous polynucleotide into a cell can be achieved by an HDR-based method. HDR is a mechanism of Double Strand Breaks (DSBs) in cellular repair DNA, and can be used to modify genomes in many organisms using a variety of gene editing systems, including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems, zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and transposases.

In some embodiments, targeted integration is performed by introducing one or more sequence-specific or targeted nucleases, including DNA-binding targeted nucleases and gene editing nucleases such as Zinc Finger Nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), as well as RNA-guided nucleases such as CRISPR-associated nucleases (Cas) systems, specifically designed to target at least one target site sequence of a target gene. Exemplary ZFNs, TALEs, and TALENs are described, for example, in Lloyd et al Frontiers in Immunology,4 (221): 1-7 (2013). In particular embodiments, targeted genetic disruption is performed at or near the target site using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins. See Sander and Joung, (2014) Nature Biotechnology,32 (4): 347-355.

Any system for gene disruption is described in section II. A.1 can be used, and when an appropriate donor template with an exogenous polynucleotide (e.g., a transgene sequence) is also introduced, a.1 can result in targeted integration of the exogenous polynucleotide at or near the genetically disrupted target site. In particular embodiments, genetic disruption is mediated using a CRISPR/Cas system containing one or more guide RNAs (grnas) and a Cas protein. Exemplary Cas proteins and grnas are described in section ii.a above, any of which may be used for HDR-mediated integration of an exogenous polynucleotide into a target locus specific for the Crispr/Cas system. The selection of the appropriate Cas nuclease and gRNA (such as according to the specific target locus and target site for cleavage and integration of the exogenous polynucleotide by HDR) is within the level of the skilled artisan. Furthermore, depending on the target locus, the skilled artisan can readily prepare appropriate donor templates, such as those described further below.

In some embodiments, the DNA editing system is an RNA-guided CRISPR/Cas system (such as an RNA-based CRISPR/Cas system), wherein the CRISPR/Cas system is capable of generating a double strand break in a target locus (e.g., a safe harbor locus) to induce insertion of a transgene into the target locus. In some embodiments, the nuclease system is a CRISPR/Cas9 system. In some embodiments, the CRISPR/Cas9 system comprises a plasmid-based Cas9. In some embodiments, the CRISPR/Cas9 system comprises RNA-based Cas9. In some embodiments, the CRISPR/Cas9 system comprises Cas9 mRNA and gRNA. In some embodiments, the CRISPR/Cas9 system comprises a protein/RNA complex, or a plasmid/RNA complex, or a protein/plasmid complex. In some embodiments, methods for generating an engineered cell are provided, the methods comprising introducing a donor template comprising a transgenic or exogenous polynucleotide sequence and a DNA nuclease system comprising a DNA nuclease system (e.g., cas 9) and a locus-specific gRNA into a source cell (e.g., a primary cell or a pluripotent stem cell, e.g., iPSC). In some embodiments, cas9 is introduced as mRNA. In some embodiments, cas9 is introduced as a ribonucleoprotein complex with a gRNA.

Typically, the donor template to be inserted will comprise at least a transgene cassette containing an exogenous polynucleotide of interest (e.g., tolerogenic factors or CARs), and optionally also a promoter. In some of these embodiments, the transgene cassette containing the exogenous polynucleotide and/or promoter to be inserted will flank in the donor template homology arms, i.e., left Homology Arm (LHA) and Right Homology Arm (RHA), having sequences homologous to sequences immediately upstream and downstream of the target cleavage site. Typically, the homology arms of the donor template are specifically designed for the target genomic locus to be used as a template for HDR. The length of each homology arm is generally dependent on the size of the insert being introduced, with larger inserts requiring longer homology arms.

In some embodiments, the donor template (e.g., recombinant donor repair template) comprises: (i) A transgene cassette comprising an exogenous polynucleotide sequence (e.g., a transgene operably linked to a promoter (e.g., a heterologous promoter); and (ii) two homology arms flanking the transgene cassette and homologous to portions of the target locus (e.g., a safe harbor locus) flanking the DNA nuclease (e.g., cas nuclease, such as Cas9 or Cas 12) cleavage site. The donor template may also comprise a selectable marker, a detectable marker, and/or a purification marker.

In some embodiments, the homology arms are the same length. In other embodiments, the homology arms are different in length. The homology arm may be at least about 10 base pairs (bp), for example at least about 10bp、15bp、20bp、25bp、30bp、35bp、45bp、55bp、65bp、75bp、85bp、95bp、100bp、150bp、200bp、250bp、300bp、350bp、400bp、450bp、500bp、550bp、600bp、650bp、700bp、750bp、800bp、850bp、900bp、950bp、1000bp、1.1 kilobases (kb)、1.2kb、1.3kb、1.4kb、1.5kb、1.6kb、1.7kb、1.8kb、1.9kb、2.0kb、2、1kb、2,2kb、2,3kb、2,4kb、2,5kb、2,6kb、2.7kb、2.8kb、2.9kb、3.0kb、3.1kb、3.2kb、3.3kb、3.4kb、3.5kb、3.6kb、3.7kb、3.8kb、3.9kb、4.0kb or longer. The homology arm may be about 10bp to about 4kb, for example about 10bp to about 20bp, about 10bp to about 50bp, about 10bp to about 100bp, about 10bp to about 200bp, about 10bp to about 500bp, about 10bp to about I kb, about 10bp to about 2kb, about 10bp to about 4kb, about 100bp to about 200bp, about 100bp to about 500bp, about 100bp to about 1kb, about 100bp to about 2kb, about 100bp to about 4kb, about 500bp to about I kb, about 500bp to about 2kb, about 500bp to about 4kb, about 1kb to about 2kb, about 1kb to about 4kb, or about 2kb to about 4kb.

In some embodiments, the donor template may be cloned into an expression vector. Conventional viral and nonviral based expression vectors known to those of ordinary skill in the art may be used.

In some embodiments, the target locus for targeted integration may be any locus where targeted integration of an exogenous polynucleotide or transgene is acceptable or desirable. Non-limiting examples of target loci include, but are not limited to, CXCR4 genes, albumin genes, SHS231 loci, F3 genes (also known as CD 142), MICA genes, MICB genes, LRP1 genes (also known as CD 91), HMGB1 genes, ABO genes, RHD genes, FUT1 genes, KDM5D genes (also known as HY), B2M genes, CIITA genes, TRAC genes, TRBC genes, CCR5 genes, F3 (i.e., CD 142) genes, MICA genes, MICB genes, LRP1 genes, HMGB1 genes, ABO genes, RHD genes, FUT1 genes, KDM5D (i.e., HY) genes, PDGFRa genes, OLIG2 genes, and/or GFAP genes. In some embodiments, the exogenous polynucleotide may be inserted into a suitable region of a target locus (e.g., a safe harbor locus), including, for example, introns, exons, and/or gene coding regions (also referred to as coding sequences, or "CDSs"). In some embodiments, the insertion occurs in one allele of the target genomic locus. In some embodiments, the insertion occurs in both alleles of the target genomic locus. In any of these embodiments, the direction of the transgene inserted into the target genomic locus may be the same or opposite to the direction of the gene in that locus.

In some embodiments, the exogenous polynucleotide is inserted into an intron, exon, or coding sequence region of a safe harbor locus. In some embodiments, the exogenous polynucleotide is inserted into an endogenous gene, wherein the insertion results in silencing or reduced expression of the endogenous gene. Exemplary genomic loci for insertion of exogenous polynucleotides are described in table 2B.

Table 2B: exemplary genomic loci for insertion of exogenous polynucleotides

In some embodiments, the target locus is a safe harbor locus. In some embodiments, the safe harbor locus is a genomic location that allows for stable expression of the integrated DNA with minimal impact on nearby or nearby endogenous genes, regulatory elements, and the like. In some cases, safe harbor genes are capable of achieving sustainable gene expression and can be targeted for genetic modification by engineered nucleases in a variety of cell types, including primary and pluripotent stem cells (including derivatives thereof) and differentiated cells thereof. Non-limiting examples of safe harbor loci include, but are not limited to, the CCR5 locus, the PPP1R12C (also known as AAVS 1) locus, the CLYBL locus, and/or the Rosa locus (e.g., rosa26 locus). In some embodiments, the safe harbor locus is selected from the group consisting of AAVS1 locus, CCR5 locus, and CLYBL locus. In some cases, SHS231 can be targeted as a safe harbor locus in many cell types. In some cases, certain loci may function as safe harbor loci in certain cell types. For example, PDGFRa is the safe harbor of Glial Progenitor Cells (GPC), OLIG2 is the safe harbor locus of oligodendrocytes, and GFAP is the safe harbor locus of astrocytes. The selection of the appropriate safe harbor locus according to the specific engineered cell type is within the level of the skilled artisan. In some cases, more than one safe harbor gene may be targeted, thereby introducing more than one transgene into the genetically modified cell.

In some embodiments, methods for generating an engineered cell are provided, the methods comprising introducing into a source cell (e.g., a primary cell or pluripotent stem cell, e.g., iPSC) a donor template comprising a transgenic or exogenous polynucleotide sequence and a DNA nuclease system comprising a DNA nuclease system (e.g., cas 9) and a locus-specific gRNA comprising a complementary portion (e.g., a gRNA targeting sequence) specific for a CCR5 locus, PPP1R12C (also known as AAVS 1) locus, CLYBL locus, and/or Rosa locus (e.g., rosa26 locus). In some embodiments, the gRNA-targeted genomic locus is within 4000bp, 3500bp, 3000bp, 2500bp, 2000bp, 1500bp, 1000bp, or 500bp of any of the loci described.

In some embodiments, the grnas herein for HDR-mediated transgene insertion comprise complementary portions that recognize a target sequence in AAVS1 (e.g., a gRNA targeting sequence). In certain of these embodiments, the target sequence is located in intron 1 of AAVS 1. AAVS1 is located on chromosome 19:55,090,918-55,117,637 reverse strand, and AAVS1 intron 1 (based on transcript ENSG 00000125503) is located on chromosome 19:55,117,222-55,112,796 reverse strand. In certain embodiments, the gRNA targets chromosome 19:55,117,222-55,112,796 4000bp, 3500bp, 3000bp, 2500bp, 2000bp, 1500bp, 1000bp or 500 bp. In certain embodiments, the gRNA targets chromosome 19: genome loci in 55,115,674 4000bp, 3500, 3000, 2500, 2000, 1500, 1000 or 500 bp. In certain embodiments, the gRNA is configured to be on chromosome 19:55,115,674 or at chromosome 19:55,115,674, 10, 15, 20, 30, 40 or 50 nucleotides within the position of the generation of cleavage site. In certain embodiments, the gRNA is GET000046, also known as "sgAAVS1-1", described in Li et al, nat. Methods 16:866-869 (2019). This gRNA comprises a complementary portion (e.g., a gRNA targeting sequence) having the nucleic acid sequence set forth in SEQ ID No. 36 (shown in table 4) and targets intron 1 of AAVS1 (also referred to as PPP1R 12C).

In some embodiments, the grnas herein for HDR-mediated transgene insertion comprise complementary portions that recognize the target sequence in CLYBL (e.g., a gRNA targeting sequence). In some of these embodiments, the target sequence is located in intron 2 of CLYBL. CLYBL located on chromosome 13:99,606,669-99,897,134 forward strand, and CLYBL intron 2 (based on transcript ENST 00000376355.7) is located on chromosome 13:99,773,011-99,858,860 forward chain. In certain embodiments, the gRNA targets chromosome 13:99,773,011-99,858,860 4000bp, 3500bp, 3000bp, 2500bp, 2000bp, 1500bp, 1000bp or 500 bp. In certain embodiments, the gRNA targets chromosome 13: genome loci in 99,822,980 4000bp, 3500, 3000, 2500, 2000, 1500, 1000 or 500 bp. In certain embodiments, the gRNA is configured to be on chromosome 13:99,822,980 or at chromosome 13:99,822,980, 10, 15, 20, 30, 40 or 50 nucleotides within the position of the generation of cleavage site. In certain embodiments, the gRNA is GET000047, which comprises a complementary portion (e.g., a gRNA targeting sequence) having the nucleic acid sequence set forth in SEQ ID NO:36 (shown in table 4) and targets intron 2 of CLYBL. The target site is similar to that of TALEN as described in Cerbini et al, PLoS One,10 (1): e 016032 (2015).

In some embodiments, the grnas herein for HDR-mediated transgene insertion comprise complementary portions that recognize a target sequence in CCR5 (e.g., a gRNA targeting sequence). In some of these embodiments, the target sequence is located in exon 3 of CCR 5. CCR5 is located on chromosome 3:46,370,854-46,376,206 forward strand, and CCR5 exon 3 (based on transcript ENST 00000292303.4) is located on chromosome 3:46,372,892-46,376,206 forward chain. In certain embodiments, the gRNA targets chromosome 3:46,372,892-46,376,2064000bp, 3500bp, 3000bp, 2500bp, 2000bp, 1500bp, 1000bp or 500bp genomic loci. In certain embodiments, the gRNA targets chromosome 3: genome loci in 46,373,180 4000bp, 3500, 3000, 2500, 2000, 1500, 1000 or 500 bp. In certain embodiments, the gRNA is configured to be on chromosome 3:46,373,180 or at chromosome 3:46,373,180, 10, 15, 20, 30, 40 or 50 nucleotides within the position of the generation of cleavage site. In certain embodiments, the gRNA is GET000048, also known as "crCCR5_D", described in Mandal et al CELL STEM CELL 15:643-652 (2014). This gRNA comprises a complementary part of the nucleic acid sequence set forth in SEQ ID NO 37 (shown in Table 4) and targets exon 3 of CCR5 (alternatively noted as exon 2 in the Ensembl genome database). See Gomez-Ospina et al, nat. Comm.10 (1): 4045 (2019).

Table 4 lists exemplary gRNA targeting sequences. In some embodiments, the gRNA targeting sequence may contain one or more thymines in the complementary partial sequences listed in table 4, substituted with uracils. It will be appreciated by those of ordinary skill in the art that uracil and thymine can both be represented by "t", rather than uracil being represented by "u" and thymine being represented by "t"; in the context of ribonucleic acids, it is to be understood that "t" is used to denote uracil unless otherwise indicated.

Table 4. Exemplary gRNA targeting sequences of CCR5

Description of the invention	Nucleic acid sequences	SEQ ID NO:
			GET000046 guide	(5'→3')accccacagtggggccacta	35
GET000047 guide	(5'→3')tgttggaaggatgaggaaat	36
			GET000048 guide	(5'→3')tcactatgctgccgcccagt	37

In some embodiments, the target locus is a locus in the cell that needs to be knocked out. In such embodiments, such a target locus is any target locus in a cell that is desired for its destruction or elimination (such as to modulate the phenotype or function of the cell). For example, any of the genetic modifications described in section ii.a that reduce expression of a target gene may be a desired target locus for targeted integration of an exogenous polynucleotide, wherein genetic disruption or knockdown of the target gene and overexpression by targeted insertion of the exogenous polynucleotide may be achieved at the same target locus or locus in the cell. For example, an HDR process can be used to cause genetic disruption to eliminate or reduce (e.g., knock out) expression of any of the target genes listed in table 1, while also integrating (e.g., knock in) an exogenous polynucleotide into the target gene by using a donor template with flanking homology arms that are homologous to the nucleic acid sequence at or near the target site of the genetic disruption.

In some embodiments, methods for generating an engineered cell are provided, the methods comprising introducing into a source cell (e.g., a primary cell or pluripotent stem cell, e.g., iPSC) a donor template comprising a transgenic or exogenous polynucleotide sequence and a DNA nuclease system comprising a DNA nuclease system (e.g., cas 9) and a locus-specific gRNA comprising a complementary portion specific for a B2M locus, CIITA locus, TRAC locus, TRBC locus. In some embodiments, the gRNA-targeted genomic locus is within 4000bp, 3500bp, 3000bp, 2500bp, 2000bp, 1500bp, 1000bp, or 500bp of any of the loci described.

In a particular embodiment, the target locus is B2M. In some embodiments, the engineered cell comprises a genetic modification that targets the B2M gene. In some embodiments, the genetic modification of the targeted B2M gene is performed by using a targeting nuclease system comprising a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the B2M gene. In some embodiments, the at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the B2M gene is selected from the group consisting of appendix 2 of WO2016/183041 or SEQ ID NO:81240-85644 of Table 15, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the exogenous polynucleotide is integrated into the disrupted B2M locus by HDR by introducing a donor template containing an exogenous polynucleotide sequence having flanking homology arms homologous to sequences adjacent to the target site targeted by the gRNA.

In a particular embodiment, the target locus is CIITA. In some embodiments, the engineered cell comprises a genetic modification that targets the CIITA gene. In some embodiments, the genetic modification of the targeted CIITA gene is performed by a targeting nuclease system comprising a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene is selected from the group consisting of appendix 1 of WO2016183041 or SEQ ID NO:5184-36352 of Table 12, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the exogenous polynucleotide is integrated into the disrupted CIITA locus by HDR by introducing a donor template containing an exogenous polynucleotide sequence having flanking homology arms homologous to sequences adjacent to the target site targeted by the gRNA.

In some embodiments, the cell is a T cell and expression of the endogenous TRAC or TRBC locus in the cell is reduced or eliminated by a gene editing method. For example, an HDR process can be used to cause genetic disruption to eliminate or reduce (e.g., knock out) expression of a TRAC or TRBC gene while also integrating (e.g., knock in) an exogenous polynucleotide into the same locus by using a donor template with flanking homology arms that are homologous to the nucleic acid sequence at or near the target site of the genetic disruption. Exemplary gRNA sequences that can be used for CRISPR/Cas-based gene targeting described herein are provided in table 2C. Such sequences can be found in US20160348073, the disclosure of which, including the sequence listing, is incorporated herein by reference in its entirety.

TABLE 2C exemplary gRNA targeting sequences useful for targeting genes

In some embodiments, the engineered cell comprises a genetic modification that targets the TRAC gene. In some embodiments, the genetic modification of the targeted TRAC gene is performed by a targeting nuclease system comprising a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the TRAC gene. In some embodiments, at least one guide ribonucleic acid sequence (e.g., a gRNA targeting sequence) for specifically targeting the TRAC gene is selected from the group consisting of SEQ ID NOs 532-609 and 9102-9797 of US20160348073, the disclosures of which are incorporated herein by reference in their entirety. In some embodiments, the exogenous polynucleotide is integrated into the disrupted TRAC locus by HDR by introducing a donor template comprising an exogenous polynucleotide sequence having flanking homology arms to sequences adjacent to the target site targeted by the gRNA.

In some embodiments, the engineered cell comprises a genetic modification that targets a TRBC gene. In some embodiments, the genetic modification of the targeted TRBC gene is performed by a targeting nuclease system comprising a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the TRBC gene. In some embodiments, at least one guide ribonucleic acid sequence (e.g., a gRNA targeting sequence) for specifically targeting the TRBC gene is selected from the group consisting of SEQ ID NOs 610-765 and 9798-10532 of US20160348073, the disclosures of which are incorporated herein by reference in their entirety. In some embodiments, the exogenous polynucleotide is integrated into the disrupted TRBC locus by HDR by introducing a donor template containing an exogenous polynucleotide sequence having flanking homology arms homologous to sequences adjacent to the target site targeted by the gRNA.

In some embodiments, it is within the level of the skilled artisan to identify new loci and/or gRNA sequences for the HDR-mediated integration method. For example, for CRISPR/Cas systems, when an existing gRNA for a particular locus (e.g., within a target gene (e.g., listed in table 1)) is known, the "inch worm (inch worming)" approach can be used to identify additional loci to target insertion transgenes by scanning flanking regions flanking the locus for PAM sequences that typically occur about once every 100 base pairs (bp) in the genome. PAM sequences will depend on the particular Cas nuclease used, as different nucleases typically have different corresponding PAM sequences. Flanking regions on both sides of the locus may be about 500 to 4000bp long, for example about 500bp, about 1000bp, about 1500bp, about 2000bp, about 2500bp, about 3000bp, about 3500bp or about 4000bp long. When PAM sequences are identified within the search, new guides can be designed based on the sequence of this locus for use in genetic disruption methods. Although the CRISPR/Cas system is described as illustrative, any of the described HDR-mediated methods can be used in this method of identifying new loci, including those using ZFNs, TALENs, meganucleases and transposases.

In some embodiments, the exogenous polynucleotide encodes an exogenous CD47 polypeptide (e.g., a human CD47 polypeptide), and the exogenous polypeptide is inserted into a safe harbor locus or safe harbor site as disclosed herein, or into a genomic locus that causes silencing or reduced expression of the endogenous gene. In some embodiments, an exogenous polynucleotide encoding CD47 is inserted into the CCR5 locus, PPP1R12C (also known as AAVS 1) locus, CLYBL locus, and/or Rosa locus (e.g., rosa26 locus). In some embodiments, the polynucleotide is inserted into the B2M, CIITA, TRAC, TRBC, PD1 or CTLA4 locus.

C. Cells

In some embodiments, the disclosure provides cells (e.g., stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, or primary cells) or populations thereof that have been engineered (or modified), wherein the genome of the cells has been modified such that expression of one or more genes (e.g., one or more genes that regulate expression of one or more MHC class I molecules or one or more MHC class II molecules) as described herein is reduced or deleted, or wherein expression of a gene or polynucleotide (e.g., a polynucleotide encoding a tolerogenic factor such as CD 47) is over-expressed or increased. In some embodiments, the application provides cells further comprising reduced or deleted CD142 expression. In some embodiments, the application provides cells that overexpress or have increased expression of one or more complement inhibitors.

In some embodiments, the engineered cell comprising the exogenous polynucleotide is a beta islet cell and comprises a first exogenous polynucleotide encoding a CD47 polypeptide. In some embodiments, the engineered β islet cells further comprise one or more additional exogenous polynucleotides encoding one or more complement inhibitors or other tolerogenic polypeptides described herein. In some embodiments, the beta islet cells comprise reduced CD142 expression and reduced expression of one or more MHC class I molecules and/or reduced expression of one or more MHC class II molecules, as described in section ii.a. In some embodiments, the first exogenous polynucleotide and the one or more additional exogenous polynucleotides are inserted into the same genomic locus. In some embodiments, the first exogenous polynucleotide and the one or more additional exogenous polynucleotides are inserted into different genomic loci. In exemplary embodiments, the engineered (e.g., low-immunogenicity) cells are primary beta islet cells or beta islet cells derived from engineered (e.g., low-immunogenicity) pluripotent cells (e.g., ipscs).

In some embodiments, the engineered cell comprising the exogenous polynucleotide is a hepatocyte and comprises a first exogenous polynucleotide encoding a CD47 polypeptide. In some embodiments, the engineered hepatocytes further comprise one or more additional exogenous polynucleotides encoding one or more complement inhibitors or other tolerogenic polypeptides described herein. In some embodiments, the beta islet cells comprise reduced CD142 expression and reduced expression of one or more MHC class I molecules and/or reduced expression of one or more MHC class II molecules, as described in section ii.a. In some embodiments, the first exogenous polynucleotide and the one or more additional exogenous polynucleotides are inserted into the same genomic locus. In some embodiments, the first exogenous polynucleotide and the one or more additional exogenous polynucleotides are inserted into different genomic loci. In exemplary embodiments, the engineered (e.g., low-immunogenicity) cells are primary hepatocytes or hepatocytes derived from engineered (e.g., low-immunogenicity) pluripotent cells (e.g., ipscs).

In some embodiments, the engineered or modified cell as provided herein is a pluripotent stem cell or a cell differentiated from a pluripotent stem cell. In some embodiments, the engineered or modified cell as provided herein is a primary cell.

The cell may be a vertebrate cell, for example a mammalian cell, such as a human cell or a mouse cell. In some embodiments, the cell is a pig (pig/pore) cell, a cow (cow/ovine) cell, or a sheep (shaep/ovine) cell. In some embodiments, the cell is a porcine cell. The cells may also be vertebrate stem cells, for example mammalian stem cells, such as human stem cells or mouse stem cells. In some embodiments, the cell or stem cell is susceptible to modification. In some cases, cells or stem cells or cells derived from such stem cells have or are considered therapeutically valuable such that cells or stem cells or cells derived from or differentiated from such stem cells can be used to treat a disease, disorder, defect, or injury in a subject in need of treatment.

In some embodiments, the cell is a stem or progenitor cell (e.g., iPSC, embryonic stem cell, hematopoietic stem cell, mesenchymal stem cell, endothelial stem cell, epithelial stem cell, adipose stem cell or progenitor cell, germ stem cell, lung stem cell or progenitor cell, breast stem cell, olfactory adult stem cell, hair follicle stem cell, multipotent stem cell, amniotic stem cell, umbilical cord blood stem cell or neural stem cell or progenitor cell). In some embodiments, the stem cell is an adult stem cell (e.g., a somatic stem cell or a tissue-specific stem cell). In some embodiments, the stem or progenitor cells are capable of differentiating (e.g., the stem cells are totipotent, pluripotent, or multipotent). In some embodiments, the cells are isolated from embryonic or neonatal tissue. In some embodiments, the cell is a mononuclear precursor fibroblast, B-cell, exocrine cell, pancreatic progenitor cell, endocrine progenitor cell, hepatoblast, myoblast, preadipocyte, progenitor cell, liver cell, chondrocyte, smooth muscle cell, K562 human erythroid leukemia cell line, bone cell, synovial cell, tendon cell, ligament cell, meniscus cell, adipocyte, dendritic cell

Or natural killer cells. In some embodiments, the cells are manipulated (e.g., transformed or differentiated) into muscle cells, erythroid megakaryocytes, eosinophils, iPS cells, macrophages, T cells, beta islet cells, neurons, cardiomyocytes, blood cells, endocrine progenitor cells, exocrine progenitor cells, ductal cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, hepatocytes, cholangiocytes, or brown adipocytes. In some embodiments, the cell is a muscle cell (e.g., skeletal muscle cell, smooth muscle cell, or cardiac muscle cell), erythroid megakaryocyte, eosinophil, iPS cell, macrophage, T cell, islet beta cell, neuron, cardiac muscle cell, blood cell (e.g., red blood cell, white blood cell, or platelet), endocrine progenitor cell, exocrine progenitor cell, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, liver cell, bile duct cell, or white or brown adipocyte. In some embodiments, the cell is a hormone-secreting cell (e.g., a cell that secretes insulin, oxytocin, endorphin, vasopressin, serotonin, somatostatin, gastrin, secretin, glucagon, thyroid hormone, bombesin, cholecystokinin, testosterone, estrogen or progesterone, renin, ghrelin, amylin or pancreatic polypeptide), an epidermal keratinocyte, an epithelial cell (e.g., a cell that secretes secretin exocrine, thyroid epithelial cell, keratinocyte, gall bladder epithelial cell or cornea, tongue, oral cavity, esophagus, anal canal, surface epithelial cells of the distal urinary tract or vagina), a kidney cell, germ cell, a bone joint synovial cell, periostin cell, a bone cell (e.g., osteoclast or osteoblast), a cartilage cell (e.g., chondroblast or chondrocyte (chondrocyte)), a chondrocyte (CARTILAGE CELL) (e.g., chondrocyte (chondrocyte)), a fibroblast, endothelial cell, pericardial cell, meninge, keratinocyte, a precursor cell, a keratinocyte, a glial stem cell, a pericyte, a plasma cell or a cell isolated from a membrane of a placenta, or a plasma cell in a body cavity such as an endomembrane, or a membrane of a body cavity such as the human body cavity).

In some embodiments, the cell is a somatic cell. In some embodiments, the cells are derived from skin or other organs, such as heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach. The cells may be from a human or other mammal (e.g., rodent, non-human primate, bovine or porcine cells).

In some embodiments, the cell is a T cell, NK cell, beta islet cell, endothelial cell, epithelial cell such as RPE, thyroid cell, skin cell, or liver cell. In some embodiments, the cell is an iPSC-derived cell that has been differentiated from an engineered iPSC. In some embodiments, the cell is an engineered cell modified by a primary cell. In some embodiments, the cells comprise reduced or eliminated CD142 expression. In some embodiments, the cell comprises over-expression or increased expression of one or more complement inhibitors.

In some embodiments, the cell is an iPSC-derived T cell engineered to contain a modification described herein (e.g., a genetic modification). In some embodiments, the cell is a primary T cell engineered to contain a modification described herein (e.g., a genetic modification). In some embodiments, the cells comprise reduced or eliminated CD142 expression. In some embodiments, the cell comprises over-expression or increased expression of one or more complement inhibitors. In some embodiments, T cells can be engineered with Chimeric Antigen Receptors (CARs), including any chimeric antigen receptor as described herein. In some embodiments, engineered (e.g., low immunogenicity) T cells can be used to treat a variety of indications using allogeneic cell therapy, including any of the indications as described herein (e.g., section IV). In some embodiments, engineered (e.g., low immunogenicity) T cells can be used to treat cancer.

In some embodiments, the cell is an iPSC-derived NK cell engineered to contain the modifications described herein (e.g., genetic modifications). In some embodiments, the cell is a primary NK cell engineered to contain the modifications described herein (e.g., genetic modifications). In some embodiments, the cells comprise reduced or eliminated CD142 expression. In some embodiments, the cell comprises over-expression or increased expression of one or more complement inhibitors. In some embodiments, NK cells can be engineered with a Chimeric Antigen Receptor (CAR), including any chimeric antigen receptor as described herein. In some embodiments, engineered (e.g., low immunogenicity) NK cells can be used to treat a variety of indications using allogeneic cell therapy, including any of the indications as described herein (e.g., section IV). In some embodiments, engineered (e.g., low immunogenicity) NK cells can be used to treat cancer.

In some embodiments, the cells are iPSC-derived beta islet cells engineered to contain the modifications described herein (e.g., genetic modifications). In some embodiments, the cell is a primary beta islet cell engineered to contain the modifications described herein (e.g., genetic modifications). In some embodiments, the cells comprise reduced or eliminated CD142 expression. In some embodiments, the cell comprises over-expression or increased expression of one or more complement inhibitors. In some embodiments, engineered (e.g., low immunogenicity) beta islet cells can be used to treat a variety of indications using allogeneic cell therapy, including any of the indications as described herein (e.g., section IV). In some embodiments, engineered (e.g., low immunogenic) beta islet cells can be used to treat diabetes, such as type I diabetes.

In some embodiments, the cells are iPSC-derived endothelial cells engineered to contain the modifications described herein (e.g., genetic modifications). In some embodiments, the cells are primary endothelial cells that are engineered to contain the modifications described herein (e.g., genetic modifications). In some embodiments, the cells comprise reduced or eliminated CD142 expression. In some embodiments, the cell comprises over-expression or increased expression of one or more complement inhibitors. In some embodiments, engineered (e.g., low immunogenicity) endothelial cells can be used to treat a variety of indications using allogeneic cell therapy, including any of the indications as described herein (e.g., section IV). In some embodiments, engineered (e.g., low immunogenicity) endothelial cells may be used to treat angiogenic diseases or ocular diseases.

In some embodiments, the cell is an iPSC-derived epithelial cell engineered to contain the modifications described herein (e.g., genetic modifications). In some embodiments, the cell is a primary epithelial cell engineered to contain the modifications described herein (e.g., genetic modifications). In some embodiments, the epithelial cell is an RPE. In some embodiments, the epithelial cell is a thyroid cell. In some embodiments, the epithelial cell is a skin cell. In some embodiments, the cells comprise reduced or eliminated CD142 expression. In some embodiments, the cell comprises over-expression or increased expression of one or more complement inhibitors. In some embodiments, engineered (e.g., low immunogenicity) epithelial cells can be used to treat a variety of indications using allogeneic cell therapy, including any of the indications as described herein (e.g., section IV). In some embodiments, engineered (e.g., low immunogenicity) epithelial cells may be used to treat thyroid disorders or skin disorders.

In some embodiments, the cell is an iPSC-derived hepatocyte engineered to contain the modifications described herein (e.g., genetic modifications). In some embodiments, the cell is a primary hepatocyte that is engineered to contain the modifications described herein (e.g., genetic modifications). In some embodiments, the cells comprise reduced or eliminated CD142 expression. In some embodiments, the cell comprises over-expression or increased expression of one or more complement inhibitors. In some embodiments, engineered (e.g., low immunogenicity) epithelial cells can be used to treat a variety of indications using allogeneic cell therapy, including any of the indications as described herein (e.g., section IV). In some embodiments, engineered (e.g., low immunogenicity) hepatocytes may be used to treat liver diseases.

In some embodiments, the engineered or modified cell as provided herein is a cell from a healthy subject (such as a subject that is unknown or not suspected of having a particular disease or disorder to be treated). For example, if beta islet cells are isolated or obtained from a donor subject, such as for treating diabetes, the donor subject is a healthy subject if the subject is not known or suspected of having diabetes or another disease or condition.

5. Primary cells

In some embodiments, an engineered cell as provided herein comprises a cell derived from a primary cell obtained or isolated from one or more individual subjects or donors. In some embodiments, the cells are derived from a pool of isolated primary cells obtained from one or more (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more) different donor subjects. In some embodiments, primary cells isolated or obtained from a plurality of different donor subjects (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more) are pooled into a batch and engineered according to the provided methods.

In some embodiments, the primary cells are from a primary cell pool of one or more donor subjects that are different from the recipient subject (e.g., the patient to whom the cells are administered). Primary cells can be obtained from 1,2,3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more donor subjects and pooled together. Primary cells may be obtained from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 50 or more, or 100 or more donor subjects and pooled together. In some embodiments, the primary cells are harvested from one or more individuals, and in some cases, the primary T cells or primary T cell bank are cultured in vitro. In some embodiments, a primary cell or primary T cell bank is engineered or modified according to the methods provided herein.

In some embodiments, the methods comprise obtaining or isolating a desired type of primary cell (e.g., T cell, NK cell, endothelial cell, beta islet cell, hepatocyte, or other primary cell as described herein) from an individual donor subject, pooling the cells to obtain a batch of primary cell types, and engineering the cells by the methods provided herein. In some embodiments, the methods comprise obtaining or isolating primary cells of a desired type (e.g., T cells, NK cells, endothelial cells, beta islet cells, hepatocytes, or other primary cells as described herein), engineering the cells of each individual donor by the methods provided herein, and pooling the engineered (modified) cells of at least two individual samples to obtain an engineered cell of a population of primary cell types.

In some embodiments, the primary cells are isolated or obtained from an individual or a primary cell bank isolated or obtained from more than one individual donor. The primary cells may be any type of primary cells described herein, including any type described in section ii.c.3. In some embodiments, the primary cell is selected from T cells, NK cells, beta islet cells, endothelial cells, epithelial cells such as RPE, thyroid cells, skin cells, or liver cells. In some embodiments, primary cells from an individual donor or an individual donor pool are engineered to contain the modifications (e.g., genetic modifications) described herein.

6. Generation of induced pluripotent stem cells

In some embodiments, the engineered cell as provided herein is an induced pluripotent stem cell or an engineered cell derived from or differentiated from an induced pluripotent stem cell. The generation of mouse and human pluripotent stem cells (commonly referred to as iPSCs; miPSC for murine cells or hiPSCs for human cells) is generally known in the art. As will be appreciated by those skilled in the art, there are a number of different methods for generating ipscs. Initial induction was performed in mouse embryonic or adult fibroblasts using viral introduction of the four transcription factors Oct3/4, sox2, c-Myc and Klf 4; see Takahashi and YAMANAKA CELL 126:663-676 (2006), which are hereby incorporated by reference in their entirety, particularly the techniques outlined therein. Since then, a number of methods have been developed; see Seki et al, world J.stem Cells 7 (1): 116-125 (2015) for review, and Lakshmidathy and Vermuri editions, methods in Molecular Biology: pluripotent STEM CELLS, methods and Protocols, springer 2013, which are hereby expressly incorporated by reference in their entirety, in particular for methods for generating hiPSCs (see, e.g., chapter 3 of the latter reference).

Typically, ipscs are produced by transiently expressing one or more reprogramming factors in a host cell, which are typically introduced using episomal vectors. Under these conditions, a small number of cells were induced to iPSC (generally, this step is inefficient because no selection markers are used). Once cells are "reprogrammed" and become pluripotent, they lose episomal vector and use endogenous genes to produce factors.

As will also be appreciated by those skilled in the art, the number of reprogramming factors that may be used or that are used may vary. In general, when fewer reprogramming factors are used, the efficiency of the cell to convert to a pluripotent state is reduced, as is the "pluripotency", e.g., fewer reprogramming factors may result in the cell not being fully pluripotent, but may only be able to differentiate into fewer cell types.

In some embodiments, a single reprogramming factor OCT4 is used. In other embodiments, two reprogramming factors OCT4 and KLF4 are used. In other embodiments, three reprogramming factors OCT4, KLF4, and SOX2 are used. In other embodiments, four reprogramming factors OCT4, KLF4, SOX2, and c-Myc are used. In other embodiments, a member selected from SOKMNLT; 5, 6 or 7 reprogramming factors for SOX2, OCT4 (POU 5F 1), KLF4, MYC, NANOG, LIN, and SV40L T antigens. Generally, these reprogramming factor genes are provided on episomal vectors, such as are known in the art and commercially available.

In some embodiments, the host cell used to transfect the one or more reprogramming factors is a non-pluripotent stem cell. Generally, ipscs are prepared from non-pluripotent cells (such as, but not limited to, blood cells, fibroblasts, etc.) by transiently expressing reprogramming factors as described herein, as is known in the art. In some embodiments, the non-pluripotent cells (such as fibroblasts) are obtained or isolated from one or more individual subjects or donors prior to reprogramming the cells. In some embodiments, ipscs are prepared from an isolated pool of non-pluripotent stem cells (e.g., obtained from one or more (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more) different donor subjects). In some embodiments, non-pluripotent cells (such as fibroblasts) are isolated or obtained from a plurality of different donor subjects (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more), pooled into a batch, reprogrammed to an iPSC, and engineered according to the provided methods.

In some embodiments, ipscs are derived from one or more donor subjects that are different from the recipient subject (e.g., a patient to whom the cells are administered), such as by transiently transfecting one or more reprogramming factors into cells from a pool of non-pluripotent cells (e.g., fibroblasts). Non-pluripotent cells (e.g., fibroblasts) to be induced as ipscs can be obtained from 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more donor subjects and pooled together. Non-pluripotent cells (e.g., fibroblasts) can be obtained from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 50 or more, or 100 or more donor subjects and pooled together. In some embodiments, non-pluripotent cells (e.g., fibroblasts) are harvested from one or more individuals, and in some cases, the non-pluripotent cells (e.g., fibroblasts) or a pool of non-pluripotent cells (e.g., fibroblasts) are cultured in vitro and transfected with one or more reprogramming factors to induce production of ipscs. In some embodiments, a library of non-pluripotent cells (e.g., fibroblasts) or non-pluripotent cells (e.g., fibroblasts) is engineered or modified according to the methods provided herein. In some embodiments, the engineered iPSC or the library of engineered ipscs is then subjected to a differentiation process to differentiate into any cells of the organism and tissue.

Once the engineered iPSC cells have been generated, their low immunogenicity and/or multipotent retention can be determined as described in WO2016183041 and WO 2018132783. In some embodiments, low immunogenicity is determined using a variety of techniques as exemplified in fig. 13 and 15 of WO 2018132783. These techniques include transplantation into an allogeneic host and monitoring the growth of low-immunogenicity pluripotent cells (e.g., teratomas) that escape the host's immune system. In some cases, the low immunogenicity multipotent cell derivative is transduced to express luciferase, which can then be tracked using bioluminescence imaging. Similarly, the host animal is tested for T cell and/or B cell responses to such cells to confirm that the cells do not elicit an immune response in the host animal. T cell responses were assessed by Elispot, ELISA, FACS, PCR or mass flow Cytometry (CYTOF). FACS or Luminex was used to assess B cell responses or antibody responses. Additionally or alternatively, the ability of a cell to avoid an innate immune response (e.g., NK cell killing) may be determined, as generally shown in fig. 14 and 15 of WO 2018132783.

In some embodiments, the immunogenicity of the cells is assessed using T cell immunoassays (such as T cell proliferation assays, T cell activation assays, and T cell killing assays) that are recognized by those of skill in the art. In some cases, the T cell proliferation assay comprises pre-treating cells with interferon-gamma and co-culturing the cells with labeled T cells, and determining the presence of a T cell population (or a proliferated T cell population) after a preselected amount of time. In some cases, the T cell activation assay comprises co-culturing T cells with the cells outlined herein, and determining the level of expression of the T cell activation marker in the T cells.

In vivo assays can be performed to assess the immunogenicity of the cells outlined herein. In some embodiments, an allogeneic humanized immunodeficiency mouse model is used to determine the viability and immunogenicity of the engineered or modified iPSC cells. In some cases, engineered or modified ipscs were transplanted into allogeneic humanized NSG-SGM3 mice and assayed for cell rejection, cell survival, and teratoma formation. In some cases, the implanted engineered ipscs or differentiated cells thereof exhibit long-term survival in a mouse model.

Additional techniques for determining immunogenicity, including low immunogenicity of cells, are described, for example, in Deuse et al, nature Biotechnology,2019,37,252-258 and Han et al, proc NATL ACAD SCI USA,2019,116 (21), 10441-10446, the disclosures of which including figures, descriptions of figures and descriptions of methods are incorporated herein by reference in their entirety.

Similarly, the pluripotency retention is tested in a variety of ways. In one embodiment, pluripotency is determined by expression of certain pluripotency-specific factors, as generally described herein and shown in figure 29 of WO 2018132783. Additionally or alternatively, differentiation of pluripotent cells into one or more cell types is used as an indication of pluripotency.

Once the engineered pluripotent stem cells have been generated (engineered ipscs), they can be maintained in an undifferentiated state, which is known for maintaining ipscs. For example, cells can be cultured on matrigel using a medium that prevents differentiation and maintains pluripotency. In addition, the cells may be in a culture medium under conditions that maintain pluripotency.

Any of the pluripotent stem cells described herein can differentiate into any cell of an organism or tissue. In one aspect, provided herein are engineered cells differentiated from ipscs into different cell types for subsequent transplantation into a recipient subject. Differentiation can be determined as known in the art, typically by assessing the presence of cell-specific markers. As will be appreciated by those skilled in the art, the differentiated engineered (e.g., low immunogenicity) pluripotent cell derivatives can be transplanted using techniques known in the art, depending on the cell type and the end use of the cells. Exemplary types of differentiated cells and methods for producing the same are described below. In some embodiments, ipscs can differentiate into any of the types of cells described herein, including any of the types described in section ii.c.3. In some embodiments, ipscs differentiate into cell types selected from T cells, NK cells, beta islet cells, endothelial cells, epithelial cells such as RPE, thyroid cells, skin cells, or hepatocytes. In some embodiments, host cells, such as non-pluripotent cells (e.g., fibroblasts) from an individual donor or an individual donor pool, are isolated or obtained to produce ipscs, wherein the ipscs are subsequently engineered to contain the modifications described herein (e.g., genetic modifications) and then differentiated into a desired cell type.

7. Cell type

O.beta.islet cells

In some embodiments, the engineered or modified cell as provided herein is a primary beta islet cell (also referred to as a pancreatic islet cell or pancreatic beta cell). In some embodiments, the primary β islet cells are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donors (e.g., subjects that are unknown or not suspected of having a disease or infection (e.g., that do not exhibit clinical signs of a disease or infection). As will be appreciated by those skilled in the art, the method of isolating or obtaining beta islet cells from an individual may be accomplished using known techniques. Provided herein are engineered primary beta islet cells containing a modification (e.g., a genetic modification) described herein for subsequent transplantation or implantation into a subject (e.g., a recipient).

In some embodiments, the beta islet cells are obtained (e.g., harvested, extracted, removed, or obtained) from a subject or individual. In some embodiments, the primary β islet cells are generated from a β islet cell bank such that the β islet cells are from one or more subjects (e.g., one or more humans, including one or more healthy humans). In some embodiments, the primary β islet cell bank is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments, the donor subject is different from the patient (e.g., the recipient to whom the therapeutic cells are administered). In some embodiments, the beta islet cell bank does not include cells from the patient. In some embodiments, one or more donor subjects from which the beta islet cell bank is obtained are different from the patient.

In some embodiments, the cells as provided herein are beta islet cells derived from engineered ipscs, which contain the modifications described herein (e.g., genetic modifications) and differentiate into beta islet cells. As will be appreciated by those skilled in the art, the method used for differentiation depends on the desired cell type using known techniques. In some embodiments, cells differentiated into various beta islet cells can be used for subsequent transplantation or implantation into a subject (e.g., a recipient). In some embodiments, pancreatic islet cells are derived from the engineered pluripotent cells described herein. Useful methods for differentiating pluripotent stem cells into beta islet cells are described, for example, in U.S. patent No. 9,683,215; U.S. patent No. 9,157,062; U.S. patent No. 8,927,280; U.S. patent publication No. 2021/0207099; hogrebe et al ,"Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells,"Nat.Biotechnol.,2020,38:460-470; and Hogrebe et al ,"Generation of insulin-producing pancreatic beta cells from multiple human stem cell lines,"Nat.Protoc.,2021, are incorporated herein by reference in their entirety.

In some embodiments, the engineered pluripotent cells described herein differentiate into β -like cells or islet organoids for transplantation to address type I diabetes (T1 DM). Cellular systems are a promising approach to address T1DM, see for example Ellis et al, nat Rev Gastroenterol hepatol.2017, month 10; 14 612-628, which are incorporated herein by reference. Furthermore, pagliuca et al (Cell, 2014,159 (2): 428-39), the contents of which are incorporated herein by reference in their entirety, report successful differentiation of beta cells from hipscs, particularly the methods and reagents for large-scale production of functional human beta cells from human pluripotent stem cells as outlined therein. Furthermore, vegas et al show that human beta cells are produced from human pluripotent stem cells and then encapsulated to avoid immune rejection by the host; vegas et al, nat Med,2016,22 (3): 306-11, which is incorporated herein by reference in its entirety, particularly the methods and reagents for large-scale production of functional human beta cells from human pluripotent stem cells as outlined therein.

In some embodiments, a method of producing an engineered pancreatic islet cell population from an engineered pluripotent cell population by in vitro differentiation comprises: (a) Culturing an engineered iPSC population in a first medium to produce an immature pancreatic islet cell population, the medium comprising one or more factors selected from the group consisting of: insulin-like growth factors, transforming growth factors, FGF, EGF, HGF, SHH, VEGF, transforming growth factor-b superfamily, BMP2, BMP7, GSK inhibitor, ALK inhibitor, BMP type 1 receptor inhibitor, and retinoic acid; and (b) culturing the population of immature pancreatic islet cells in a second medium, different from the first medium, to produce the population of engineered pancreatic islet cells. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of GSK inhibitor ranges from about 2mM to about 10mM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or variant thereof. In some cases, the concentration of ALK inhibitor ranges from about 1pM to about 10pM. In some embodiments, the first medium and/or the second medium is free of animal serum.

Differentiation is typically determined by assessing the presence of beta cell-related or specific markers, including but not limited to insulin, as is known in the art. Differentiation can also be measured functionally, such as measuring glucose metabolism, see generally Muraro et al, cell syst.2016, 10, 26; 3 (4): 385-394.E3, which is hereby incorporated by reference in its entirety, particularly the biomarkers outlined therein. Once the beta cells are produced, they can be transplanted (as a cell suspension or within a gel matrix as discussed herein) into the portal vein/liver, omentum, gastrointestinal mucosa, bone marrow, muscle, or subcutaneous sac.

Additional description including pancreatic islet cells for use in the present technology can be found in WO2020/018615, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, an engineered β islet cell population, such as primary β islet cells isolated from one or more individual donors (e.g., healthy donors) or endothelial cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), is maintained in a medium, in some cases expanded prior to administration. In certain embodiments, the engineered β islet cell population is cryopreserved prior to administration.

Exemplary pancreatic islet cell types include, but are not limited to, pancreatic islet progenitor cells, immature pancreatic islet cells, mature pancreatic islet cells, and the like. In some embodiments, pancreatic cells described herein are administered to a subject to treat diabetes.

In some embodiments, engineered pancreatic islet cells as disclosed herein, such as primary beta islet cells isolated from one or more individual donors (e.g., healthy donors) or beta islet cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), secrete insulin. In some embodiments, pancreatic islet cells exhibit at least two characteristics of endogenous pancreatic islet cells, such as, but not limited to, secretion of insulin and expression of a beta cell marker in response to glucose.

Exemplary β cell markers or β cell progenitor cell markers include, but are not limited to, c-peptide, pdxl, glucose transporter 2 (Glut 2), HNF6, VEGF, glucokinase (GCK), prohormone convertase (PC 1/3), cdcpl, neuroD, ngn3, nkx2.2, nkx6.1, nkx6.2, pax4, pax6, ptfla, isll, sox9, sox17, and FoxA2.

In some embodiments, pancreatic islet cells, such as primary beta islet cells isolated from one or more individual donors (e.g., healthy donors) or beta islet cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), produce insulin in response to an increase in glucose. In various embodiments, pancreatic islet cells secrete insulin in response to increased glucose. In some embodiments, the cells have a unique morphology, such as a cobblestone cell morphology and/or a diameter of about 17pm to about 25 pm.

In some embodiments, the present technology relates to the expression of or lack of engineered beta islet cells, such as primary beta islet cells isolated from one or more individual donors (e.g., healthy donors) or beta islet cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), that overexpress tolerogenic factors (e.g., CD 47), have reduced expression of or lack of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigens and/or one or more MHC class II human leukocyte antigens), and have reduced expression of CD 142. In some embodiments, the beta islet cells further express one or more complement inhibitors. In certain embodiments, the engineered β islet cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications in the B2M gene and have reduced CD142 expression. In some embodiments, the beta islet cells further express one or more complement inhibitors. In some embodiments, the engineered β islet cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications in the CIITA gene and have reduced CD142 expression. In some embodiments, the beta islet cells further express one or more complement inhibitors. In some embodiments, the beta islet cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications that disrupt one or more of the following genes: B2M, CIITA and CD142 genes.

In some embodiments, the engineered β islet cells provided evade immune recognition. In some embodiments, engineered beta islet cells described herein, such as primary beta islet cells isolated from one or more individual donors (e.g., healthy donors) or beta islet cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), do not activate an immune response in a patient (e.g., post-administration recipient). Methods of treating a disease by administering an engineered beta islet cell population described herein to a subject (e.g., a recipient) or patient in need thereof are provided.

In some embodiments, the dose of the cell number administered is lower than the dose required for an immunogenic cell (e.g., a population of cells of the same or similar cell type or phenotype of an engineered cell but without modification (e.g., genetic modification), e.g., having endogenous levels of CD142, MHC class I, and/or MHC class II expression and without increased (e.g., exogenous) CD47 expression). In some embodiments, the dose of the cell number administered is lower than the dose required for the immunogenic cells (e.g., the same or similar cell type or phenotype of the engineered cells but without modification (e.g., genetic modification) of the cell population, e.g., with endogenous levels of CD142, MHC class I and/or MHC class II expression and without increased (e.g., exogenous) CD47 expression) to reduce immune rejection.

P. liver cells

In some embodiments, the engineered or modified cell as provided herein is a primary hepatocyte. In some embodiments, the primary hepatocytes are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donors (e.g., subjects that are unknown or not suspected of having a disease or infection (e.g., that do not exhibit clinical signs of a disease or infection)). As will be appreciated by those skilled in the art, the method of isolating or obtaining hepatocytes from an individual may be accomplished using known techniques. Provided herein are engineered primary hepatocytes containing modifications (e.g., genetic modifications) described herein for subsequent transplantation or implantation into a subject (e.g., a recipient). In some embodiments, the engineered primary hepatocytes may be administered as a cell therapy to address loss of hepatocyte function or cirrhosis.

In some embodiments, the primary hepatocytes are obtained (e.g., harvested, extracted, removed, or otherwise obtained) from a subject or individual. In some embodiments, the primary hepatocytes are generated from a hepatocyte pool such that the hepatocytes are from one or more subjects (e.g., one or more humans, including one or more healthy humans). In some embodiments, the primary hepatocyte pool is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments, the donor subject is different from the patient (e.g., the recipient to whom the therapeutic cells are administered). In some embodiments, the hepatocyte pool does not include cells from the patient. In some embodiments, one or more donor subjects from which the hepatocyte pool is obtained are different from the patient.

In some embodiments, the cells as provided herein are hepatocytes differentiated from engineered ipscs, which contain the modifications described herein (e.g., genetic modifications) and differentiate into hepatocytes. As will be appreciated by those skilled in the art, the method used for differentiation depends on the desired cell type using known techniques. In some embodiments, cells differentiated into hepatocytes may be used for subsequent transplantation or implantation into a subject (e.g., a recipient). In some embodiments, engineered hepatocytes differentiated from pluripotent stem cells may be administered as cell therapies to address hepatocyte loss of function or cirrhosis.

In some embodiments, an engineered pluripotent cell containing a modification described herein is differentiated into a hepatocyte. There are a number of techniques that can be used to differentiate engineered pluripotent cells into hepatocytes; see, e.g., pettinato et al, doi:10.1038/spre32888, snykers et al, methods Mol Biol,2011 698:305-314, si-Tayeb et al, hepatology,2010,51:297-305 and Asgari et al, STEM CELL REV,2013,9 (4): 493-504, all of which are incorporated herein by reference in their entirety, particularly for Methods and reagents for differentiation. Differentiation may be determined, as known in the art, typically by assessing the presence of hepatocyte-related and/or specific markers including, but not limited to, albumin, alpha fetoprotein and fibrinogen. Differentiation can also be measured functionally (such as ammonia metabolism, LDL storage and uptake, ICG uptake and release, and glycogen storage).

In some embodiments, an engineered population of hepatocytes, such as primary hepatocytes isolated from one or more individual donors (e.g., healthy donors) or hepatocytes differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), is maintained in a medium, in some cases expanded prior to administration. In certain embodiments, the population of hepatocytes is cryopreserved prior to administration.

In some embodiments, the present technology relates to the expression of or lack of engineered hepatocytes, such as primary hepatocytes isolated from one or more individual donors (e.g., healthy donors) or hepatocytes differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), that overexpress tolerogenic factors (e.g., CD 47), and have reduced expression of or lack of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules), and have reduced expression of CD 142. In some embodiments, the hepatocyte also expresses one or more complement inhibitors. In certain embodiments, the engineered hepatocyte over-expresses tolerogenic factors (e.g., CD 47) and has genomic modifications in the B2M gene and reduced CD142 expression. In some embodiments, the hepatocyte also expresses one or more complement inhibitors. In some embodiments, the engineered hepatocyte over-expresses tolerogenic factors (e.g., CD 47) and has genomic modifications in the CIITA gene. In some embodiments, the engineered hepatocyte has reduced CD142 expression. In some embodiments, the hepatocyte also expresses one or more complement inhibitors. In some embodiments, the engineered hepatocyte overexpresses tolerogenic factors (e.g., CD 47) with genomic modifications that disrupt one or more of the following genes: B2M and CIITA genes, and has reduced CD142 expression. In some embodiments, the hepatocyte also expresses one or more complement inhibitors.

In some embodiments, the engineered hepatocytes provided evade immune recognition. In some embodiments, the engineered hepatocytes described herein, such as primary hepatocytes isolated from one or more individual donors (e.g., healthy donors) or hepatocytes differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), do not activate an immune response in a patient (e.g., post-administration recipient). Methods of treating a disease by administering an engineered hepatocyte population described herein to a subject (e.g., a recipient) or patient in need thereof are provided.

Q.T cells

In some embodiments, the engineered or modified cell as provided herein is a primary T lymphocyte (also referred to as a T cell). In some embodiments, primary T lymphocytes are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donors (e.g., subjects that are unknown or not suspected of having a disease or infection (e.g., that do not exhibit clinical signs of a disease or infection). In some cases, the T cells are a population or subpopulation of primary T cells from one or more individuals. As will be appreciated by those skilled in the art, the method of isolating or obtaining T lymphocytes from an individual may be accomplished using known techniques. Provided herein are engineered primary T lymphocytes that contain a modification (e.g., a genetic modification) described herein for subsequent transplantation or implantation into a subject (e.g., a recipient).

In some embodiments, the primary T cells are obtained (e.g., harvested, extracted, removed, or obtained) from a subject or individual. In some embodiments, the primary T cells are generated from a T cell pool such that the T cells are from one or more subjects (e.g., one or more humans, including one or more healthy humans). In some embodiments, the primary T cell repertoire is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments, the donor subject is different from the patient (e.g., the recipient to whom the therapeutic cells are administered). In some embodiments, the T cell repertoire does not include cells from a patient. In some embodiments, one or more donor subjects from which the T cell repertoire is obtained are different from the patient.

In some embodiments, the cells as provided herein are T lymphocytes differentiated from an engineered pluripotent cell, which contain a modification described herein (e.g., a genetic modification) and differentiate into T lymphocytes. As will be appreciated by those skilled in the art, the method used for differentiation depends on the desired cell type using known techniques. In some embodiments, cells differentiated into T lymphocytes can be used for subsequent transplantation or implantation into a subject (e.g., a recipient).

Methods for generating T cells from pluripotent stem cells (e.g., ipscs) are described, for example, in Iriguchi et al, nature Communications, 430 (2021); themeli et al 16 (4): 357-366 (2015); themeli et al, nature Biotechnology, 31:928-933 (2013).

Non-limiting examples of primary T cells include cd3+ T cells, cd4+ T cells, cd8+ T cells, non-primed T cells, regulatory T (Treg) cells, non-regulatory T cells, th1 cells, th2 cells, th9 cells, th17 cells, T follicular helper (Tfh) cells, cytotoxic T Lymphocytes (CTLs), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem) cells, effector memory T cells expressing CD45RA (TEMRA) cells, tissue resident memory (Trm) cells, virtual memory T cells, innate memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cells. In some embodiments, the primary T cells are selected from the group consisting of cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tumor infiltrating lymphocytes, and combinations thereof.

Exemplary T cells of the disclosure are selected from the group consisting of: cytotoxic T cells, helper T cells, memory T cells, central memory T cells, effector memory RA T cells, regulatory T cells, tissue infiltrating lymphocytes, and combinations thereof. In many embodiments, the T cells express CCR7, CD27, CD28, and CD45RA. In some embodiments, the central T cell expresses CCR7, CD27, CD28, and CD45RO. In other embodiments, effector memory T cells express PD-1, CD27, CD28, and CD45RO. In other embodiments, effector memory RA T cells express PD-1, CD57, and CD45RA.

In some embodiments, an engineered T cell described herein, such as a primary T cell isolated from one or more individual donors (e.g., healthy donors) or a T cell differentiated from an iPSC (derived from one or more individual donors (e.g., healthy donors)), comprises a T cell engineered (e.g., modified) to express a chimeric antigen receptor, including but not limited to the chimeric antigen receptors described herein. Any suitable CAR may be included in the T cell, including the CARs described herein. In some embodiments, the engineered T-cells express at least one chimeric antigen receptor that binds to an antigen or epitope of interest expressed on the surface of at least one of the following cells: damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutant cells, and combinations thereof. In other cases, the engineered T-cells comprise modifications that cause the cells to express at least one protein that modulates a biological effect of interest in an adjacent cell, tissue or organ when the cell is in proximity to the adjacent cell, tissue or organ. Modifications useful for T cells, including primary T cells, are described in detail in U.S. Pat. No. 2016/0348073 and WO2020/018620, the disclosures of which are incorporated herein in their entirety.

In some embodiments, the T cell comprises a polynucleotide encoding a CAR, wherein the polynucleotide is inserted into a genomic locus. The CAR can be inserted into the genomic locus of the T cell using any suitable method, including lentiviral-based transduction methods or gene editing methods described herein (e.g., CRISPR/Cas system). In some embodiments, the polynucleotide is inserted into a safe harbor locus (such as, but not limited to, AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD 142), MICA, MICB, LRP1 (also known as CD 91), HMGB1, ABO, RHD, FUT1, or KDM5D locus). In some embodiments, the polynucleotide is inserted into the B2M, CIITA, TRAC, TRBC, PD1 or CTLA4 gene.

In some embodiments, a T cell described herein (such as an engineered or modified T cell) comprises reduced expression of an endogenous T cell receptor. In some embodiments, the TRAC or TRBC locus is disrupted or eliminated in the cell, such as by the gene editing methods described herein (e.g., CRISPR/Cas system). In some embodiments, an exogenous polynucleotide or transgene (such as a polynucleotide encoding a CAR or the other polynucleotide) is inserted into the disrupted TRAC or TRBC locus.

In some embodiments, the T cells described herein, such as engineered or modified T cells, comprise reduced expression of cytotoxic T lymphocyte-associated protein 4 (CTLA 4). In some embodiments, the CTLA-4 locus is disrupted or eliminated in the cell, such as by a gene editing method described herein (e.g., CRISPR/Cas system). In some embodiments, an exogenous polynucleotide or transgene (such as a polynucleotide encoding a CAR or the other exogenous polynucleotide) is inserted into the disrupted CTLA-4 locus.

In other embodiments, a T cell described herein, such as an engineered or modified T cell, comprises reduced expression of programmed cell death (PD 1). In some embodiments, the PD1 locus is disrupted or eliminated in the cell, such as by a gene editing method described herein (e.g., CRISPR/Cas system). In some embodiments, an exogenous polynucleotide or transgene (such as a polynucleotide encoding a CAR or the other exogenous polynucleotide) is inserted into the disrupted PD1 locus. In certain embodiments, T cells described herein (such as engineered or modified T cells) comprise reduced expression of CTLA4 and PD 1.

In certain embodiments, a T cell described herein (such as an engineered or modified T cell) comprises enhanced expression of PD-L1. In some embodiments, the PD-L1 locus is disrupted or eliminated in the cell, such as by a gene editing method described herein (e.g., CRISPR/Cas system). In some embodiments, an exogenous polynucleotide or transgene (such as a polynucleotide encoding a CAR or the other exogenous polynucleotide) is inserted into the disrupted PD-L1 locus.

In some embodiments, the present technology relates to the expression of or lack of engineered T cells, such as primary T cells isolated from one or more individual donors (e.g., healthy donors) or T cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), that overexpress tolerogenic factors (e.g., CD 47), and have reduced expression of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules), and have reduced expression of CD 142. In some embodiments, the engineered T cells also express one or more complement inhibitors. In certain embodiments, the engineered T cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications in the B2M gene with reduced CD142 expression. In some embodiments, the engineered T cells also express one or more complement inhibitors. In some embodiments, the engineered T cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications in the CIITA gene and have reduced CD142 expression. In some embodiments, the engineered T cells also express one or more complement inhibitors. In some embodiments, the engineered T cells are also engineered to express a CAR. In some embodiments, the engineered T cells have reduced expression or lack thereof of a TCR complex molecule, such as by genomic modification (e.g., gene disruption) in the TRAC gene or the TRBC gene. In some embodiments, the T cells overexpress tolerogenic factors (e.g., CD 47) and CARs, and have genomic modifications that disrupt one or more of the following genes: B2M, CIITA, TRAC and TRBC genes, and has reduced CD142 expression. In some embodiments, the engineered T cells also express one or more complement inhibitors.

In some embodiments, an engineered T cell is provided that evades immune recognition. In some embodiments, an engineered T cell described herein, such as a primary T cell isolated from one or more individual donors (e.g., healthy donors) or a T cell differentiated from an iPSC (derived from one or more individual donors (e.g., healthy donors)), does not activate an immune response in a patient (e.g., post-administration recipient). Methods of treating a disease by administering an engineered T cell population described herein to a subject (e.g., a recipient) or patient in need thereof are provided.

T cells provided herein can be used to treat suitable cancers, including but not limited to B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous lymphoblastic leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

R, natural killer cells

In some embodiments, the engineered or modified cell as provided herein is a Natural Killer (NK) cell. In some embodiments, NK cells are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donors (e.g., subjects that are unknown or not suspected of having a disease or infection (e.g., that do not exhibit clinical signs of a disease or infection)). In some cases, the NK cells are NK cell populations or subpopulations from one or more individuals. As will be appreciated by those skilled in the art, the method of isolating or obtaining NK cells from an individual may be accomplished using known techniques. Provided herein are engineered primary NK cells containing a modification (e.g., a genetic modification) described herein for subsequent transplantation or implantation into a subject (e.g., a recipient). For example, the engineered T cells are administered to a subject (e.g., a recipient such as a patient) by infusing the engineered NK cells into the subject.

In some embodiments, the cells as provided herein are NK cells differentiated from engineered pluripotent cells that contain the modifications described herein (e.g., genetic modifications) and differentiate into NK cells. As will be appreciated by those skilled in the art, the method used for differentiation depends on the desired cell type using known techniques. In some embodiments, the cells differentiated into NK cells can be used for subsequent administration to a subject (e.g., a recipient such as a patient), such as by infusion of the differentiated NK cells into the subject.

Methods for generating NK cells from pluripotent stem cells (e.g., ipscs) are described, for example, in U.S. patent No. 10626373; shankar et al STEM CELL RES ter 2020;11:234; euchner et al Frontiers in Immunology,2021;12,Article 640672.doi = 10.3389/fimmu.2021.640672.

In some embodiments, NK cells are obtained (e.g., harvested, extracted, removed, or retrieved) from a subject or individual. In some embodiments, the NK cells are generated from a NK cell bank such that the NK cells are from one or more subjects (e.g., one or more humans, including one or more healthy humans). In some embodiments, the primary NK cell pool is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments, the donor subject is different from the patient (e.g., recipient administered engineered NK cells). In some embodiments, the NK cell bank does not include cells from the patient. In some embodiments, one or more donor subjects from which the NK cell repertoire is obtained are different from the patient.

In some embodiments, NK cells (including primary NK cells isolated from one or more individual donors (e.g., healthy donors) or NK cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)) express CD56 (e.g., CD56 ^dim or CD56 ^bright) and lack CD3 (e.g., CD3 ^neg). In some embodiments, NK cells as described herein may also express the low affinity fcγ receptor CD16 that mediates ADCC. In some embodiments, the NK cells also express one or more natural killer cell receptors NKG2A and NKG2D or one or more natural cytotoxic receptors NKp46, NKp44, NKp30. For example, in the case of primary NK cells, primary cells may be isolated in particular cases from an initial source of NK cells, such as a sample containing Peripheral Blood Mononuclear Cells (PBMCs), by depletion of CD3, CD14 and/or CD19 positive cells. For example, immunomagnetic beads to which antibodies directed against CD3, CD14 and/or CD19, respectively, are attached may be used to deplete cells, thereby generating an enriched NK cell population. In other cases, primary NK cells may be isolated from a starting source that is a mixed population (e.g., PBMCs) by selecting whether the cells are present with one or more markers on NK cells, such as CD56, CD16, NKp46, and/or NKG 2D.

In some embodiments, NK cells (such as isolated primary NK cells) may undergo one or more expansion or activation steps prior to engineering as described herein. In some embodiments, expansion may be achieved by culturing NK cells with feeder cells (such as antigen presenting cells that may or may not be irradiated). The ratio of NK cells to Antigen Presenting Cells (APC) in the expansion step may be a number such as, for example, 1:1, 1:1.5, 1:2 or 1:3. In certain aspects, APCs are engineered to express membrane-bound IL-21 (mblL-21). In particular aspects, APCs can alternatively or additionally be engineered to express IL-21, IL-15, and/or IL-2. In certain embodiments, the medium in which the amplification step occurs comprises one or more agents that promote amplification, such as one or more recombinant cytokines. In particular embodiments, the medium comprises one or more recombinant cytokines from IL-2, IL-15, IL-18, and/or IL-21. In some embodiments, the step of engineering NK cells by introducing a modification as described herein is performed 2-12 days after initiation of amplification, such as at or about day 2, 3,4, 5, 6, 7, 8, 9, 10, 11 or 12, or about day 2, 3,4, 5, 6, 7, 8, 9, 10, 11 or 12.

In some embodiments, an engineered NK cell described herein, such as a primary NK cell isolated from one or more individual donors (e.g., healthy donors), comprises an NK cell engineered (e.g., modified) to express a chimeric antigen receptor, including but not limited to the chimeric antigen receptor described herein. Any suitable CAR may be included in the NK cells, including the CARs described herein. In some embodiments, the engineered NK cells express at least one chimeric antigen receptor that binds to an antigen or epitope of interest expressed on the surface of at least one of the following cells: damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutant cells, and combinations thereof. In other cases, the engineered NK cells comprise modifications that cause the cells to express at least one protein that modulates a biological effect of interest in an adjacent cell, tissue, or organ when the cell is in proximity to the adjacent cell, tissue, or organ.

In some embodiments, the NK cells comprise a polynucleotide encoding a CAR, wherein the polynucleotide is inserted into a genomic locus. The CAR can be inserted into the genomic locus of the NK cell using any suitable method, including lentiviral-based transduction methods or gene editing methods described herein (e.g., CRISPR/Cas system). In some embodiments, the polynucleotide is inserted into a safe harbor locus (such as, but not limited to, AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD 142), MICA, MICB, LRP1 (also known as CD 91), HMGB1, ABO, RHD, FUT1, or KDM5D locus).

In some embodiments, the present technology relates to the expression of or lack of engineered NK cells, such as primary NK cells isolated from one or more individual donors (e.g., healthy donors) or NK cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), that overexpress tolerogenic factors (e.g., CD 47), have reduced expression or lack of expression of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules), and have reduced expression or lack of CD142 expression. In some embodiments, the engineered NK cells express one or more complement inhibitors selected from CD46, CD59, and CD 55. In certain embodiments, the engineered NK cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications in the B2M gene. In some embodiments, the engineered NK cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications in the CIITA gene. In some embodiments, the engineered NK cells are also engineered to express a CAR.

In some embodiments, an engineered NK cell is provided that evades immune recognition. In some embodiments, an engineered NK cell described herein, such as a primary NK cell isolated from one or more individual donors (e.g., healthy donors), does not activate an immune response in a patient (e.g., post-administration recipient). Methods of treating a disease by administering an engineered NK cell population described herein to a subject (e.g., a recipient) or patient in need thereof are provided.

NK cells provided herein can be used to treat suitable cancers, including but not limited to B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous lymphoblastic leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

S, endothelial cells

In some embodiments, the engineered or modified cell as provided herein is a primary endothelial cell. In some embodiments, the primary endothelial cells are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donors (e.g., subjects that are unknown or not suspected of having a disease or infection (e.g., that do not exhibit clinical signs of a disease or infection)). As will be appreciated by those skilled in the art, the method of isolating or obtaining endothelial cells from an individual may be accomplished using known techniques. Provided herein are engineered primary endothelial cell types containing modifications (e.g., genetic modifications) described herein for subsequent transplantation or implantation into a subject (e.g., a recipient).

In some embodiments, the primary endothelial cells are obtained (e.g., harvested, extracted, removed, or otherwise obtained) from the subject or individual. In some embodiments, the primary endothelial cells are produced from a pool of endothelial cells such that the endothelial cells are from one or more subjects (e.g., one or more humans, including one or more healthy humans). In some embodiments, the primary endothelial cell pool is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments, the donor subject is different from the patient (e.g., the recipient to whom the therapeutic cells are administered). In some embodiments, the endothelial cell bank does not include cells from the patient. In some embodiments, one or more donor subjects from which the endothelial cell bank is obtained are different from the patient.

In some embodiments, the cells as provided herein are endothelial cells differentiated from engineered ipscs, which contain the modifications described herein (e.g., genetic modifications) and differentiate into endothelial cell types. As will be appreciated by those skilled in the art, the method used for differentiation depends on the desired cell type using known techniques. In some embodiments, cells differentiated into various endothelial cell types can be used for subsequent transplantation or implantation into a subject (e.g., a recipient).

In some embodiments, the engineered pluripotent cells described herein differentiate into Endothelial Colony Forming Cells (ECFCs) to form new blood vessels, thereby addressing peripheral arterial disease. Techniques for differentiating endothelial cells are known. See, e.g., prasain et al, doi:10.1038/nbt.3048, which is incorporated herein by reference in its entirety, particularly for methods and reagents for the production of endothelial cells from human pluripotent stem cells, as well as for transplantation techniques. Differentiation can be determined, as known in the art, typically by assessing the presence of endothelial cell-related or specific markers or by functional measurements.

In some embodiments, a method of producing an engineered endothelial cell population from an engineered pluripotent cell population by in vitro differentiation comprises: (a) Culturing a population of engineered iPSC cells in a first medium comprising a GSK inhibitor; (b) Culturing the population of engineered iPSC cells in a second medium comprising VEGF and bFGF to produce a population of pre-endothelial cells; and (c) culturing the population of pre-endothelial cells in a third medium comprising a ROCK inhibitor and an ALK inhibitor to produce a population of differentiated endothelial cells engineered to contain the modifications described herein.

In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of GSK inhibitor ranges from about 1mM to about 10mM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some cases, the concentration of ROCK inhibitor ranges from about 1pM to about 20pM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or variant thereof. In some cases, the concentration of ALK inhibitor ranges from about 0.5pM to about 10pM.

In some embodiments, the first medium comprises CHIR-99021 of from 2pM to about 10 pM. In some embodiments, the second medium comprises 50ng/ml VEGF and 10ng/ml bFGF. In other embodiments, the second medium further comprises Y-27632 and SB-431542. In various embodiments, the third medium comprises 10pM Y-27632 and 1pM SB-431542. In certain embodiments, the third medium further comprises VEGF and bFGF. In certain cases, the first medium and/or the second medium is free of insulin.

The cells provided herein can be cultured on a surface (such as a synthetic surface) to support and/or promote differentiation of pluripotent cells into endothelial cells. In some embodiments, the surface comprises a polymeric material including, but not limited to, homopolymers or copolymers of selected one or more acrylate monomers. Non-limiting examples of acrylate monomers and methacrylate monomers include tetra (ethylene glycol) diacrylate, glycerol dimethacrylate, 1, 4-butanediol dimethacrylate, poly (ethylene glycol) diacrylate, di (ethylene glycol) dimethacrylate, tetra (ethylene glycol) dimethacrylate, 1, 6-hexanediol propoxylate diacrylate, neopentyl glycol diacrylate, trimethylolpropane benzoate diacrylate, ethoxylated trimethylolpropane (1 EO/QH) methyl ester, tricyclo [5.2.1.0 ^2,6 ] decane dimethanol diacrylate, neopentyl glycol deoxidizing diacrylate (neopentyl glycol exhoxylate diacrylate) and trimethylolpropane triacrylate. Acrylates are synthesized in a manner known in the art or are obtained from commercial suppliers such as Polysciences, inc., SIGMA ALDRICH, inc.

In some embodiments, endothelial cells may be seeded onto the polymer matrix. In some cases, the polymer matrix is biodegradable. Suitable biodegradable matrices are well known in the art and include collagen-GAGs, collagen, fibrin, PLA, PGA and PLA/PGA copolymers. Additional biodegradable materials include poly (anhydride), poly (hydroxy acid), poly (orthoester), poly (propyl fumarate), poly (caprolactone), polyamides, polyamino acids, polyacetals, biodegradable polycyanoacrylates, biodegradable polyurethanes, and polysaccharides.

Non-biodegradable polymers may also be used. Other non-biodegradable but biocompatible polymers include polypyrrole, polyaniline, polythiophene, polystyrene, polyester, non-biodegradable polyurethane, polyurea, poly (ethylene vinyl acetate), polypropylene, polymethacrylate, polyethylene, polycarbonate, and poly (ethylene oxide). The polymer matrix may be formed into any shape, such as particles, sponges, tubes, spheres, wires, wound wires, capillary networks, films, fibers, webs, or sheets. The polymer matrix may be modified to include natural or synthetic extracellular matrix materials and factors.

The polymeric material may be dispersed on the surface of the support material. Useful support materials suitable for culturing cells include ceramic substances, glass, plastics, polymers or copolymers, any combination thereof, or coatings of one material on another. In some cases, the glass includes soda lime glass, heat resistant glass, high silica glass, quartz glass, silicon, derivatives of these glasses, or the like.

In some cases, the plastic or polymer comprising the dendritic polymer comprises poly (vinyl chloride), poly (vinyl alcohol), poly (methyl methacrylate), poly (vinyl acetate-maleic anhydride), poly (dimethylsiloxane) monomethacrylate, cyclic olefin polymers, fluorocarbon polymers, polystyrene, polypropylene, polyethyleneimine or derivatives of these, and the like. In some cases, the copolymer includes poly (vinyl acetate-co-maleic anhydride), poly (styrene-co-maleic anhydride), poly (ethylene-co-acrylic acid), derivatives of these, or the like.

Additional description of endothelial cells and differentiation thereof for use in the methods provided herein can be found in WO2020/018615, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, an engineered population of endothelial cells, such as primary endothelial cells isolated from one or more individual donors (e.g., healthy donors) or endothelial cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), is maintained in a medium, and in some cases expanded prior to administration. In certain embodiments, the population of endothelial cells is cryopreserved prior to administration.

In some embodiments, the present technology relates to the expression of or lack of engineered endothelial cells, such as primary endothelial cells isolated from one or more individual donors (e.g., healthy donors) or endothelial cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), that overexpress tolerogenic factors (e.g., CD 47), have reduced expression of or lack of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules), and have reduced expression of CD142. In some embodiments, the endothelial cells also express one or more complement inhibitors. In certain embodiments, the engineered endothelial cells overexpress tolerogenic factors (e.g., CD 47), have genomic modifications in the B2M gene, and have reduced CD142 expression. In some embodiments, the endothelial cells also express one or more complement inhibitors. In some embodiments, the engineered endothelial cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications in the CIITA gene and CD142 gene. In some embodiments, the engineered endothelial cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications that disrupt one or more of the following genes: B2M, CIITA gene and CD142.

In some embodiments, engineered endothelial cells are provided that evade immune recognition. In some embodiments, the engineered endothelial cells described herein, such as primary endothelial cells isolated from one or more individual donors (e.g., healthy donors) or endothelial cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), do not activate an immune response in a patient (e.g., post-administration recipient). Methods of treating a disease by administering an engineered endothelial cell population described herein to a subject (e.g., a recipient) or patient in need thereof are provided.

In some embodiments, engineered endothelial cells, such as primary endothelial cells isolated from one or more individual donors (e.g., healthy donors) or endothelial cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), are administered to a patient (e.g., a human patient in need thereof). The engineered endothelial cells may be administered to a patient suffering from a disease or disorder such as, but not limited to, cardiovascular disease, vascular disease, peripheral vascular disease, ischemic disease, myocardial infarction, congestive heart failure, peripheral vascular occlusive disease, stroke, reperfusion injury, limb ischemia, neuropathy (e.g., peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver failure, kidney failure, etc.), diabetes, rheumatoid arthritis, osteoporosis, vascular injury, tissue damage, hypertension, angina and myocardial infarction caused by coronary artery disease, renal vascular hypertension, renal failure caused by renal arterial stenosis, lameness of the lower limb, and the like. In certain embodiments, the patient has had or is suffering from a transient ischemic attack or stroke, which in some cases may be due to cerebrovascular disease. In some embodiments, the engineered endothelial cells are administered to treat tissue ischemia (e.g., tissue ischemia that occurs in atherosclerosis, myocardial infarction, and limb ischemia), and repair damaged blood vessels. In some cases, the cells are used for bioengineering of the implant.

For example, engineered endothelial cells can be used in cell therapies to repair ischemic tissue, form blood vessels and heart valves, engineer vascular prostheses, repair damaged blood vessels, and induce the formation of blood vessels in engineered tissue (e.g., prior to implantation). In addition, endothelial cells can be further modified to deliver agents to target and treat tumors.

In many embodiments, provided herein is a method of repairing or replacing tissue in need of vascular cells or vascularization. The methods involve administering to a human patient in need of such treatment a composition containing engineered endothelial cells (such as isolated primary endothelial cells or differentiated endothelial cells) to promote angiogenesis in such tissues. The tissue requiring vascular cells or vascularization may be heart tissue, liver tissue, pancreatic tissue, kidney tissue, muscle tissue, nerve tissue, bone tissue, etc., which may be damaged and characterized by excessive cell death, tissue at risk of damage, or artificially engineered tissue.

In some embodiments, vascular diseases that may be associated with heart diseases or conditions may be treated by administering endothelial cells, such as, but not limited to, shaped vascular endothelial cells and endocardial endothelial cells derived as described herein. Such vascular diseases include, but are not limited to, coronary artery disease, cerebrovascular disease, aortic stenosis, aortic aneurysm, peripheral arterial disease, atherosclerosis, varicose veins, vascular disease, heart infarct zone lacking coronary perfusion, non-healing wounds, diabetes or non-diabetic ulcers, or any other disease or condition in which induction of angiogenesis is desired.

In certain embodiments, endothelial cells are used to improve prosthetic implants (e.g., blood vessels made of synthetic materials such as Dacron and Gortex) used in vascular reconstructive surgery. For example, prosthetic arterial implants are commonly used to replace diseased arteries perfusing vital organs or limbs. In other embodiments, engineered endothelial cells are used to cover the surface of the prosthetic heart valve to reduce the risk of embolic formation by making the valve surface less prone to thrombosis.

The outlined endothelial cells may be transplanted into a patient using well known surgical techniques to transplant tissue and/or isolated cells into blood vessels. In some embodiments, the cells are introduced into the heart tissue of the patient by injection (e.g., intramyocardial injection, intracoronary injection, endocardial injection, epicardial injection, percutaneous injection), infusion, transplantation, and implantation.

Administration (delivery) of endothelial cells includes, but is not limited to, subcutaneous or parenteral administration, including intravenous, intra-arterial (e.g., intra-coronary), intramuscular, intraperitoneal, intramyocardial, endocardial, epicardial, intranasal administration, and intrathecal administration, and infusion techniques.

As will be appreciated by those skilled in the art, the cells are transplanted using techniques known in the art, depending on the cell type and the end use of the cells. In some embodiments, the cells provided herein are transplanted to a particular location within a patient intravenously or by injection. When transplanted to a specific location, cells may be suspended in a gel matrix to prevent them from dispersing upon fixation.

Exemplary endothelial cell types include, but are not limited to, capillary endothelial cells, vascular endothelial cells, aortic endothelial cells, arterial endothelial cells, venous endothelial cells, renal endothelial cells, brain endothelial cells, hepatic endothelial cells, and the like.

The endothelial cells (such as isolated primary endothelial cells or differentiated endothelial cells) outlined herein may express one or more endothelial cell markers. Non-limiting examples of such markers include VE-cadherin (CD 144), ACE (angiotensin converting enzyme) (CD 143), BNH9/BNF13, CD31, CD34, CD54 (ICAM-l), CD62E (E-selectin), CD105 (Endoglin), CD146, endocan (ESM-l), endoglyx-l, endostatin (endostatin), eotaxin-3, EPAS1 (endothelial PAS domain protein 1), factor VIII related antigen, FLI-l, flk-l (KDR, VEGFR-2), FLT-l (VEGFR-l), GATA2, GBP-l (guanylate binding protein-l), GRO-alpha, HEX) ICAM-2 (intercellular adhesion molecule 2), LM02, LYVE-l, MRB (magic round robin), nucleolin, PAL-E (patho-anatomical Leiden-endothelium (pathologische anatomie Leiden-endothelium)), RTK, sVCAM-l, TALI, TEM1 (tumor endothelial marker 1), TEM5 (tumor endothelial marker 5), TEM7 (tumor endothelial marker 7), thrombomodulin (TM, CD 141), VCAM-l (vascular cell adhesion molecule-1) (CD 106), VEGF, vWF (Feng Wei Rich factor (von Willebrand factor)), ZO-l, endothelial cell selective adhesion molecule (ESAM), CD102, CD93, CD184, CD304 and DLL4.

In some embodiments, the endothelial cells are further genetically modified to express exogenous genes encoding proteins of interest (such as, but not limited to, enzymes, hormones, receptors, ligands, or drugs) useful in treating or ameliorating symptoms of the disorder/condition. Standard methods for genetically modifying endothelial cells are described, for example, in US5,674,722.

Such endothelial cells can be used to provide constitutive synthesis and delivery of polypeptides or proteins useful in the prevention or treatment of diseases. In this way, the polypeptide is secreted directly into the blood stream or other region of the body (e.g., the central nervous system) of the individual. In some embodiments, endothelial cells may be modified to secrete insulin, clotting factors (e.g., factor VIII or von willebrand factor (von Willebrand Factor)), alpha-l antitrypsin, adenosine deaminase, tissue plasminogen activator, interleukins (e.g., IL-l, IL-2, IL-3), and the like.

In certain embodiments, the endothelial cells may be modified in a manner that improves their performance in the context of the transplanted implant. Non-limiting illustrative examples include secretion or expression of thrombolytic agents to prevent intraluminal clot formation, secretion of smooth muscle proliferation inhibitors to prevent luminal narrowing due to smooth muscle hypertrophy, and expression and/or secretion of endothelial cell mitogens or autocrine factors to stimulate endothelial cell proliferation and improve the extent or duration of implant luminal endothelial cell lining.

In some embodiments, the engineered endothelial cells are used to deliver therapeutic levels of secretory products to a specific organ or limb. For example, an in vitro engineered (transduced) endothelial cell lined vascular implant can be transplanted into a particular organ or limb. The secretory products of the transduced endothelial cells will be delivered to the perfused tissue in high concentrations to achieve the desired effect of targeting the anatomical site.

In other embodiments, the endothelial cells are further genetically modified to contain genes that disrupt or inhibit angiogenesis when expressed by the endothelial cells in the vascularized tumor. In some cases, endothelial cells may also be genetically modified to express any of the selective suicide genes described herein, which allow for negative selection of the implanted endothelial cells after tumor treatment is completed.

In some embodiments, endothelial cells described herein (such as isolated primary endothelial cells or differentiated endothelial cells) are administered to the subject to treat a vascular disorder selected from the group consisting of: vascular injury, cardiovascular disease, vascular disease, peripheral vascular disease, ischemic disease, myocardial infarction, congestive heart failure, peripheral vascular obstructive disease, hypertension, ischemic tissue injury, reperfusion injury, limb ischemia, stroke, neuropathy (e.g., peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver failure, kidney failure, etc.), diabetes, rheumatoid arthritis, osteoporosis, cerebrovascular disease, hypertension, myocardial infarction due to angina pectoris, renal vascular hypertension, renal failure due to renal arterial stenosis, other vascular disorders or diseases.

T. epithelial cells

3) Retinal Pigment Epithelial (RPE) cells

In some embodiments, the engineered or modified cell as provided herein is a primary Retinal Pigment Epithelium (RPE) cell. In some embodiments, the primary RPE cells are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donors (e.g., subjects that are unknown or not suspected of having a disease or infection (e.g., that do not exhibit clinical signs of a disease or infection). As will be appreciated by those skilled in the art, the method of isolating or obtaining RPE cells from an individual may be accomplished using known techniques. Provided herein are engineered primary RPE cells containing a modification (e.g., a genetic modification) described herein for subsequent transplantation or implantation into a subject (e.g., a recipient).

In some embodiments, the primary RPE cells are obtained (e.g., harvested, extracted, removed, or obtained) from a subject or individual. In some embodiments, the primary RPE cells are generated from a pool of RPE cells such that the RPE cells are from one or more subjects (e.g., one or more humans, including one or more healthy humans). In some embodiments, the primary RPE cell pool is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments, the donor subject is different from the patient (e.g., the recipient to whom the therapeutic cells are administered). In some embodiments, the RPE cell bank does not include cells from the patient. In some embodiments, one or more donor subjects from which the RPE cell pool is obtained are different from the patient.

In some embodiments, the cells as provided herein are RPE cells differentiated from engineered ipscs, which contain the modifications described herein (e.g., genetic modifications) and differentiate into RPE cells. As will be appreciated by those skilled in the art, the method used for differentiation depends on the desired cell type using known techniques. In some embodiments, cells differentiated into RPE cells may be used for subsequent transplantation or implantation into a subject (e.g., a recipient).

Useful methods for differentiating pluripotent stem cells into RPE cells are described, for example, in US9,458,428 and US9,850,463, the disclosures of which are incorporated herein by reference in their entirety, including the specification. Additional methods for the production of RPE cells from human induced pluripotent stem cells can be found, for example, in Lamba et al, PNAS,2006,103 (34): 12769-12774; mellough et al, STEM CELLS,2012,30 (4): 673-686; idelson et al, CELL STEM CELL,2009,5 (4): 396-408; rowland et al Journal of Cellular Physiology,2012,227 (2): 457-466, buchholz et al STEM CELLS TRANS MED,2013,2 (5): 384-393 and da Cruz et al, nat Biotech,2018,36:328-337.

Human pluripotent stem cells have been differentiated into RPE cells using the techniques outlined in Kamao et al, stem Cell Reports 2014:2:205-18 (which is hereby incorporated by reference in its entirety, in particular the methods and reagents for differentiation techniques and reagents outlined therein); see also Mandai et al, N Engl J Med,2017,376:1038-1046, the contents of which are incorporated in their entirety for techniques for producing RPE cell sheets and transplanting into patients. Differentiation can be determined, as known in the art, typically by assessing the presence of RPE-related and/or specific markers or by functional measurements. See, e.g., kamao et al, stem Cell Reports,2014,2 (2): 205-18, the contents of which are incorporated by reference in their entirety, particularly the markers outlined in the first paragraph of the results section.

In some embodiments, a method of producing an engineered Retinal Pigment Epithelium (RPE) cell population from an engineered pluripotent cell population by in vitro differentiation comprises: (a) Culturing an engineered pluripotent cell population in a first medium comprising any one factor selected from the group consisting of: activin A, bFGF, BMP/7, DKK1, IGF1, noggin, BMP inhibitor, ALK inhibitor, ROCK inhibitor, and VEGFR inhibitor to produce a population of pre-RPE cells; and (b) culturing the population of pre-RPE cells in a second medium different from the first medium to produce an engineered population of RPE cells. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or variant thereof. In some cases, the concentration of ALK inhibitor ranges from about 2mM to about 10pM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some cases, the concentration of ROCK inhibitor ranges from about 1pM to about 10pM. In some embodiments, the first medium and/or the second medium is free of animal serum.

Differentiation can be determined, as known in the art, typically by assessing the presence of RPE-related and/or specific markers or by functional measurements. See, e.g., kamao et al, stem Cell Reports,2014,2 (2): 205-18, the contents of which are incorporated by reference in their entirety, particularly the results section.

Additional description of RPE cells, including methods for their differentiation and methods used in the present technology, can be found in WO2020/018615, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, an engineered RPE cell population, such as primary RPE cells isolated from one or more individual donors (e.g., healthy donors) or RPE cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), is maintained in culture medium, and in some cases expanded prior to administration. In certain embodiments, the RPE cell population is cryopreserved prior to administration.

Exemplary RPE cell types include, but are not limited to, retinal Pigment Epithelial (RPE) cells, RPE progenitor cells, immature RPE cells, mature RPE cells, functional RPE cells, and the like.

In some embodiments, RPE cells, such as primary RPE cells isolated from one or more individual donors (e.g., healthy donors) or RPE cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), have a similar or substantially similar genetic expression profile as native RPE cells. Such RPE cells may have a polygonal, planar lamellar morphology of native RPE cells when grown to confluence on planar substrates.

In some embodiments, the present technology relates to the expression of or lack of engineered RPE cells, such as primary RPE cells isolated from one or more individual donors (e.g., healthy donors) or RPE cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), that overexpress tolerogenic factors (e.g., CD 47), have reduced expression of or lack of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules), and have reduced expression of CD 142. In some embodiments, the RPE cells also express one or more complement inhibitors. In certain embodiments, the engineered RPE cells overexpress tolerogenic factors (e.g., CD 47), have genomic modifications in the B2M gene, and have reduced CD142 expression. In some embodiments, the RPE cells also express one or more complement inhibitors. In some embodiments, the engineered RPE cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications in the CIITA gene and have reduced CD142 expression. In some embodiments, the RPE cells also express one or more complement inhibitors. In some embodiments, the engineered RPE cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications that disrupt one or more of the following genes: B2M, CIITA and CD142 genes.

In some embodiments, an engineered RPE cell is provided that evades immune recognition. In some embodiments, an engineered RPE cell described herein, such as a primary RPE cell isolated from one or more individual donors (e.g., healthy donors) or an RPE cell differentiated from an iPSC (derived from one or more individual donors (e.g., healthy donors)), does not activate an immune response in a patient (e.g., a recipient after administration). Methods of treating a disease by administering an engineered RPE cell population described herein to a subject (e.g., a recipient) or patient in need thereof are provided.

RPE cells may be implanted in patients with macular degeneration or patients with compromised RPE cells. In some embodiments, the patient has age-related macular degeneration (AMD), early AMD, intermediate AMD, advanced AMD, non-neovascular age-related macular degeneration, dry macular degeneration (dry age-related macular degeneration), wet macular degeneration (wet age-related macular degeneration), juvenile Macular Degeneration (JMD) (e.g., stargardt disease (STARGARDT DISEASE), bedset disease (Best disease), and juvenile retinal cleavage), leber's congenital amaurosis (Leber's Congenital Ameurosis), or retinitis pigmentosa. In other embodiments, the patient has retinal detachment.

For therapeutic applications, cells prepared according to the disclosed methods may generally be provided in the form of pharmaceutical compositions comprising isotonic excipients and prepared under conditions sufficiently sterile for administration to humans. For general principles of pharmaceutical formulation of cellular compositions, see "CELL THERAPY: stem Cell Transplantation, GENE THERAPY, and Cellular Immunotherapy," editions Morstyn and Sheridan, cambridge University Press,1996; and "Hematopic STEM CELL THERAPY," E.D.ball, J.Lister and P.Law, churchill Livingstone,2000. The cells may be packaged in a device or container suitable for dispensing or clinical use.

4) Thyroid cells

In some embodiments, the engineered or modified cell as provided herein is a primary thyroid cell. In some embodiments, the primary thyroid cells are isolated or obtained from one or more individual donor subjects, such as one or more individual healthy donors (e.g., subjects that are unknown or not suspected of having a disease or infection (e.g., that do not exhibit clinical signs of a disease or infection)). As will be appreciated by those skilled in the art, the method of isolating or obtaining thyroid cells from an individual may be accomplished using known techniques. Provided herein are engineered primary thyroid cells containing a modification (e.g., a genetic modification) described herein for subsequent transplantation or implantation into a subject (e.g., a recipient).

In some embodiments, the primary thyroid cells are obtained (e.g., harvested, extracted, removed, or obtained) from a subject or individual. In some embodiments, the primary thyroid cells are generated from a thyroid cell bank such that the thyroid cells are from one or more subjects (e.g., one or more humans, including one or more healthy humans). In some embodiments, the primary thyroid cell pool is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments, the donor subject is different from the patient (e.g., the recipient to whom the therapeutic cells are administered). In some embodiments, the thyroid cell bank does not include cells from the patient. In some embodiments, one or more donor subjects from which the thyroid cell library is obtained are different from the patient.

In some embodiments, the cells as provided herein are thyroid cells differentiated from engineered ipscs, which contain the modifications described herein (e.g., genetic modifications) and differentiate into thyroid cells. As will be appreciated by those skilled in the art, the method used for differentiation depends on the desired cell type using known techniques. In some embodiments, cells differentiated into thyroid cells can be used for subsequent transplantation or implantation into a subject (e.g., a recipient).

In some embodiments, the engineered pluripotent cells containing modifications described herein differentiate into thyroid progenitor cells and thyroid follicular organoids, which can secrete thyroid hormones to address autoimmune thyroiditis. Techniques for differentiating thyroid cells are known in the art. See, e.g., kurmann et al, CELL STEM CELL,2015, 11, 5; 17 527-42, which are incorporated herein by reference in their entirety, in particular methods and reagents for generating thyroid cells from human pluripotent stem cells and also for transplantation techniques. Differentiation can be determined, as known in the art, typically by assessing the presence of thyroid cell associated or specific markers or by functional measurements.

In some embodiments, an engineered thyroid cell population, such as primary thyroid cells isolated from one or more individual donors (e.g., healthy donors) or thyroid cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), is maintained in culture medium, and in some cases expanded prior to administration. In certain embodiments, the thyroid cell population is cryopreserved prior to administration.

In some embodiments, the present technology relates to engineering the expression or lack thereof of thyroid cells, such as primary thyroid cells isolated from one or more individual donors (e.g., healthy donors) or thyroid cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), that overexpress tolerogenic factors (e.g., CD 47), and have reduced expression of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules), and have reduced expression of CD 142. In some embodiments, the thyroid cells also express one or more complement inhibitors. In certain embodiments, the engineered thyroid cells overexpress tolerogenic factors (e.g., CD 47), have genomic modifications in the B2M gene, and have reduced CD142 expression. In some embodiments, the thyroid cells also express one or more complement inhibitors. In some embodiments, the engineered thyroid cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications in the CIITA gene and in the CD142 gene. In some embodiments, the engineered thyroid cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications that disrupt one or more of the following genes: B2M and CIITA genes, and CD146 gene.

In some embodiments, an engineered thyroid cell is provided that evades immune recognition. In some embodiments, an engineered thyroid cell described herein, such as a primary thyroid cell isolated from one or more individual donors (e.g., healthy donors) or a beta islet cell differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), does not activate an immune response in a patient (e.g., post-administration recipient). Methods of treating a disease by administering an engineered endothelial cell population described herein to a subject (e.g., a recipient) or patient in need thereof are provided.

U.Heart cells

Provided herein are cardiac cell types differentiated from HIP cells for subsequent transplantation or implantation into a subject (e.g., a recipient). As will be appreciated by those skilled in the art, the method used for differentiation depends on the desired cell type using known techniques. Exemplary cardiac cell types include, but are not limited to, cardiomyocytes, nodular cardiomyocytes, conducting cardiomyocytes, working cardiomyocytes, cardiomyocyte precursor cells, cardiac muscle progenitor cells, cardiac stem cells, cardiac myocytes, atrial cardiac stem cells, ventricular cardiac stem cells, epicardial cells, hematopoietic cells, vascular endothelial cells, endocardial endothelial cells, cardiac valve mesenchymal cells, cardiac pacing cells, and the like.

In some embodiments, the cardiac cells described herein are administered to a recipient subject to treat a cardiac disorder selected from the group consisting of: pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, perinatal cardiomyopathy, inflammatory cardiomyopathy, idiopathic cardiomyopathy, other cardiomyopathy, myocardial ischemia reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary heart disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart disease, arterial inflammation, cardiovascular disease, myocardial infarction, myocardial ischemia, congestive heart failure, myocardial infarction, myocardial ischemia, cardiac injury, myocardial ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, coronary artery disease, dysfunction of the conduction system, coronary artery dysfunction, pulmonary hypertension, arrhythmia, muscular dystrophy, abnormal muscle mass, muscle degeneration, myocarditis, infectious myocarditis, drug or toxin induced muscle abnormalities, allergic myocarditis and autoimmune endocarditis.

Accordingly, provided herein are methods for treating and preventing cardiac injury or heart disease or cardiac disorder in a subject in need thereof. The methods described herein may be used to treat, ameliorate, prevent or slow the progression of a variety of heart diseases or symptoms thereof, such as those that result in pathological damage to heart structure and/or function. The terms "heart disease," "heart condition," and "heart injury" are used interchangeably herein and refer to conditions and/or disorders associated with the heart (including valves, endothelium, infarct zone, or other components or structures of the heart). Such heart diseases or heart related diseases include, but are not limited to, myocardial infarction, heart failure, cardiomyopathy, congenital heart defects, heart valve diseases or dysfunction, endocarditis, rheumatic fever, mitral valve prolapse, infectious endocarditis, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis, heart enlargement, and/or mitral insufficiency, etc.

In some embodiments, the cardiomyocyte precursor comprises a cell capable of producing a progeny comprising a mature (terminal) cardiomyocyte. Cardiomyocyte precursor cells can be identified generally using one or more markers selected from the GATA-4, nkx2.5 and MEF-2 transcription factor families. In some cases, cardiomyocytes refer to immature cardiomyocytes or mature cardiomyocytes that express one or more markers (sometimes at least 2,3,4, or 5 markers) from the following list: cardiac troponin I (cTnl), cardiac troponin T (cTnT), sarcomere Myosin Heavy Chain (MHC), GATA-4, nkx2.5, N-cadherin, beta 2-adrenoceptor, ANF, MEF-2 transcription factor family, creatine kinase MB (CK-MB), myoglobin and Atrial Natriuretic Factor (ANF). In some embodiments, the cardiac cells exhibit spontaneous periodic contractile activity. In some cases, when cardiac cells are cultured in a suitable tissue culture environment with appropriate Ca ²⁺ concentrations and electrolyte balance, the cells are observed to shrink in a periodic fashion along one axis of the cells without adding any additional components to the medium, and then released from the shrink. In some embodiments, the cardiac cell is a hypoimmunogenic cardiac cell.

In some embodiments, a method of generating a population of low-immunogenicity cardiac cells from a population of low-immunogenicity pluripotent (HIP) cells by in vitro differentiation comprises: (a) Culturing a population of HIP cells in a medium comprising a GSK inhibitor; (b) Culturing the HIP cell population in a medium comprising a WNT antagonist to produce a pre-cardiac cell population; and (c) culturing the pre-cardiac cell population in a medium comprising insulin to produce a low immune cardiac cell population. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of GSK inhibitor ranges from about 2mM to about 10mM. In some embodiments, the WNT antagonist is IWR1, a derivative or variant thereof. In some cases, the concentration of WNT antagonist ranges from about 2mM to about 10mM.

In some embodiments, the population of low-immunogenicity cardiac cells is separated from non-cardiac cells. In some embodiments, the isolated population of low-immunogenicity cardiac cells is expanded prior to administration. In certain embodiments, the isolated population of low-immunogenicity cardiac cells is expanded and cryopreserved prior to administration.

Other useful methods for differentiating induced pluripotent stem cells or multipotent stem cells into cardiac cells are described, for example, in US2017/0152485; US2017/0058263; US2017/0002325; US2016/0362661; US2016/0068814; US9,062,289; US7,897,389; and US7,452,718. Additional methods for generating cardiac cells from induced pluripotent stem cells or multipotent stem cells are described, for example, in Xu et al, STEM CELLS AND Development,2006,15 (5): 631-9, burridge et al, CELL STEM CELL,2012,10:16-28 and Chen et al, STEM CELL RES,2015, l5 (2): 365-375.

In various embodiments, the hypoimmunogenic cardiac cells can be cultured in a medium comprising: BMP pathway inhibitors, WNT signaling activators, WNT signaling inhibitors, WNT agonists, WNT antagonists, src inhibitors, EGFR inhibitors, PCK activators, cytokines, growth factors, myocardial agents, compounds, and the like.

WNT signaling activators include, but are not limited to CHIR99021.PCK activators include, but are not limited to, PMA. Inhibitors of WNT signaling include, but are not limited to, compounds selected from KY02111, SO3031 (KY 01-I), SO2031 (KY 02-I) and SO3042 (KY 03-I), and XAV939. Src inhibitors include, but are not limited to, a419259.EGFR inhibitors include, but are not limited to AG1478.

Non-limiting examples of agents for producing cardiac cells from ipscs include activin A, BMP4, wnt3a, VEGF, soluble frizzled, cyclosporin a, angiotensin II, phenylephrine, ascorbic acid, dimethyl sulfoxide, 5-aza-2' -deoxycytidine, and the like.

The cells provided herein can be cultured on a surface, such as a synthetic surface, to support and/or promote differentiation of the low-immunogenicity pluripotent cells into cardiac cells. In some embodiments, the surface comprises a polymeric material including, but not limited to, homopolymers or copolymers of selected one or more acrylate monomers. Non-limiting examples of acrylate monomers and methacrylate monomers include tetra (ethylene glycol) diacrylate, glycerol dimethacrylate, 1, 4-butanediol dimethacrylate, poly (ethylene glycol) diacrylate, di (ethylene glycol) dimethacrylate, tetra (ethylene glycol) dimethacrylate, 1, 6-hexanediol propoxylate diacrylate, neopentyl glycol diacrylate, trimethylolpropane benzoic acid diacrylate, ethoxylated trimethylolpropane (1 EO/QH) methyl ester, tricyclo [5.2.1.0 ^2,6 ] decane dimethanol diacrylate, neopentyl glycol deoxidizing diacrylate and trimethylolpropane triacrylate. Acrylates are synthesized in a manner known in the art or are obtained from commercial suppliers such as Polysciences, inc., SIGMA ALDRICH, inc.

The efficacy of heart cells prepared as described herein can be assessed in an animal model of cardiac freeze injury that results in 55% of left ventricular wall tissue becoming scar tissue untreated (Li et al, ann. Thorac. Surg.62:654,1996; sakai et al, ann. Thorac. Surg.8:2074,1999, sakai et al, thorac. Cardiovasc. Surg.118:715,1999). Successful treatment may reduce scar area, limit scar dilation, and improve cardiac function (as determined by systolic, diastolic, and developing pressures). Embolic coils in the distal portion of the left anterior descending branch can also be used to model cardiac injury (Watanabe et al, cell Transplant.7:239,1998), and therapeutic efficacy can be assessed by histology and cardiac function.

In some embodiments, an engineered population of cardiac cells, such as cardiac cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), is maintained in culture, and in some cases expanded prior to administration. In certain embodiments, the population of cardiac cells is cryopreserved prior to administration.

In some embodiments, the present technology relates to engineered cardiac cells, such as cardiac cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), that overexpress tolerogenic factors (e.g., CD 47), have reduced expression or lack expression of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules), and have reduced expression of CD 142. In some embodiments, the cardiac cells also express one or more complement inhibitors. In certain embodiments, the engineered cardiac cells overexpress tolerogenic factors (e.g., CD 47), have genomic modifications in the B2M gene, and have reduced CD142 expression. In some embodiments, the cardiac cells also express one or more complement inhibitors. In some embodiments, the engineered cardiac cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications in the CIITA gene and have reduced CD142 expression. In some embodiments, the cardiac cells also express one or more complement inhibitors. In some embodiments, the engineered cardiac cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications that disrupt one or more of the following genes: B2M and CIITA genes, and CD142 gene.

In some embodiments, an engineered cardiac cell is provided that evades immune recognition. In some embodiments, the engineered cardiac cells described herein, such as cardiac cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), do not activate an immune response in a patient (e.g., post-administration recipient). Methods of treating a disease by administering an engineered cardiac cell population described herein to a subject (e.g., a recipient) or patient in need thereof are provided.

In some embodiments, administering comprises implanting heart tissue, intravenous injection, intra-arterial injection, intra-coronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, endocardial injection, epicardial injection, or infusion in the subject.

In some embodiments, the patient administered the engineered cardiac cells is also administered a cardiac drug. Illustrative examples of cardiac drugs suitable for combination therapy include, but are not limited to, growth factors, polynucleotides encoding growth factors, angiogenic agents, calcium channel blockers, antihypertensives, antimitotics, inotropic agents, anti-atherosclerosis agents, anticoagulants, beta-blockers, antiarrhythmic agents, anti-inflammatory agents, vasodilators, thrombolytics, cardiac glycosides, antibiotics, antiviral agents, antifungal agents, protozoan inhibitors, nitrates, angiotensin Converting Enzyme (ACE) inhibitors, angiotensin II receptor antagonists, brain Natriuretic Peptides (BNP); antitumor agents, steroids, and the like.

The therapeutic effect according to the methods provided herein can be monitored in a variety of ways. For example, an Electrocardiogram (ECG) or Hott monitor (holier monitor) may be utilized to determine treatment efficacy. ECG is a measure of heart rhythm and electrical impulses and is a very effective and non-invasive way to determine whether a treatment improves or maintains, prevents or slows the degradation of the subject's cardiac electrical conduction. Monitoring cardiac abnormalities, arrhythmia conditions, and the like using a portable ECG hall monitor that can be worn for extended periods of time is also a reliable method of assessing the effectiveness of a treatment. ECG or nuclear studies can be used to determine improvement in ventricular function.

V. nerve cells

Provided herein are different neural cell types differentiated from the engineered pluripotent cells (e.g., ipscs) that can be used for subsequent transplantation or implantation into a recipient subject. As will be appreciated by those skilled in the art, the method used for differentiation depends on the desired cell type using known techniques. Exemplary types of neural cells include, but are not limited to, brain endothelial cells, neurons (e.g., dopaminergic neurons), glial cells, glial progenitor cells, and the like.

In some embodiments, differentiation of the induced pluripotent stem cells is performed by exposing the cells to or contacting the cells with specific factors known to produce a specific cell lineage in order to target differentiation thereof to a specific, desired lineage and/or cell type of interest. In some embodiments, terminally differentiated cells exhibit a particular phenotypic characteristic or trait. In certain embodiments, the stem cells described herein differentiate into a population of neuroectodermal, neuronal, neuroendocrine, dopaminergic, cholinergic, serotonergic (5-HT), glutamatergic, GABAergic, adrenergic, noradrenergic, sympathetic, parasympathetic, sympathetic peripheral, or glial cells. In some cases, the population of glial cells includes a population of microglial (e.g., anamorphic, branched, activated phagocytic, and activated non-phagocytic) cells or macroglial (central nervous system cells: astrocytes, oligodendrocytes, ependymal cells, and radial glial cells; and peripheral nervous system cells: schwann cells (SCHWANN CELL) and satellite cells) cells, or precursor and progenitor cells of any of the foregoing.

Protocols for producing different types of neural cells are described in PCT application No. WO2010144696, U.S. patent No. 9,057,053;9,376,664; and 10,233,422. Additional description of methods for differentiating low immunogenicity pluripotent cells can be found in Deuse et al, nature Biotechnology,2019,37,252-258 and Han et al, proc NATL ACAD SCI USA,2019,116 (21), 10441-10446, for example. Methods for determining the effect of neural cell transplantation in animal models of neurological disorders or conditions are described in the following references: for spinal cord injuries, curtis et al CELL STEM CELL,2018,22,941-950; for Parkinson's disease (Parkinson's disease), kikuchi et al, nature,2017,548:592-596; for ALS, izrael et al, STEM CELL RESEARCH,2018,9 (1): 152 and Izrael et al, interchOpen, DOI: 10.5772/intelchopen.72862; for epilepsy, upadhya et al, PNAS,2019,116 (1): 287-296.

In some embodiments, an engineered neural cell population, such as neural cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), is maintained in culture medium, and in some cases expanded prior to administration. In certain embodiments, the population of neural cells is cryopreserved prior to administration.

In some embodiments, the present technology relates to engineered neural cells, such as neural cells differentiated from ipscs (derived from one or more individual donors (e.g., healthy donors)), that overexpress tolerogenic factors (e.g., CD 47) and have reduced expression or lack the expression of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules) and have reduced expression of CD 142. In some embodiments, the neural cells also express one or more complement inhibitors. In certain embodiments, the engineered neural cells overexpress tolerogenic factors (e.g., CD 47), have genomic modifications in the B2M gene, and have reduced CD142 expression. In some embodiments, the neural cells also express one or more complement inhibitors. In some embodiments, the engineered neural cells overexpress tolerogenic factors (e.g., CD 47) and have genomic modifications in the CIITA gene and have reduced CD142 expression. In some embodiments, the neural cells also express one or more complement inhibitors. In some embodiments, the engineered neural cell overexpresses a tolerogenic factor (e.g., CD 47) with genomic modifications that disrupt one or more of the following genes: B2M and CIITA genes, and has reduced CD142 expression. In some embodiments, the neural cells also express one or more complement inhibitors.

In some embodiments, the engineered neural cells provided evade immune recognition. In some embodiments, an engineered neural cell described herein, such as a neural cell differentiated from an iPSC (derived from one or more individual donors (e.g., healthy donors)), does not activate an immune response in a patient (e.g., a post-administration recipient). Methods of treating a disease by administering an engineered neural cell population described herein to a subject (e.g., a recipient) or patient in need thereof are provided.

In some embodiments, the neural cells are administered to a subject to treat parkinson's disease, huntington's disease (Huntington disease), multiple sclerosis, other neurodegenerative diseases or disorders, attention Deficit Hyperactivity Disorder (ADHD), tourette's Syndrome (TS), schizophrenia, psychosis, depression, other neuropsychiatric disorders. In some embodiments, the neural cells described herein are administered to a subject to treat or ameliorate stroke. In some embodiments, neurons and glial cells are administered to a subject suffering from Amyotrophic Lateral Sclerosis (ALS).

1) Brain endothelial cells

In some embodiments, brain Endothelial Cells (ECs), precursors and progenitors thereof are differentiated from pluripotent stem cells (e.g., induced pluripotent stem cells) on the surface by culturing the cells in a medium comprising one or more factors that promote brain EC or neurogenesis. In some cases, the medium comprises one or more of the following: CHIR-99021, VEGF, basic FGF (bFGF) and Y-27632. In some embodiments, the culture medium comprises a supplement designed to promote survival and functionality of the neural cells.

In some embodiments, brain Endothelial Cells (ECs), precursors and progenitors thereof differentiate from pluripotent stem cells on the surface by culturing the cells in a non-conditioned or conditioned medium. In some cases, the culture medium comprises factors or small molecules that promote or contribute to differentiation. In some embodiments, the culture medium comprises one or more factors or small molecules selected from the group consisting of: VEGR, FGF, SDF-1, CHIR-99021, Y-27632, SB 431542, and any combination thereof. In some embodiments, the surface for differentiation comprises one or more extracellular matrix proteins. The surface may be coated with one or more extracellular matrix proteins. Cells may be differentiated in suspension and then placed into a gel matrix form (such as matrigel, gelatin, or fibrin/thrombin form) to promote cell survival. In some cases, differentiation is typically determined by assessing the presence of cell-specific markers, as is known in the art.

In some embodiments, the brain endothelial cells express or secrete a factor selected from the group consisting of CD31, VE cadherin, and combinations thereof. In certain embodiments, the brain endothelial cells express or secrete one or more factors selected from the group consisting of: CD31, CD34, CD45, CD117 (c-kit), CD146, CXCR4, VEGF, SDF-1, PDGF, GLUT-1, PECAM-1, eNOS, blocking protein-5, blocking protein, ZO-1, p-glycoprotein, feng Wei Rich factor, VE-cadherin, low Density lipoprotein receptor LDLR, low Density lipoprotein receptor-related protein 1LRP1, insulin receptor INSR, leptin receptor LEPR, basal cell adhesion molecule BCAM, transferrin receptor TFRC, late glycosylation end product specific receptor AGER, retinol uptake receptor STRA6, large neutral amino acid transporter small subunit 1SLC7A5, excitatory amino acid transporter 3SLC1A1, sodium-coupled neutral amino acid transporter 5SLC38A5, solute carrier family 16 member 1SLC16A1, ATP-dependent translocase ABCB1, ATP-ABCC2 binding cassette ABCG2, multi-drug-related protein transporter 1 CC1, small anion transporter Guan Duote, multi-drug-resistant ABCC2, and multi-drug-related protein ABCC 4.

In some embodiments, the brain EC is characterized by having one or more characteristics selected from the group consisting of: tightly linked high expression, high resistance, low fenestration, small perivascular space, ubiquitous presence of insulin and transferrin receptors, and high mitochondrial numbers.

In some embodiments, a positive selection strategy is used to select or purify brain ECs. In some cases, brain ECs are sorted according to endothelial cell markers such as, but not limited to, CD 31. In other words, CD31 positive brain ECs were isolated. In some embodiments, a negative selection strategy is used to select or purify brain ECs. In some embodiments, undifferentiated or pluripotent stem cells are removed by selecting cells that express a pluripotency marker (including, but not limited to TRA-1-60 and SSEA-1).

In some embodiments, brain endothelial cells are administered to alleviate symptoms or effects of cerebral hemorrhage. In some embodiments, the dopaminergic neurons are administered to patients suffering from parkinson's disease. In some embodiments, the noradrenergic neurons, gabaergic interneurons are administered to a patient who has experienced an epileptic seizure. In some embodiments, motor neurons, interneurons, schwann cells, oligodendrocytes, and microglia are administered to a patient experiencing spinal cord injury.

2) Dopaminergic neurons

In some embodiments, HIP cells described herein differentiate into dopaminergic neurons, including neuronal stem cells, neuronal progenitor cells, immature dopaminergic neurons, and mature dopaminergic neurons.

In some cases, the term "dopaminergic neuron" includes a neuronal cell that expresses Tyrosine Hydroxylase (TH), which is the rate-limiting enzyme for dopamine synthesis. In some embodiments, the dopaminergic neurons secrete the neurotransmitter dopamine, and little or no dopamine hydroxylase is expressed. Dopaminergic (DA) neurons may express one or more of the following markers: neuron-specific enolase (NSE), 1-aromatic amino acid decarboxylase, vesicle monoamine transporter 2, dopamine transporter, nurr-l, and dopamine 2 receptor (D2 receptor). In certain instances, the term "neural stem cell" includes a population of pluripotent cells that partially differentiate along a neural cell pathway and express one or more neural markers (including, for example, nestin). Neural stem cells can differentiate into neurons or glial cells (e.g., astrocytes and oligodendrocytes). The term "neural progenitor cells" includes cultured cells that express FOXA2 and low levels of b-tubulin but do not express tyrosine hydroxylase. Such neural progenitor cells have the ability to differentiate into multiple neuronal subtypes upon culturing an appropriate factor such as those described herein; in particular the ability of various dopaminergic neuron subtypes.

In some embodiments, the DA neurons from HIP cells are administered to a patient, e.g., a human patient, to treat a neurodegenerative disease or disorder. In some cases, the neurodegenerative disease or disorder is selected from the group consisting of parkinson's disease, huntington's disease, and multiple sclerosis. In other embodiments, the DA neurons are used to treat or ameliorate one or more symptoms of neuropsychiatric disorders, such as Attention Deficit Hyperactivity Disorder (ADHD), tourette Syndrome (TS), schizophrenia, psychosis, and depression. In yet other embodiments, the DA neurons are used to treat patients with impaired DA neurons.

In some embodiments, the DA neurons, precursors and progenitors thereof are differentiated from pluripotent stem cells by culturing the stem cells in a medium comprising one or more factors or additives. Useful factors and additives that promote DA neuronal differentiation, growth, expansion, maintenance and/or maturation include, but are not limited to, wntl, FGF2, FGF8a, sonic hedgehog (SHH), brain Derived Neurotrophic Factor (BDNF), transforming growth factor a (TGF-a), TGF-B, interleukin 1 beta, glial cell line derived neurotrophic factor (GDNF), GSK-3 inhibitors (e.g., CHIR-99021), TGF-B inhibitors (e.g., SB-431542), B-27 supplements, doxomorphin, puromorphine, noggin (noggin), retinoic acid, cAMP, ascorbic acid, neurorank protein (neurturin), knockout serum substitutes, N-acetylcysteine, c-kit ligands, modified forms thereof, mimetics thereof, analogues thereof, and variants thereof. In some embodiments, the DA neurons differentiate in the presence of one or more factors that activate or inhibit WNT pathway, NOTCH pathway, SHH pathway, BMP pathway, FGF pathway, and the like. Differentiation protocols and detailed descriptions thereof are provided, for example, in US9,968,637, US7,674,620, kim et al, nature,2002,418,50-56; bjorklund et al, PNAS,2002,99 (4), 2344-2349; the disclosures of Grow et al STEM CELLS TRANSL Med.2016,5 (9): 1133-44 and Cho et al PNAS,2008,105:3392-3397, including detailed descriptions of examples, methods, figures and results, are incorporated herein by reference in their entirety.

In some embodiments, the population of hypoimmunogenic dopaminergic neurons is isolated from non-neuronal cells. In some embodiments, the isolated population of hypoimmunogenic dopaminergic neurons is amplified prior to administration. In certain embodiments, the isolated population of hypoimmunogenic dopaminergic neurons is amplified and cryopreserved prior to administration.

To characterize and monitor DA differentiation and evaluate DA phenotype, the expression of any number of molecules and genetic markers can be evaluated. For example, the presence of a genetic marker may be determined by various methods known to those skilled in the art. Expression of the molecular markers may be determined by quantitative methods such as, but not limited to, qPCR-based assays, immunoassays, immunocytochemical assays, immunoblot assays, and the like. Exemplary markers for DA neurons include, but are not limited to, TH, B-tubulin, pax6, insulin gene-enhanced protein (Isl 1), nestin, diaminobenzidine (DAB), G-protein activated inward rectifier potassium channel 2 (GIRK 2), microtubule-associated protein 2 (MAP-2), NURR1, dopamine transporter (DAT), fork box protein A2 (FOXA 2), FOX3, diproteins, and LIM homeobox transcription factor l-beta (LMX 1B), and the like. In some embodiments, the DA neuron expresses one or more markers selected from the group consisting of corin, FOXA2, tuJ1, NURR1, and any combination thereof.

In some embodiments, the DA neurons are evaluated based on cellular electrophysiological activity. The electrophysiology of a cell can be assessed by using assays known to those skilled in the art. For example, whole cell and perforated patch clamp, assays for detecting cell electrophysiological activity, assays for measuring cell action potential magnitude and duration, and functional assays for detecting dopamine production by DA cells.

In some embodiments, DA neuron differentiation is characterized by spontaneous rhythmic action potentials and high frequency action potentials with spike frequency adaptation after injection of depolarization currents. In other embodiments, the DA differentiation is characterized by the production of dopamine. The level of dopamine produced is calculated by measuring the width of the action potential at half its maximum amplitude (peak half maximum width).

In some embodiments, the differentiated DA neurons are transplanted to a specific location in the patient intravenously or by injection. In some embodiments, differentiated DA cells are transplanted into the substantia nigra of the brain (particularly in or near the dense region), ventral Tegmental Area (VTA), caudate nucleus, putamen, nucleus accumbens, subthalamic nucleus, or any combination thereof, in place of DA neurons whose degeneration leads to parkinson's disease. Differentiated DA cells may be injected as a cell suspension into the target area. Or when included in such delivery devices, the differentiated DA cells may be embedded in a supporting matrix or scaffold. In some embodiments, the scaffold is biodegradable. In other embodiments, the scaffold is non-biodegradable. The scaffold may comprise natural or synthetic (artificial) materials.

Delivery of the DA neurons may be achieved by using a suitable vehicle such as, but not limited to, liposomes, microparticles, or microcapsules. In other embodiments, the differentiated DA neurons are administered in the form of a pharmaceutical composition comprising an isotonic excipient. The pharmaceutical composition is prepared under conditions sufficiently sterile for human administration. In some embodiments, the DA neurons differentiated from HIP cells are provided in the form of a pharmaceutical composition. General principles for therapeutic formulation of cell compositions can be found in CELL THERAPY: stem Cell Transplantation, GENE THERAPY, and Cellular Immunotherapy, G.Morstyn and W.Shredan editions, cambridge University Press,1996 and Hematopic STEM CELL THERAPY, E.BALL, J.Lister and P.Law, churchill Livingstone,2000, the disclosures of which are incorporated herein by reference.

Useful descriptions of stem Cell-derived neurons and methods of their preparation can be found, for example, in Kirkeby et al, cell Rep,2012,1:703-714; kriks et al, nature,2011,480:547-551; wang et al ,Stem Cell Reports,2018,11(1):171-182;Lorenz Studer,"Chapter 8-Strategies for Bringing Stem Cell-Derived Dopamine Neurons to the clinic-The NYSTEM Trial"in Progress in Brain Research,2017,, volume 230, pages 191-212; liu et al Nat Protoc,2013,8:1670-1679; upadhya et al, curr Protoc Stem Cell Biol,38,2d.7.1-2d.7.47; U.S. published application number 20160115448 and US8,252,586; US8,273,570; US9,487,752 and US10,093,897, the contents of which are incorporated herein by reference in their entirety.

In addition to DA neurons, other neuronal cells, precursors and progenitors thereof can also differentiate from HIP cells outlined herein by culturing the cells in a medium comprising one or more factors or additives. Non-limiting examples of factors and additives include GDNF, BDNF, GM-CSF, B27, basic FGF, basic EGF, NGF, CNTF, SMAD inhibitor, wnt antagonists, SHH signaling activators, and any combinations thereof. In some embodiments, the SMAD inhibitor is selected from the group consisting of ：SB431542、LDN-193189、Noggin PD169316、SB203580、LY364947、A77-01、A-83-01、BMP4、GW788388、GW6604、SB-505124、 Le Demu mab (lerdelimumab), a mertemumab (metelimumab)、GC-I008、AP-12009、AP-110I4、LY550410、LY580276、LY364947、LY2109761、SB-505124、E-616452(RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, K26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, dorsomorphin dihydrochloride, and derivatives thereof. In some embodiments, the Wnt antagonist is selected from the group consisting of ：XAV939、DKK1、DKK-2、DKK-3、DKK-4、SFRP-1、SFRP-2、SFRP-3、SFRP-4、SFRP-5、WIF-1、Soggy、IWP-2、IWR1、ICG-001、KY0211、Wnt-059、LGK974、IWP-L6 and derivatives thereof. In some embodiments, the SHH signaling activator is selected from the group consisting of: smooth Agonists (SAG), SAG analogues, SHH, C25-SHH, C24-SHH, purmorphamine, hg-Ag and/or derivatives thereof.

In some embodiments, the neuron expresses one or more markers selected from the group consisting of: glutamate ion receptor NMDA subunit 1GRIN1, glutamate decarboxylase 1GAD1, gamma aminobutyric acid GABA, tyrosine hydroxylase TH, LIM homeobox transcription factor 1-alpha LMX1A, fork box protein O1 FOXO1, fork box protein A2 FOXA2, fork box protein O4 FOXO4, FOXG1, 2',3' -cyclic nucleotide 3' -phosphodiesterase CNP, myelin basic protein MBP, tubulin beta chain 3TUB3, tubulin beta chain 3NEUN, solute carrier family 1 member 6SLC1A6, SST, PV, calbindin 、RAX、LHX6、LHX8、DLX1、DLX2、DLX5、DLX6、SOX6、MAFB、NPAS1、ASCL1、SIX6、OLIG2、NKX2.1、NKX2.2、NKX6.2、VGLUT1、MAP2、CTIP2、SATB2、TBR1、DLX2、ASCL1、ChAT、NGFI-B、c-fos、CRF、RAX、POMC、 subhill secretin, NADPH, NGF, ach, VAChT, PAX, EMX2p75, CORIN, TUJ1, NURR1 and/or any combination thereof.

3) Glial cells

In some embodiments, the neural cells include glial cells, such as, but not limited to, microglial cells, astrocytes, oligodendrocytes, ependymal cells and schwann cells, glial precursors, and glial progenitor cells thereof are generated by differentiating pluripotent stem cells into therapeutically effective glial cells, and the like. Differentiation of the low-immunogenicity pluripotent stem cells produces low-immunogenicity neural cells, such as low-immunogenicity glial cells.

In some embodiments, the glial cells, precursors and progenitors thereof are produced by culturing pluripotent stem cells in a medium comprising one or more agents selected from the group consisting of: retinoic acid, IL-34, M-CSF, FLT3 ligand, GM-CSF, CCL2, TGF beta inhibitor, BMP signaling inhibitor, SHH signaling activator, FGF, platelet derived growth factor PDGF, PDGFR-alpha, HGF, IGF1, noggin, SHH, dorsomorphin, noggin, and any combination thereof. In certain instances, the BMP signaling inhibitor is LDN193189, SB431542, or a combination thereof. In some embodiments, the glial cell expresses NKX2.2, PAX6, SOX10, brain derived neurotrophic factor BDNF, neutrophil trophic factor-3 NT-3, NT-4, EGF, ciliary neurotrophic factor CNTF, nerve growth factor NGF, FGF8, EGFR, OLIG1, OLIG2, myelin basic protein MBP, GAP-43, LNGFR, nestin, GFAP, CD b, CD11c, CX3CR1, P2RY12, IBA-1, TMEM119, CD45, and any combination thereof. Exemplary differentiation media may include any particular factor and/or small molecule that may promote or be capable of producing glial cell types as recognized by one of skill in the art.

To determine whether cells produced according to an in vitro differentiation protocol exhibit the characteristics and features of glial cells, the cells can be transplanted into an animal model. In some embodiments, the glial cells are injected into an immunocompromised mouse, e.g., an immunocompromised shiverer mouse. Glial cells were administered to the brains of mice and the implanted cells were evaluated after a preselected time. In some cases, the implanted cells in the brain are visualized by using immunostaining and imaging methods. In some embodiments, glial cells are determined to express a known glial cell biomarker.

Useful methods for producing glial cells, precursors and progenitors thereof from stem cells can be found, for example, in US7,579,188;US7,595,194;US8,263,402;US8,206,699;US8,252,586;US9,193,951;US9,862,925;US8,227,247;US9,709,553;US2018/0187148;US2017/0198255;US2017/0183627;US2017/0182097;US2017/253856;US2018/0236004;WO2017/172976; and WO2018/093681. Methods for differentiating pluripotent stem cells are described, for example, in Kikuchi et al, nature,2017,548,592-596; kriks et al, nature,2011,547-551; doi et al Stem Cell Reports,2014,2,337-50; perrier et al, proc NATL ACAD SCI USA,2004,101,12543-12548; chambers et al, nat Biotechnol,2009,27,275-280; and Kirkeby et al, cell Reports,2012,1,703-714.

The efficacy of neural cell transplantation for spinal cord injury can be assessed, for example, in a rat model of acute spinal cord injury, as described by McDonald et al, nat.med.,1999, 5:1410) and Kim et al, nature,2002, 418:50. For example, successful transplantation may show the presence of graft-derived cells at the foci after 2-5 weeks, differentiation into astrocytes, oligodendrocytes and/or neurons, and migration from the focal end along the spinal cord, gait, coordination and load bearing capacity improvement. A particular animal model is selected based on the type of neural cell and the neurological disease or condition to be treated.

Neural cells can be administered in a manner that allows them to be implanted into the desired tissue site and to reconstruct or regenerate the functionally defective area. For example, depending on the disease being treated, the nerve cells may be transplanted directly into a parenchymal or intrathecal site of the central nervous system. In some embodiments, any of the neural cells described herein (including brain endothelial cells, neurons, dopaminergic neurons, ependymal cells, astrocytes, microglia, oligodendrocytes, and schwann cells) are injected into a patient by intravenous, intraspinal, intraventricular, intrathecal, intraarterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, intraabdominal, intraocular, retrobulbar, and combinations thereof. In some embodiments, the cells are injected or deposited in the form of a bolus or continuous infusion. In certain embodiments, the neural cells are administered by injection into the brain, near the brain, and combinations thereof. For example, the injection may be performed through a drill hole opened in the skull of the subject. Suitable sites for administering neural cells to the brain include, but are not limited to, ventricles, lateral ventricles, greater pools, putamen, basal nuclei, hippocampal cortex, striatum, caudate region, and combinations thereof.

Additional description of neural cells including dopaminergic neurons for use in the present technology can be found in WO2020/018615, the disclosure of which is incorporated herein by reference in its entirety.

W, hematopoietic Stem Cells (HSC)

In some embodiments, the engineered cell is a hematopoietic stem cell. In some cases, hematopoietic stem cells are immature cells that can develop into all types of blood cells, including leukocytes, erythrocytes, and platelets. Hematopoietic Stem Cells (HSCs) are present in peripheral blood and bone marrow. In some cases, hematopoietic stem cells are isolated from peripheral blood or bone marrow.

In some embodiments, the engineered HSCs or populations comprising the engineered HSCs are administered to treat a hematopoietic disease or disorder. In some embodiments, the hematopoietic disease or disorder is spinal cord dysplasia, aplastic anemia, fanconi anemia (Fanconi anemia), paroxysmal sleep hemoglobinuria, sickle cell disease, congenital pure erythrocyte aplastic anemia (Diamond Blackfan anemia), schwann-Dai Mengde disease (SCHACHMAN DIAMOND DISORDER), ke Shiwen syndrome (Kostmann's syndrome), chronic granulomatosis, adrenoleukodystrophy, leukocyte adhesion deficiency, hemophilia, thalassemia, beta-thalassemia, leukemias such as Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), adult lymphoblastic leukemia, chronic Lymphoblastic Leukemia (CLL), B-cell chronic lymphoblastic leukemia (B-CLL), chronic Myelogenous Leukemia (CML), juvenile Chronic Myelogenous Leukemia (CML) and juvenile myelomonocytic leukemia (JMML), severe combined immunodeficiency disease (d), X-linked severe immunodeficiency, wilt-Aldrich syndrome (wilt-Aldrich), hodgkin's-lymphomatosis (pandyk-Aldrich), chronic lymphomatosis (panda-Aldrich) or hodgkin-deficiency (panda-Aldrich, hodgkin-ldh-disease).

In some embodiments, the engineered HSC or population comprising the engineered HSC is administered to treat a cellular defect associated with leukemia or myeloma, or to treat leukemia or myeloma.

In some embodiments, the engineered HSCs or populations comprising the engineered HSCs are administered to treat a cell defect associated with or treat an autoimmune disease or disorder. in some embodiments, the autoimmune disease or disorder is acute disseminated encephalomyelitis, acute hemorrhagic white matter encephalitis, addison's disease, agaropectinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, anti-synthetase syndrome, atopic allergy, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, Autoimmune polycycloadenosis syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, barorosis (Balo disease), baloc concentric sclerosis (Balo concentric sclerosis), behcet's syndrome (Bechets syndrome), berger's disease, beckrstamen encephalitis (Bickertaff 'SENCEPHALITIS), blau syndrome (Blau syndrome), behcet's syndrome (Balo concentric sclerosis), Bullous pemphigoid, cancer, castleman's disease, celiac disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, chager-Schtreus syndrome, cicatricial pemphigoid, crohn syndrome, condensed collectinopathy, complement component 2 deficiency, craniofacial arteritis, CREST syndrome, crohn's disease, cushing's syndrome, cutaneous leucocyte-disrupting vasculitis, degome disease (Dego's disease), dekkera disease (Dercum's disease), dermatitis herpetiformis, dermatomyositis, type 1 diabetes, diffuse systemic sclerosis of the skin, derwler syndrome (Dressler's syndrome), discoid lupus erythematosus, eczema, attachment point associated arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, acquired epidermolysis bullosa, erythema nodosum, primary mixed condensed globulinemia, ewens syndrome (Evan ' ssyndrome), progressive ossified fibrotic insufficiency, inflammatory bowel disease (Evan ' ssyndrome), fibroalveolar inflammation, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, nephritis syndrome (goodpasture 'ssyndrome), grave's disease, guillain-Barre syndrome (GBS), hashimoto 'S ENCEPHALITIS, hashimoto's thyroiditis, hemolytic anemia, allergic purpura, herpes gestation, hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, igA nephropathy, inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, kawasaki's disease, lanbert-Earthwang's disease (Lambert-Eaton myasthenic syndrome), white blood cell disruption vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), gray's disease (Lou Gehrig's diseas), Lupus hepatitis, lupus erythematosus, ma Jide syndrome (Majeed syndrome), meniere's disease, microscopic polyangiitis, miller-Fei Xuezeng syndrome, mixed connective tissue disease, scleroderma, acute acne-like lichen furuncle (Mucha-Habermann disease), multiple sclerosis, myasthenia gravis, myositis, neuromyelitis optica, neuromuscular rigidity, ocular cicatricial pemphigoid, strabismus ocular clonus myoclonus syndrome, Thyroiditis, recurrent rheumatism, paraneoplastic cerebellar degeneration, paroxysmal sleep hemoglobinuria (PNH), pa Luo Zeng syndrome (Parry Romberg syndrome), parsen-Techner syndrome (Parsonnage-Turner syndrome), platycodon grandis, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, rheumatalgia polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, Psoriasis arthritis, pyoderma gangrenosum, pure red cell aplasia, las Mu Sen encephalitis (Rasmussen ' S ENCEPHALITIS), raynaud's phenomenon (Raynaud phenomenon), recurrent polychondritis, reiter's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatoid heat, sarcoidosis, schmidt syndrome (Schmidt syndrome), schmidt syndrome (Schnitzler syndrome), scleritis, Scleroderma, sjogren's syndrome, spondyloarthropathies, still's disease, stiff man's syndrome, subacute bacterial endocarditis, susaxophone's syndrome (Susac's syndrome), sjogren's syndrome (Sweet's syndrome), siemens ' chorea (Sydenham chorea), sympathogenic ophthalmia, large arteritis (Takayasu ' S ARTERITIS), temporal arteritis, painful oculoparalysis syndrome (Tolosa-Hunt syndrome), transverse myelitis, Ulcerative colitis, undifferentiated connective tissue diseases, undifferentiated spondyloarthropathies, vasculitis, vitiligo or Wegener's granulomatosis (Wegener's disease).

ABO blood group and Rh antigen expression

Blood products can be divided into different groups depending on the presence or absence of antigen (ABO blood group) on the surface of each red blood cell in the human body. A. B, AB and Al antigens are determined by oligosaccharide sequences on the erythrocyte glycoprotein. Genes in the blood group antigen group provide instructions for the production of antigenic proteins. Blood group antigen proteins perform a variety of functions within the cell membrane of erythrocytes. These protein functions include transporting other proteins and molecules into and out of cells, maintaining cell structure, attaching to other cells and molecules, and participating in chemical reactions.

Rhesus factor (Rh) blood group is the second major blood group system next to ABO blood group system. The Rh blood group system consisted of 49 defined blood group antigens, of which five antigens D, C, c, E and e are the most important. The Rh (D) status of an individual is generally described by a positive or negative suffix following ABO type. The terms "Rh factor", "Rh positive" and "Rh negative" refer only to Rh (D) antigen. Antibodies to Rh antigens may be involved in hemolytic transfusion reactions and antibodies to Rh (D) and Rh (c) antigens may present significant hemolytic disease risks to fetuses and newborns. ABO antibodies are produced early in the life of everyone. However, rh-rhesus antibodies in humans are usually only produced when humans are sensitive. This may occur, for example, by giving up rh+ infants or by receiving rh+ transfusion.

A. b, H and Rh antigens are the main determinants of the histocompatibility between blood, tissue and cell transplant donors and recipients. The glycosyltransferase activity encoded by the ABO gene is responsible for the production of A, B, AB, O tissue blood group antigens, which are displayed on the cell surface. Type a individuals encode the ABO gene product, have specificity for producing alpha (1, 3) N-acetylgalactosamine transferase activity, and type B individuals have specificity for producing alpha (1, 3) galactosyl transferase activity. Individuals of type O do not produce a functional galactosyltransferase at all and therefore do not produce any modification. Individuals of the AB type each possess one copy and produce two types of modifications. The enzyme product of the ABO gene acts as a substrate on the H antigen, so that individuals of type O lacking ABO activity present unmodified H antigen and are therefore commonly referred to as type O (H).

The H antigen itself is the product of an a (l, 2) fucosyltransferase encoded by the FUTI gene. In very rare individuals, the H antigen is completely lost due to disruption of the FUTI gene and there is no substrate for ABO to produce a or B tissue blood group. These individuals are said to belong to the tissue blood group of Montely (Bombay). The Rh antigen is encoded by the RHD gene and Rh-negative individuals carry a deletion or disruption of the RHD gene.

In some embodiments, a cell or cell population provided herein is ABO type Rh factor negative. In some embodiments, the ABO-type Rh factor negative cells described herein are derived from an ABO-type Rh factor negative donor. In some embodiments, ABO-type Rh factor negative cells described herein are engineered to lack presentation of ABO a-type, ABO B-type, or Rh factor antigens. In some embodiments, the ABO-type and/or Rh-negative cells comprise partial or complete inactivation of the ABO gene (e.g., by a deleterious variant of the ABO gene or by insertion of the exon 6 258delg variant of the ABO gene), and/or expression of the RHD gene is partially or completely inactivated by a deleterious variant of the RHD gene. In some embodiments, the ABO type Rh negative cells comprise partial or complete inactivation of the FUT1 gene, and/or expression of the RHD gene is partially or completely inactivated by deleterious variants of the RHD gene. In some embodiments, engineered ABO O-type and/or Rh factor negative cells are generated using gene editing, for example, to modify a-type cells to O-type cells, B-type cells to O-type cells, AB-type cells to O-type cells, a+ type cells to O-type cells, a-type cells to O-type cells, ab+ type cells to O-type cells, AB-type cells to O-type cells, b+ type cells to O-type cells, and B-type cells to O-type cells. Exemplary engineered ABO O-type Rh factor negative cells and methods of producing the same are described in WO2021/146222, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the cell or population of cells provided herein comprising increased CD46 and CD59 expression is ABO type a, ABO type B, or ABO type AB, and/or the cell or population of cells provided herein comprising increased CD46 and CD59 expression is Rh factor positive. In some embodiments, cells comprising increased CD46 and CD59 expression may be administered to a recipient patient that is not compatible with ABO and/or Rh factors without eliciting a CDC response.

9. Sex chromosome

In certain aspects, cells with sex chromosomes can express certain antigens (e.g., Y antigens), and the receptors can have pre-existing sensitivity to such antigens. For example, in some embodiments, a woman carrying a male fetus may exclude cells from a male donor. Thus, in some embodiments, the donor is male and the recipient is male. In some embodiments, the donor is a female and the recipient is a female. In some embodiments, the engineered cells comprise modifications that reduce the expression of antigens (such as tropocadherin Y and/or glial protein Y). In some embodiments, the gene encoding tropocadherin Y (PCDH 11Y; ensembl ID ENSG 00000099715) is reduced or eliminated, e.g., knocked out, in the engineered cells. In some embodiments, the gene encoding glial protein Y (NLGN 4Y; ensembl ID ENSG 00000165246) is reduced or eliminated, e.g., knocked out, in the engineered cell. Any method for reducing or eliminating gene expression, such as any of the methods described herein, may be used. In some embodiments, PCDH11Y and/or NLGN4Y in the engineered cells are reduced or eliminated by nuclease-mediated gene editing methods (such as using a CRISPR/Cas system).

D. Exemplary embodiments of engineered cells

In some embodiments, cells (e.g., engineered beta islet cells, hepatocytes, or other cell types that come into contact with blood during transplantation) and populations thereof exhibit increased CD47 expression, reduced CD142 expression, and reduced expression of one or more molecules of the MHC class I complex. In some embodiments, the cells and populations thereof exhibit increased CD47 expression, reduced CD142 expression, and reduced expression of one or more molecules of MHC class I and MHC class II complexes. In some embodiments, the engineered cells express one or more exogenous complement inhibitor polypeptides selected from the group consisting of CD46, CD59, CD55, and any combination thereof.

In some embodiments, the cells and populations thereof exhibit increased CD47 expression, decreased CD142 expression, and decreased B2M expression. In some embodiments, the cells and populations thereof exhibit increased CD47 expression, decreased CD142 expression, and decreased CIITA expression. In some embodiments, the cells and populations thereof exhibit increased CD47 expression, decreased CD142 expression, and decreased NLRC5 expression. In some embodiments, the cells and populations thereof exhibit increased CD47 expression, decreased CD142 expression, and decreased expression of one or more molecules of B2M and CIITA. In some embodiments, the cells and populations thereof exhibit increased CD47 expression, decreased CD142 expression, and decreased expression of one or more molecules of B2M and NLRC 5. In some embodiments, the cells and populations thereof exhibit increased CD47 expression, decreased CD142 expression, and decreased expression of one or more molecules of B2M, CIITA and NLRC 5. Any of the cells described herein may also exhibit increased expression of one or more factors selected from the group including, but not limited to: DUX4, CD24, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, IL-39, fasL, CCL21, CCL22, mfge and Serpinb9. In some embodiments, the engineered cells express one or more exogenous complement inhibitor polypeptides selected from the group consisting of CD46, CD59, CD55, and any combination thereof.

In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, decreased expression of CD142, and decreased expression of one or more molecules of the MHC class I complex. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, decreased expression of CD142, and decreased expression of one or more molecules of the MHC class II complex. in some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, decreased expression of CD142, and decreased expression of one or more molecules of MHC class II and MHC class II complexes. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, decreased expression of CD142, and decreased expression of B2M. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, decreased expression of CD142, and decreased expression of CIITA. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, decreased expression of CD142, and decreased expression of NLRC 5. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, decreased expression of CD142, and decreased expression of one or more molecules of B2M and CIITA. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, decreased expression of CD142, and decreased expression of one or more molecules of B2M and NLRC 5. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, decreased expression of CD142, and decreased expression of one or more molecules of CIITA and NLRC 5. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, decreased expression of CD142, and decreased expression of one or more molecules of B2M, CIITA and NLRC 5.

Those skilled in the art will appreciate that expression levels (such as increased or decreased expression of a gene, protein, or molecule) may be referenced to or compared with comparable cells. In some embodiments, an engineered cell (e.g., an engineered beta islet cell or liver cell) having increased expression of a protein (e.g., CD46, CD59, CD55, CD47, or any other tolerogenic factor) refers to a modified cell (e.g., an engineered beta islet cell or liver cell) having a higher level of protein than an unmodified cell. In some embodiments, an engineered cell (e.g., an engineered β islet cell or liver cell) having increased expression of a protein (e.g., CD46, CD59, CD55, CD47, or any other tolerogenic factor) is a cell comprising a modification, wherein the cell comprising the modification has a higher level of the protein than a cell without the modification (e.g., a stem cell without the modification may comprise other modifications). In some embodiments, an engineered cell (e.g., an engineered β islet cell or liver cell) having reduced expression of a protein (e.g., CD142, B2M, or CIITA) is a cell comprising a modification, wherein the cell comprising the modification has a lower level of protein or RNA than a cell without the modification (e.g., a stem cell without the modification may comprise other modifications). In some embodiments, the engineered cells express one or more exogenous complement inhibitor polypeptides selected from the group consisting of CD46, CD59, CD55, and any combination thereof.

In one embodiment, provided herein are engineered cells (e.g., engineered beta islet cells or liver cells) that express exogenous CD47 polypeptides and have reduced CD142 expression and reduced expression of one or more MHC class I complex proteins, one or more MHC class II complex proteins, or any combination of MHC class I and class II complex proteins. In another embodiment, the cells express exogenous CD47 polypeptides and express reduced levels of CD142, B2M, and CIITA polypeptides. In some embodiments, the cells express exogenous CD47 polypeptides and have modifications of CD142, B2M, and CIITA genes. In some cases, the modification inactivates B2M and CIITA genes. In some embodiments, the engineered cells express one or more exogenous complement inhibitor polypeptides selected from the group consisting of CD46, CD59, CD55, and any combination thereof.

In some embodiments, cells (e.g., engineered beta islet cells, hepatocytes, or other cell types that come into contact with blood during transplantation) and populations thereof exhibit increased CD47 expression, as well as reduced expression of one or more molecules of the MHC class I complex, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT), and/or activated protein C). In some embodiments, the cells and populations thereof exhibit increased CD47 expression and reduced expression of one or more molecules of MHC class I and MHC class II complexes, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT) and/or activated protein C). In some embodiments, the engineered cells express one or more exogenous complement inhibitor polypeptides selected from the group consisting of CD46, CD59, CD55, and any combination thereof.

In some embodiments, the cells and populations thereof exhibit increased CD47 expression and decreased B2M expression, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT) and/or activated protein C). In some embodiments, the cells and populations thereof exhibit increased CD47 expression and decreased CIITA expression, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT) and/or activated protein C). In some embodiments, the cells and populations thereof exhibit increased CD47 expression and decreased NLRC5 expression, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT) and/or activated protein C). In some embodiments, the cells and populations thereof exhibit increased CD47 expression and reduced expression of one or more molecules of B2M and CIITA, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT) and/or activated protein C). In some embodiments, the cells and populations thereof exhibit increased CD47 expression and reduced expression of one or more molecules of B2M and NLRC5, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT) and/or activated protein C). In some embodiments, the cells and populations thereof exhibit increased CD47 expression and reduced expression of one or more molecules of B2M, CIITA and NLRC5, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT) and/or activated protein C). Any of the cells described herein may also exhibit increased expression of one or more factors selected from the group including, but not limited to: DUX4, CD24, CD27, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, IL-39, fasL, CCL21, CCL22, mfge8 and Serpinb9. In some embodiments, the engineered cells express one or more exogenous complement inhibitor polypeptides selected from the group consisting of CD46, CD59, CD55, and any combination thereof.

In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and decreased expression of one or more molecules of the MHC class I complex, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT), and/or activated protein C). In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and decreased expression of one or more molecules of the MHC class II complex, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT), and/or activated protein C). In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and decreased expression of one or more molecules of MHC class II and MHC class II complexes, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT), and/or activated protein C). In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and reduced expression of B2M, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT), and/or activated protein C). In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and reduced expression of CIITA, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT), and/or activated protein C). In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one complement inhibitor selected from the group consisting of CD46, CD59, CD55, and any combination thereof, and decreased expression of one or more molecules of B2M and CIITA, and are administered in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT), and/or activated protein C).

In one embodiment, provided herein are engineered cells (e.g., engineered beta islet cells or liver cells) that express exogenous CD47 polypeptides and have reduced expression of one or more MHC class I complex proteins, one or more MHC class II complex proteins, or any combination of MHC class I and class II complex proteins. In another embodiment, the cell expresses an exogenous CD47 polypeptide and expresses reduced levels of B2M and CIITA polypeptides. In some embodiments, the cells express exogenous CD47 polypeptides and have modifications of B2M and CIITA genes. In some cases, the modification inactivates B2M and CIITA genes. In some embodiments, the engineered cells express one or more exogenous complement inhibitor polypeptides selected from the group consisting of CD46, CD59, CD55, and any combination thereof. In some embodiments, the engineered cells are administered to the patient in combination with an anticoagulant (e.g., heparin, melagatran, LMW-DS, N-acetylcysteine, alpha-1 antitrypsin (AAT), and/or activated protein C).

E. determination of the Low immunogenicity phenotype

In some embodiments, the provided engineered cells are modified such that they are capable of evading immune recognition and response when administered to a patient (e.g., a recipient subject). Cells can evade killing of immune cells in vitro and in vivo. In some embodiments, the cells evade killing of macrophages and NK cells. In some embodiments, the cells are ignored by the immune cells or the immune system of the subject. In other words, cells administered according to the methods described herein are not detectable by immune cells of the immune system. In some embodiments, the cells are masked and thus immune rejection is avoided.

Methods for determining whether an engineered cell provided herein evades immune recognition include, but are not limited to, IFN- γ Elispot assays, microglial killing assays, cell implantation animal models, cytokine release assays, ELISA, killing assays using bioluminescence imaging or chromium release assays or Xcelligence assays, mixed lymphocyte reactions, immunofluorescence assays, and the like.

In some embodiments, the immunogenicity of the cells is assessed in a Complement Dependent Cytotoxicity (CDC) assay. CDC can be determined in vitro by incubating cells with IgG or IgM antibodies directed against HLA-independent antigens expressed on the cell surface in the presence of serum containing complement and analyzing cell killing. In some embodiments, CDC may be determined by incubating cells with ABO blood group incompatible serum, wherein the cells comprise an a antigen or a B antigen and the serum comprises antibodies to the a antigen and/or the B antigen of the cells.

In some embodiments, once the engineered cells are modified or generated as described herein, their low immunogenicity can be determined. Any of a variety of assays can be used to assess whether a cell is hypoimmunogenic or can evade the immune system. Exemplary assays include any of the assays described in WO2016183041 and WO 2018132783.

In some embodiments, an engineered cell described herein survives in a host without stimulating the host immune response for one week or more (e.g., one week, two weeks, one month, two months, three months, six months, one year, two years, three years, four years, five years, or more, e.g., the lifetime of the cell and/or its progeny). As long as the cell survives the host, the cell will maintain the expression of the transgene and/or the expression of the target gene in the cell will be deleted or reduced. In some aspects, the engineered cells may be removed by the immune system of the host when the transgene is no longer expressed and/or when the target gene is expressed. In some embodiments, the persistence or survival of an engineered cell can be monitored after administration to a recipient by further expressing a transgene encoding a protein (e.g., a fluorescent protein such as GFP, truncated receptor, or other surrogate or other detectable marker) that allows the cell to be detected in vivo.

The hypoimmunogenic cells are administered in a manner that allows them to be implanted into the desired tissue site and to reconstitute or regenerate the functionally defective region. In some embodiments, implantation (e.g., successful implantation) of the low-immunogenicity cells is determined. In some embodiments, the engraftment of the low-immunogenicity cells is evaluated after a preselected amount of time. In some embodiments, the cell survival of the implanted cells is monitored. For example, cell survival can be monitored via bioluminescence imaging (BLI), wherein cells are transduced with a luciferase expression construct to monitor cell survival. In some embodiments, the implanted cells are visualized by immunostaining and imaging methods known in the art. In some embodiments, the implanted cells express a known biomarker that can be detected to determine successful implantation. For example, flow cytometry may be used to determine the surface expression of a particular biomarker. In some embodiments, the low-immunogenicity cells are implanted at the desired tissue site as desired (e.g., successful implantation of the low-immunogenicity cells). In some embodiments, the hypoimmunogenic cells are implanted as desired to a tissue site, such as a cell defect site. In some embodiments, the low-immunogenicity cells are implanted into the desired tissue site in the same manner as the same type of cells that do not contain the modification.

In some embodiments, administering an engineered cell population (e.g., comprising modified engineered beta islet cells or hepatocytes comprising reduced CD142 expression) or a combination (e.g., administering an engineered cell population in combination with an anticoagulant) improves survival and implantation by allowing the cells to avoid or reduce IBMIR that occurs as a result of cell exposure to blood during transplantation. In some embodiments, the reduction in IBMIR reduces the amount of cell loss (e.g., loss of transplanted islets or hepatocytes) that occurs during transplantation.

In some embodiments, the function of the hypoimmunogenic cells is determined. In some embodiments, the function of the hypoimmunogenic cells is determined prior to implantation into the desired tissue site. In some embodiments, the function of the hypoimmunogenic cells is determined after implantation into the desired tissue site. In some embodiments, the function of the hypoimmunogenic cells is assessed after a preselected amount. In some embodiments, the function of the implanted cells is assessed by the ability of the cells to produce a detectable phenotype. For example, implanted beta islet cell function can be evaluated based on the restoration of glucose control lost due to diabetes. In some embodiments, the function of the low-immunogenicity cells is as intended (e.g., the low-immunogenicity cells function successfully while avoiding antibody-mediated rejection). In some embodiments, the function of the hypoimmunogenic cells is as desired, such as having sufficient function at the site of cell defect while avoiding antibody-mediated rejection. In some embodiments, the engineered cells function in the same manner as non-engineered cells of the same type.

10. Immediate blood-mediated inflammatory response

In some embodiments, the engineered cells provided herein evade immediate blood-mediated inflammatory responses. One major cause of poor clinical islet transplantation is the occurrence of a destructive immediate blood-mediated inflammatory response (IBMIR), which results in loss of transplanted tissue when islets encounter blood in the portal vein (Bennet et al, (1995) Diabetes 48:1907-1914; moberg et al, (2002) Lancet 360:2039-2045). This response is triggered by Tissue Factor (TF) expression of endocrine cells of the islets in combination with a series of other pro-inflammatory events such as the expression of MCP-1 (Piemonti et al, (2002) Diabetes 51:55-65), IL-8 and MIF (Waeber et al, (1997) Proc NATL ACAD SCI USA 94:4782-4787; johansson et al, (2006) Am J Transplantation (2): 305).

An immediate blood-mediated inflammatory response (IBMIR) is a non-specific inflammatory and thrombotic response that can occur when CD142 expressing cells are contacted with blood. IBMIR is rapidly initiated by human blood exposure to the portal vein. Which is characterized by activation of complement, platelets, and the coagulation pathway, which in turn leads to recruitment of neutrophils. IBMIR results in significant loss of transplanted islets or hepatocytes. In some embodiments, provided herein are compositions (e.g., engineered cells comprising reduced expression of CD142 in combination with one or more other modifications described herein), combinations (e.g., combinations comprising any of the engineered cell populations described herein and anticoagulants that reduce blood coagulation), and methods of reducing IBMIR associated with cell transplantation or cell exposure to blood (e.g., methods of treating a patient comprising administering any of the engineered cell populations described herein and anticoagulants that reduce blood coagulation).

In some embodiments, IBMIR can be determined in vitro, for example in the IBMIR's extracorporeal tube circulation model (in vitro tubing loop model), which has been previously described in U.S. patent No. 7,045,502, which is incorporated by reference herein in its entirety.

In some embodiments, IBMIR can be determined in vivo (e.g., in a mammal or in a human patient) by taking a blood sample during peri-transplantation and assessing the plasma levels of: thrombin-antithrombin III complex (TAT), C peptide, XIa factor-antithrombin (FXIa-AT), XIIa factor-antithrombin (FXIIa-AT), thrombin-antithrombin (TAT), plasmin- α2 antiplasmin (PAP) and/or complement C3a. In some embodiments, IBMIR is associated with increased levels of TAT, C-peptide, FXIa-AT, FXIIa-AT, PAP, and/or complement C3a during infusion of transplanted cells and/or during a period of time following transplantation (e.g., up to 3, 5, 10, or more than 10 hours following transplantation). In some embodiments, IBMIR can be determined by monitoring the count of free circulating platelets, wherein a decrease in platelet count during or after transplantation is correlated with IBMIR (e.g., correlated with IBMIR-induced platelet consumption).

11. Complement dependent cytotoxicity

In some embodiments, the engineered cells provided herein (e.g., beta islets) evade Complement Dependent Cytotoxicity (CDC). In some embodiments, CDC is secondary to IBMIR's thrombotic reaction. In some embodiments, CDC occurs independent of IBMIR.

In some embodiments, the sensitivity of the cells to CDC may be analyzed in vitro according to standard protocols understood by one of ordinary skill in the art. In some embodiments, CDC may be analyzed in vitro by mixing serum (e.g., human serum) comprising components of the complement system with target cells to which antibodies (e.g., igG or IgM antibodies) bind, and then determining cell death. In some embodiments, the sensitivity of cells to CDC may be analyzed in vitro by incubating the cells in the presence of ABO-incompatible or Rh factor-incompatible serum comprising components of the complement system and antibodies to ABO type a, ABO type B, and/or Rh factor antigens of the cells.

Common CDC assays determine cell death via preloading of radioactive compounds on target cells. When the cells die, the radioactive compound is released therefrom. Thus, the efficacy of antibodies in mediating cell death was determined by radioactivity levels. Unlike radioactive CDC assays, non-radioactive CDC assays typically use fluorescent or luminescent assays to determine the release of a large number of cellular components (such as GAPDH). In some embodiments, a label-free platform, such as xcelligent ^TM (Agilent), may be used to analyze CDC-induced cell killing.

Engineered cell populations and pharmaceutical compositions

Provided herein are engineered cell populations comprising a plurality of the provided engineered cells.

In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise a set of modifications described herein. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise a set of modifications that reduce the expression of MHC class I molecules and/or one or more MHC class II molecules, increase the expression of one or more tolerogenic factors, and reduce the expression of CD 142. In some embodiments, the one or more tolerogenic factors are one or more of the following: DUX4, B2M-HLA-E, CD, CD52, CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8, SERPINB9, CD35, IL-39, CD16 Fc receptor, IL15-RF and H2-M3 or any combination thereof. In some embodiments, the one or more tolerogenic factors is CD47. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise an exogenous polynucleotide encoding CD 46. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise an exogenous polynucleotide encoding CD 59. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise an exogenous polynucleotide encoding CD 55.

In some embodiments, at least 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise one or more alterations that inactivate both alleles of the B2M gene. In some embodiments, at least 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise one or more alterations that inactivate both alleles of the CIITA gene. In some embodiments, at least 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of the cells in the population comprise one or more alterations that inactivate both alleles of the CD142 gene.

Also provided herein are compositions comprising the engineered cells or engineered cell populations. In some embodiments, the composition is a pharmaceutical composition.

In some embodiments, the pharmaceutical compositions provided herein further comprise a pharmaceutically acceptable excipient or carrier. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexahydrocarbon quaternary ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins such as serum albumin, gelatin, or immunoglobulins, and the like; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zn-protein complexes); and/or nonionic surfactants such as polysorbates (TWEEN ^TM), poloxamers (PLURONICS ^TM), or polyethylene glycols (PEG). In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable buffer (e.g., neutral buffered saline or phosphate buffered saline). In some embodiments, the pharmaceutical composition may contain one or more excipients for altering, maintaining or maintaining, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeation of the composition. In some aspects, the skilled artisan will appreciate that the pharmaceutical composition comprising the cell may be different from the pharmaceutical composition comprising the protein.

The term "pharmaceutical formulation" refers to a formulation in a form that allows for the biological activity of the active ingredient contained therein to be effective, and which does not contain other components that have unacceptable toxicity to the subject to whom the formulation is to be administered.

By "pharmaceutically acceptable carrier" is meant an ingredient other than the active ingredient in a pharmaceutical formulation that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to: buffers, excipients, stabilizers or preservatives.

In some embodiments the pharmaceutical composition contains an amount (such as a therapeutically effective amount or a prophylactically effective amount) of an engineered cell as described herein effective to treat or prevent a disease or disorder. In some embodiments, the pharmaceutical composition contains an amount (such as a therapeutically effective amount or a prophylactically effective amount) of an engineered cell as described herein effective to treat or prevent a disease or disorder. In some embodiments, the treatment or prevention efficacy is monitored by periodic assessment of the subject being treated. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and may be determined. The desired dose may be delivered by a single bolus administration of the composition, by multiple bolus administration of the composition, or by continuous infusion administration of the composition.

In some embodiments, the engineered cells as described herein are administered using standard administration techniques, formulations, and/or devices. In some embodiments, the engineered cells as described herein are administered using standard administration techniques, formulations, and/or devices. Formulations and devices, such as syringes and vials, for storing and administering the compositions are provided. The engineered cells may be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When a therapeutic composition (e.g., a pharmaceutical composition containing engineered cells) is administered, it is typically formulated in unit dose injectable form (solution, suspension, emulsion).

Formulations include those for intravenous, intraperitoneal or subcutaneous administration. In some embodiments, the population of cells is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, the population of cells is administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

In some embodiments, the compositions are provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, or dispersions, which in some aspects may be buffered to a selected pH. Liquid compositions are somewhat more convenient to administer, particularly by injection. The liquid composition may comprise a carrier, which may be a solvent or dispersion medium containing, for example, water, brine, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof. Sterile injectable solutions may be prepared by incorporating the cells in a solvent, such as with a suitable carrier, diluent or excipient (such as sterile water, physiological saline, dextrose, or the like).

In some embodiments, pharmaceutically acceptable carriers may include all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like (Gennaro,2000,Remington:The science and practice of pharmacy,Lippincott,Williams&Wilkins,Philadelphia,PA). examples of such carriers or diluents include, but are not limited to, water, saline, ringer' ssolution, dextrose solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. Supplementary active compounds may also be incorporated into the compositions. The drug carrier should be one suitable for engineering cells, such as saline solution, dextrose solution, or a solution comprising human serum albumin. In some embodiments, the pharmaceutically acceptable carrier or vehicle of such compositions is any non-toxic aqueous solution in which the engineered cells can remain or remain viable for a time sufficient to allow administration of the viable cells. For example, the pharmaceutically acceptable carrier or vehicle may be an aqueous saline solution or a buffered aqueous saline solution.

In some embodiments, the composition (including pharmaceutical compositions) is sterile. In some embodiments, the isolation, enrichment, or culture of the cells is performed in a closed or sterile environment (e.g., in a sterile culture bag) to minimize errors, user handling, and/or contamination. In some embodiments, sterility can be readily achieved, for example, by filtration through a sterile filtration membrane. In some embodiments, the culturing is performed using a gas-permeable culture vessel. In some embodiments, the culturing is performed using a bioreactor.

Also provided herein are compositions suitable for cryopreserving the provided engineered cells. In some embodiments, the provided engineered cells are cryopreserved in a cryopreservation medium. In some embodiments, the cryopreservation medium is a serum-free cryopreservation medium. In some embodiments, the composition comprises a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and/or glycerol. In some embodiments, the cryopreservation medium is DMSO (volume/volume) between 5% or about 5% and 10% or about 10%. In some embodiments, the cryopreservation medium is or is about 5% DMSO (volume/volume). In some embodiments, the cryopreservation medium is or is about 6% DMSO (volume/volume). In some embodiments, the cryopreservation medium is or is about 7% DMSO (volume/volume). In some embodiments, the cryopreservation medium is or is about 7.5% DMSO (volume/volume). In some embodiments, the cryopreservation medium is or is about 8% DMSO (volume/volume). In some embodiments, the cryopreservation medium is or is about 9% DMSO (volume/volume). In some embodiments, the cryopreservation medium is or is about 10% DMSO (volume/volume). In some embodiments, the cryopreservation media comprises a commercially available cryopreservation solution (CryoStor ^TMCS10).CryoStor^TM CS10 is a cryopreservation media comprising 10% dimethyl sulfoxide (DMSO). In some embodiments, compositions formulated for cryopreservation may be stored at low temperatures, such as ultra low temperatures, for example, at a temperature range of-40 ℃ to-150 ℃ (such as or about 80 ℃ + -6.0 ℃).

In some embodiments, the pharmaceutical composition comprises an engineered cell described herein and a pharmaceutically acceptable carrier comprising 31.25% (v/v) Plasma-Lyte a, 31.25% (v/v) 5% dextrose/0.45% sodium chloride, 10% dextran 40 (LMD)/5% dextrose, 20% (v/v) 25% Human Serum Albumin (HSA), and 7.5% (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the cryopreserved engineered cells are prepared for thawing administration. In some cases, the engineered cells can be administered to the subject immediately after thawing. In such embodiments, the composition may be used without any further processing. In other cases, the engineered cells are further processed after thawing (such as by resuspension with a pharmaceutically acceptable carrier, incubation with an activator or stimulator), or are activated and washed and resuspended in a pharmaceutically acceptable buffer prior to administration to a subject.

IV kits, components and articles of manufacture

In some aspects, provided herein are kits, components, and compositions (such as consumables) of the methods, devices, and systems described herein. In some embodiments, the kit includes instructions for use according to the disclosure herein.

In some embodiments, provided herein is a kit or combination comprising an engineered cell population described herein and an anticoagulant or cell coating that reduces blood coagulation. In some embodiments, provided herein is a kit or combination comprising: (a) A cell population comprising a plurality of engineered cells, wherein the engineered cells comprise a modification that (I) increases expression of CD47, and (II) decreases expression of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules), wherein increased expression of (I) and decreased expression of (II) are relative to a cell of the same cell type that does not comprise the modification; and (b) an anticoagulant. In some embodiments, provided herein is a kit or combination comprising: (a) A cell population comprising a plurality of engineered cells, wherein the engineered cells comprise a modification that (i) increases expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD 55; (ii) Increasing expression of CD47, and (iii) decreasing expression of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules), wherein increased expression of (I) and (II) and decreased expression of (iii) are relative to a cell of the same cell type that does not comprise the modification; and (b) an anticoagulant.

In some embodiments, the anticoagulant is selected from the group consisting of heparin, an antithrombin activator, a factor II (fhi) inhibitor, a factor VII (fhi) inhibitor, and a factor X (fX) inhibitor. In some embodiments, the anticoagulant is heparin. In some embodiments, heparin is plain heparin. In some embodiments, heparin is low molecular weight heparin. In some embodiments, heparin is soluble heparin. In some embodiments, the anticoagulant is melagatran or LMW-DS. In some embodiments, wherein the anticoagulant is N-acetylcysteine (NAC). In some embodiments, the anticoagulant is alpha-1 antitrypsin (AAT) and/or activated protein C. In some embodiments, the anticoagulant is an antibody directed against CD 142.

In some embodiments of the invention, articles of manufacture are provided that contain materials useful in clinical transplantation therapies, including cell therapies. In some embodiments, the article of manufacture contains materials useful for treating cellular defects such as, but not limited to, diabetes (e.g., type I diabetes), vascular disorders or diseases, autoimmune thyroiditis, liver diseases (e.g., liver cirrhosis), corneal diseases (e.g., fuchs dystrophy or congenital genetic endothelial dystrophy), kidney diseases and cancers (e.g., B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous lymphoblastic leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer). The article of manufacture may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like (e.g., glass or plastic containers). Typically, the container contains a composition that is effective for allogeneic cell therapy, and may have a sterile access port (e.g., the container may be an intravenous fluid bag (intravenous solution bag) or a vial having a stopper pierceable by a hypodermic injection needle).

In some aspects, the kits or articles of manufacture provided herein include an engineered cell, such as any of the engineered cells provided herein (e.g., engineered beta islet cells or hepatocytes). In some embodiments, the kit or article of manufacture comprises a composition comprising a population of engineered beta islet cells, wherein the engineered beta islet cells comprise: (i) a transgene comprising an exogenous polynucleotide encoding CD47, (ii) inactivation or disruption of both alleles of the CD142 gene, and (iii) inactivation or disruption of both alleles of the B2M gene. In some embodiments, the engineered β -cells further comprise inactivation or disruption of both alleles of the CIITA gene. In some embodiments, the kit or article of manufacture comprises a composition comprising an engineered population of hepatocytes, wherein the engineered hepatocytes comprise: (i) a transgene comprising an exogenous polynucleotide encoding CD47, (ii) inactivation or disruption of both alleles of the CD142 gene, and (iii) inactivation or disruption of both alleles of the B2M gene. In some embodiments, the engineered hepatocyte comprises inactivation or disruption of both alleles of the CIITA gene.

The label or package insert indicates that the composition is to be used to treat a particular disorder. The label or package insert will also include instructions for administering the pharmaceutical composition to a patient. In some embodiments, the article of manufacture comprises a combination therapy.

The article of manufacture and/or kit may further comprise packaging instructions. The instructions refer to instructions, typically included in commercial packages of therapeutic products, that contain information regarding the indication, usage, dosage, administration, contraindications, and/or warnings of using such therapeutic products.

V. therapeutic methods

Provided herein are compositions and methods relating to provided cell compositions comprising an engineered cell population as described herein for use in treating a disease or disorder in a subject. Provided herein are methods of treating a patient by administering the engineered cell populations described herein. In some embodiments, the population of cells is formulated for administration in a pharmaceutical composition, such as any of the pharmaceutical compositions described herein. Such methods and uses include therapeutic methods and uses, for example, involving administering an engineered cell population or a composition containing the same to a subject suffering from a disease, disorder, or condition. It is within the level of one skilled in the art to select the appropriate engineered cells for a particular disease indication as provided herein. In some embodiments, the cells or pharmaceutical compositions thereof are administered in an amount effective to effect treatment of the disease or disorder. Uses include the use of engineered cells or pharmaceutical compositions thereof in such methods and treatments, and the use in the manufacture of medicaments for performing such methods of treatment. In some embodiments, the method thereby treats a disease or disorder or condition in a subject.

The engineered cells provided herein can be administered to any suitable patient, including, for example, candidates for cell therapies for treating a disease or disorder. Candidates for cell therapy include any patient suffering from a disease or disorder that may benefit from the therapeutic effects of the subject engineered cells provided herein. In some embodiments, the patient is an allogeneic recipient of the administered cells. In some embodiments, the engineered cells provided are effective for use in allogeneic cell therapy. Candidates that benefit from the therapeutic effects of the subject engineered cells provided herein exhibit elimination, reduction, or amelioration of a disease or disorder.

In some embodiments, engineered cells as provided herein (including those produced by any of the methods provided herein) can be used in cell therapies. The therapeutic cells outlined herein may be used to treat disorders such as, but not limited to, cancer, genetic disorders, chronic infectious diseases, autoimmune disorders, neurological disorders, and the like.

In some embodiments, the patient has a cellular defect. As used herein, "cell defect" refers to any disease or condition that causes dysfunction or loss of a cell population in a patient, wherein the patient is unable to naturally replace or regenerate the cell population. Exemplary cellular defects include, but are not limited to, autoimmune diseases (e.g., multiple sclerosis, myasthenia gravis, rheumatoid arthritis, diabetes, systemic lupus erythematosus), neurodegenerative diseases (e.g., huntington's disease and parkinson's disease), cardiovascular disorders and diseases, vascular disorders and diseases, corneal disorders and diseases, liver disorders and diseases, thyroid disorders and diseases, and kidney disorders and diseases. In some embodiments, the patient to whom the engineered cells are administered has cancer. Exemplary cancers that can be treated by the engineered cells provided herein include, but are not limited to, B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous lymphoblastic leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer. In certain embodiments, a cancer patient is treated by administering the engineered CAR T cells provided herein.

In some embodiments, provided herein is a method of administering an engineered cell population to a patient in need thereof, wherein the engineered cell is contacted with blood during or after administration, and wherein the engineered cell comprises a modification that prevents or reduces IBMIR when the engineered cell is contacted with blood. In some embodiments, the engineered cells are administered intravenously or via intramuscular injection. In some embodiments, the engineered cells overexpress tolerogenic factors (e.g., CD 47), have reduced expression or lack of expression of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules), and have reduced expression of CD 142. In some embodiments, the engineered cell is a beta islet cell or a liver cell. In some embodiments, the engineered cells further comprise overexpression of one or more complement inhibitors.

In some embodiments, provided herein is a method of administering an engineered population of cells to a patient in need thereof, wherein the engineered cells are contacted with blood during or after administration, wherein the engineered cells overexpress tolerogenic factors (e.g., CD 47) and have reduced expression or lack of expression of one or more MHC class I molecules and/or one or more MHC class II molecules (e.g., one or more MHC class I human leukocyte antigen molecules and/or one or more MHC class II human leukocyte antigen molecules), and wherein the method further comprises administering an anticoagulant to the patient. In some embodiments, the engineered cell further comprises overexpression of one or more complement inhibitors selected from CD46, CD59, and CD 55. In some embodiments, the engineered cell comprises overexpression of CD46 and CD 59. In some embodiments, the engineered cells comprise CD46, CD59, and overexpression of CD 59.

In some embodiments, the cell defect is associated with diabetes, or the cell therapy is used to treat diabetes, optionally wherein the diabetes is type I diabetes. In some embodiments, the engineered cell population is a population of islet cells (including beta islet cells). In some embodiments, the islet cells are selected from the group consisting of islet progenitor cells, immature islet cells, and mature islet cells. In some embodiments, the method comprises administering to the patient a composition comprising a population of engineered beta islet cells, wherein the engineered beta islet cells comprise: (i) a transgene comprising an exogenous polynucleotide encoding CD47, (ii) inactivation or disruption of both alleles of the CD142 gene, and (iii) inactivation or disruption of both alleles of the B2M gene. In some embodiments, the method comprises administering to the patient a composition comprising a population of engineered beta islet cells, wherein the engineered beta islet cells comprise: (i) A transgene comprising an exogenous polynucleotide encoding CD47, and (ii) inactivation or disruption of both alleles of a B2M gene, wherein the method further comprises administering an anticoagulant (e.g., heparin or any anticoagulant described in section V.C) to the patient. In some embodiments, the engineered β -cell comprises inactivation or disruption of both alleles of the CIITA gene. In some embodiments, the transgene comprising a polynucleotide encoding CD47 is a polycistronic vector, and the transgene further comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59. In other embodiments, the beta islet cells further comprise a single polycistronic vector, wherein the polycistronic vector comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59.

In some embodiments, the cell defect is associated with a liver disease, or the cell therapy is used to treat a liver disease. In some embodiments, the liver disease comprises liver cirrhosis. In some embodiments, the population of cells is a population of hepatocytes or hepatic progenitors. In some embodiments, the method comprises administering to the patient a composition comprising an engineered population of hepatocytes, wherein the engineered hepatocytes comprise: (i) a transgene comprising an exogenous polynucleotide encoding CD47, (ii) inactivation or disruption of both alleles of the CD142 gene, and (iii) inactivation or disruption of both alleles of the B2M gene. In some embodiments, the method comprises administering to the patient a composition comprising an engineered population of hepatocytes, wherein the engineered hepatocytes comprise: (i) A transgene comprising an exogenous polynucleotide encoding CD47, and (ii) inactivation or disruption of both alleles of a B2M gene, wherein the method further comprises administering an anticoagulant (e.g., heparin or any anticoagulant described in section V.C) to the patient. In some embodiments, the engineered hepatocyte comprises inactivation or disruption of both alleles of the CIITA gene. In some embodiments, the transgene comprising a polynucleotide encoding CD47 is a polycistronic vector, and the transgene further comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59. In other embodiments, the hepatocyte further comprises a single polycistronic vector, wherein the polycistronic vector comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59.

In some embodiments, the engineered cells provided herein, or compositions containing the same, can be used to treat patients sensitized with one or more antigens present in a previous graft (such as, for example, a cell graft, a blood transfusion, a tissue graft, or an organ graft). In certain embodiments, the previous graft is an allograft and the patient is sensitive to one or more autoantigens from the allograft. Allografts include, but are not limited to, allograft cell grafts, allograft blood transfusion, allograft tissue grafts or allograft organ grafts. In some embodiments, the patient is a pregnant or already pregnant sensitized patient (e.g., is or has been alloimmunized during pregnancy). In certain embodiments, the patient is sensitized with one or more antigens contained in a previous graft, wherein the previous graft is a modified human cell, tissue or organ. In some embodiments, the modified human cell, tissue or organ is a modified autologous human cell, tissue or organ. In some embodiments, the previous graft is a non-human cell, tissue or organ. In exemplary embodiments, the previous graft is a modified non-human cell, tissue or organ. In certain embodiments, the previous graft is a chimeric comprising a human component. In certain embodiments, the previous graft is a CAR T cell. In certain embodiments, the previous graft is an autograft and the patient is sensitive to one or more autoantigens from the autograft. In certain embodiments, the previous graft is an autologous cell, tissue or organ. In certain embodiments, the sensitized patient suffers from allergy and is sensitive to one or more allergens. In exemplary embodiments, the patient has hay fever, food allergy, insect allergy, drug allergy, or atopic dermatitis.

In some embodiments, a patient undergoing treatment with the provided engineered cells or compositions containing the same receives prior treatment. In some embodiments, the engineered cells or compositions containing the same are used to treat the same disorder as the previous treatment. In certain embodiments, the engineered cells or compositions containing the same are used to treat a condition different from a previous treatment. In some embodiments, the engineered cells administered to a patient or a composition containing the same exhibit enhanced therapeutic effects for treating the same condition or disease treated by a previous treatment. In certain embodiments, the engineered cells or compositions containing the same administered exhibit a longer therapeutic effect for treating a disorder or disease in a patient than previous treatments. In exemplary embodiments, the administered cells exhibit enhanced efficacy, and/or specificity against cancer cells as compared to previous treatments. In particular embodiments, the engineered cell is a CAR T cell for use in treating cancer.

The methods provided herein can be used as a two-wire treatment for a particular condition or disease after failure of a first-wire treatment. In some embodiments, the previous treatment is a therapeutically ineffective treatment. As used herein, a "treatment ineffective" treatment refers to a treatment that produces less than the desired clinical outcome in the patient. For example, in the case of treatment of a cellular defect, a therapeutically ineffective treatment may refer to a treatment that does not achieve the desired level of functional cells and/or cellular activity to replace the defective cells in the patient, and/or lacks persistence of the treatment. For cancer treatment, treatment-ineffective treatment refers to treatment that does not reach the desired level of efficacy, and/or specificity. The therapeutic effect may be measured using any suitable technique known in the art. In some embodiments, the patient responds to the previous therapy. In some embodiments, the previous treatment is a cell, tissue or organ implant that is rejected by the patient. In some embodiments, the prior treatment comprises mechanical assistance treatment. In some embodiments, the mechanical assist treatment comprises hemodialysis or ventricular assist devices. In some embodiments, the patient responds to mechanical assistance therapy. In some embodiments, the previous treatment includes a population of therapeutic cells that includes a safety switch that can cause the therapeutic cells to die if they grow and divide in an undesired manner. In certain embodiments, the patient develops an immune response as the safety switch induces therapeutic cell death. In certain embodiments, the patient is sensitized to a previous treatment. In exemplary embodiments, the patient is not sensitized with engineered cells as provided herein.

In some embodiments, the provided engineered cells or compositions containing the same are administered prior to providing a tissue, organ or partial organ transplant to a patient in need thereof. In particular embodiments, the patient does not exhibit an immune response to the engineered cells. In certain embodiments, the engineered cells are administered to a patient to treat a cellular defect in a particular tissue or organ, and the patient then receives a tissue or organ transplant of the same particular tissue or organ. In such embodiments, the engineered cell therapy functions as a transitional therapy to the final tissue or organ replacement. For example, in some embodiments, the patient has a liver disorder and receives an engineered hepatocyte therapy as provided herein prior to receiving a liver transplant. In certain embodiments, the engineered cells are administered to a patient to treat a cellular defect in a particular tissue or organ, and the patient then receives a tissue or organ transplant of a different tissue or organ. For example, in some embodiments, the patient is a diabetic patient treated with engineered pancreatic β cells as provided herein prior to receiving a kidney transplant. In some embodiments, the methods are for treating a cellular defect. In exemplary embodiments, the tissue or organ transplant is a heart transplant, a lung transplant, a kidney transplant, a liver transplant, a pancreas transplant, an intestine transplant, a stomach transplant, a cornea transplant, a bone marrow transplant, a blood vessel transplant, a heart valve transplant, or a bone transplant.

Methods of treating a patient are generally by administering an engineered cell or composition containing the same as provided herein. It will be appreciated that for all of the various embodiments described herein in connection with cells and/or timing of therapy, administration of the cells is accomplished by a method or pathway that results in at least partial localization of the introduced cells to the desired site. The cells may be implanted directly into the desired site, or administered by any suitable route that results in delivery to the desired site in the subject, at which site at least a portion of the implanted cells or cellular components remain viable. In some embodiments, the cells are administered to treat a disease or disorder, such as any disease, disorder, condition, or symptom thereof that can be alleviated by cell therapy.

In some embodiments, the engineered cell population or composition containing the same is administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, or at least 1 month or more after the patient is sensitized. In some embodiments, the engineered cell population or composition containing the same is administered for at least 1 week (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, or more) or longer after the patient is sensitized or exhibits the sensitized characteristics or properties. In some embodiments, the engineered cell population or composition containing the same is administered for at least 1 month (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months or more) or longer after the patient has received a transplant (e.g., an allograft), has been pregnant (e.g., has undergone or has undergone alloimmunization during pregnancy), or has been sensitized or has exhibited the characteristic or property of sensitization.

In some embodiments, a dosing regimen is administered to a patient who has received a transplant, has been pregnant (e.g., has been immunized during pregnancy or has been immunized with an allo), and/or is susceptible to an antigen (e.g., alloantigen), comprising a first dose administration of an engineered cell population described herein, a recovery period following the first dose, and a second dose administration of the engineered cell population. In some embodiments, the complexes of cell types present in the first cell population and the second cell population are different. In certain embodiments, the complexes of cell types present in the first engineered cell population and the second engineered cell population are the same or substantially identical. In many embodiments, the first engineered cell population and the second engineered cell population comprise the same cell type. In some embodiments, the first engineered cell population and the second engineered cell population comprise different cell types. In some embodiments, the first engineered cell population and the second engineered cell population comprise the same percentage of cell types. In other embodiments, the first engineered cell population and the second cell population comprise different percentages of cell types.

In some embodiments, the recovery period begins after the first administration of the engineered cell population or composition containing the same, and ends when such cells are no longer present or detectable in the patient. In some embodiments, the duration of the recovery period after the initial administration of the cells is at least 1 week (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, or more) or longer. In some embodiments, the duration of the recovery period after the initial administration of the cells is at least 1 month (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, or more) or longer.

In some embodiments, upon administration to a subject, the population of engineered cells administered or the composition containing the same is hypoimmunogenic. In some embodiments, the engineered cells are hyperimmune. In some embodiments, the immune response against the engineered cells is reduced or reduced by at least 5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％ or 99% as compared to the level of immune response generated by administration of an immunogenic cell (e.g., a population of cells of the same or similar cell type or phenotype but without modification (e.g., genetic modification) of the engineered cells). In some embodiments, the population of engineered cells or the composition containing the same administered is incapable of eliciting an immune response against the engineered cells in the patient.

In some embodiments, the administered engineered cell population or composition containing the same elicits reduced or lower levels of systemic TH1 activation in the patient. In some cases, the level of systemic TH1 activation elicited by the cells is at least 5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％ or 99% lower than the level of systemic TH1 activation produced by administration of an immunogenic cell (e.g., a population of cells of the same or similar cell type or phenotype but without modification (e.g., genetic modification) of the engineered cell). In some embodiments, the administered engineered cell population or composition containing the same is incapable of eliciting systemic TH1 activation in a patient.

In some embodiments, the administered engineered cell population or composition containing the same elicits reduced or lower levels of immune activation of Peripheral Blood Mononuclear Cells (PBMCs) in the patient. In some cases, the cells elicit at least 5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％ or 99% lower levels of immune activation of PBMCs as compared to the levels of immune activation of PBMCs produced by administration of immunogenic cells (e.g., a population of cells of the same or similar cell type or phenotype but without modification of the engineered cells (e.g., genetic modification)). In some embodiments, the administered engineered cell population or composition containing the same is incapable of eliciting immune activation of PBMCs in the patient.

In some embodiments, the administered engineered cell population or composition containing the same elicits reduced or lower levels of donor-specific IgG antibodies in the patient. In some cases, the cell elicits a donor-specific IgG antibody level that is at least 5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％ or 99% lower than a donor-specific IgG antibody level produced by administration of an immunogenic cell (e.g., a population of cells of the same or similar cell type or phenotype but without modification (e.g., genetic modification) of an engineered cell). In some embodiments, the population of engineered cells administered is incapable of eliciting donor-specific IgG antibodies in the patient.

In some embodiments, the administered engineered cell population or composition containing the same elicits reduced or lower levels of IgM and IgG antibody production in the patient. In some cases, the cells elicit IgM and IgG antibody production levels that are at least 5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％ or 99% lower than IgM and IgG antibody production levels produced by administration of immunogenic cells (e.g., cell populations of the same or similar cell types or phenotypes but without modification (e.g., genetic modification) of the engineered cells). In some embodiments, the administered engineered cell population or composition containing the same is incapable of eliciting IgM and IgG antibody production in a patient.

In some embodiments, the administered engineered cell population or composition containing the same induces reduced or lower levels of cytotoxic T cell killing in the patient. In some cases, the level of cytotoxic T cell killing induced by the cells is at least 5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％ or 99% lower than the level of cytotoxic T cell killing produced by administration of an immunogenic cell (e.g., a population of cells of the same or similar cell type or phenotype but without modification (e.g., genetic modification) of the engineered cell). In some embodiments, the administered engineered cell population or composition containing the same is incapable of eliciting cytotoxic T cell killing in a patient.

As described above, provided herein are cells that, in certain embodiments, can be administered to a patient that is sensitive to an alloantigen, such as MHC class I and/or MHC class II molecules (e.g., human leukocyte antigens). In some embodiments, the patient is or has been pregnant, for example, having alloimmunity during pregnancy (e.g., fetal and neonatal Hemolysis (HDFN), neonatal Alloimmune Neutropenia (NAN), or fetal and neonatal alloimmune thrombocytopenia (FNAIT)). In other words, the patient has or has had a disorder or condition associated with alloimmunity during pregnancy such as, but not limited to, fetal and neonatal Hemolysis (HDFN), neonatal Alloimmune Neutropenia (NAN), and fetal and neonatal alloimmune thrombocytopenia (FNAIT). In some embodiments, the patient has received an allogeneic transplant, such as, but not limited to, an allogeneic cell transplant, an allogeneic blood transfusion, an allogeneic tissue transplant, or an allogeneic organ transplant. In some embodiments, the patient exhibits memory B cells directed against the alloantigen. In some embodiments, the patient exhibits memory T cells directed against the alloantigen. Such patients may exhibit memory B cells and memory T cells directed against alloantigens.

After administration of the cells, the patient does not exhibit a systemic immune response or a reduced level of systemic immune response compared to a response to non-hypoimmunogenic cells. In some embodiments, the patient does not exhibit or has reduced level of adaptive immune response as compared to response to non-hypoimmunogenic cells. In some embodiments, the patient does not exhibit an innate immune response or a reduced level of an innate immune response as compared to a response to cells that are not hypoimmunogenic. In some embodiments, the patient does not exhibit a T cell immune response or a reduced level of T cell immune response compared to a response to a non-hypoimmunogenic cell. In some embodiments, the patient does not exhibit a B cell immune response or a reduced level of B cell immune response compared to a response to a non-hypoimmunogenic cell.

A. Dosing and dosage regimen

Any therapeutically effective amount of the cells described herein may be included in the pharmaceutical composition, depending on the indication being treated. Non-limiting examples of cells include primary cells (e.g., primary beta islet cells) and cells differentiated from the engineered induced pluripotent stem cells (e.g., beta islet cells or hepatocytes differentiated from ipscs). In some embodiments, the pharmaceutical composition comprises at least about 1x10²、5x10²、1x10³、5x10³、1x10⁴、5x10⁴、1x10⁵、5x10⁵、1x10⁶、5x10⁶、1x10⁷、5x10⁷、1x10⁸、5x10⁸、1x10⁹、5x10⁹、1x10¹⁰ or 5x10 ¹⁰ cells. In some embodiments, the pharmaceutical composition comprises up to about 1x10²、5x10²、1x10³、5x10³、1x10⁴、5x10⁴、1x10⁵、5x10⁵、1x10⁶、5x10⁶、1x10⁷、5x10⁷、1x10⁸、5x10⁸、1x10⁹、5x10⁹、1x10¹⁰ or 5x10 ¹⁰ cells. In some embodiments, the pharmaceutical composition comprises up to about 6.0x10 ⁸ cells. In some embodiments, the pharmaceutical composition comprises up to about 8.0x10 ⁸ cells. In some embodiments, the pharmaceutical composition comprises at least about 1x10²-5x10²、5x10²-1x10³、1x10³-5x10³、5x10³-1x10⁴、1x10⁴-5x10⁴、5x10⁴-1x10⁵、1x10⁵-5x10⁵、5x10⁵-1x10⁶、1x10⁶-5x10⁶、5x10⁶-1x10⁷、1x10⁷-5x10⁷、5x10⁷-1x10⁸、1x10⁸-5x10⁸、5x10⁸-1x10⁹、1x10⁹-5x10⁹、5x10⁹-1x10¹⁰ or 1x10 ¹⁰-5x10¹⁰ cells. In an exemplary embodiment, the pharmaceutical composition comprises about 1.0x10 ⁶ to about 2.5x10 ⁸ cells.

In some embodiments, the pharmaceutical composition has a volume of at least 5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、150、160、170、180、190、200、250、300、350、400 or 500 ml. In exemplary embodiments, the pharmaceutical composition has a volume of up to about 5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、150、160、170、180、190、200、250、300、350、400 or 500 ml. In an exemplary embodiment, the pharmaceutical composition has a volume of about 5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、150、160、170、180、190、200、250、300、350、400 or 500 ml. In some embodiments, the pharmaceutical composition has a volume of about 1-50ml, 50-100ml, 100-150ml, 150-200ml, 200-250ml, 250-300ml, 300-350ml, 350-400ml, 400-450ml, or 450-500 ml. In some embodiments, the pharmaceutical composition has a volume of about 1-50ml, 50-100ml, 100-150ml, 150-200ml, 200-250ml, 250-300ml, 300-350ml, 350-400ml, 400-450ml, or 450-500 ml. In some embodiments, the pharmaceutical composition has a volume of about 1-10ml, 10-20ml, 20-30ml, 30-40ml, 40-50ml, 50-60ml, 60-70ml, 70-80ml, 80-90ml, or 90-100 ml. In some embodiments, the pharmaceutical composition has a volume ranging from about 5ml to about 80 ml. In exemplary embodiments, the pharmaceutical composition has a volume ranging from about 10ml to about 70 ml. In many embodiments, the pharmaceutical composition has a volume ranging from about 10ml to about 50 ml.

The specific amount/dosage regimen will vary depending on the following factors: the weight, sex, age and health of the individual; formulation, biochemical properties, biological activity, bioavailability, and side effects of cells, and the number and nature of cells in the complete treatment regimen.

In some embodiments, the dose of the pharmaceutical composition comprises about 1.0x10 ⁵ to about 2.5x10 ⁸ cells in a volume of about 10mL to 50mL, and the pharmaceutical composition is administered as a single dose.

In many embodiments, the cells are T cells and the pharmaceutical composition comprises from about 2.0x10 ⁶ to about 2.0x10 ⁸ cells, such as, but not limited to, primary T cells, T cells differentiated from engineered induced pluripotent stem cells. In some cases, the dose comprises about 1.0x10 ⁵ to about 2.5x10 ⁸ primary T cells described herein in a volume of about 10ml to 50 ml. In several cases, the dose includes from about 1.0x10 ⁵ to about 2.5x10 ⁸ primary T cells, which have been described above, in a volume of from about 10ml to 50 ml. In various cases, the dose includes from about 1.0x10 ⁵ to about 2.5x10 ⁸ T cells differentiated from the engineered induced pluripotent stem cells described herein in a volume of from about 10ml to 50 ml. In other cases, the dose ranges from less than about 1.0x10 ⁵ to about 2.5x10 ⁸ T cells (including primary T cells or T cells differentiated from engineered induced pluripotent stem cells). In still other cases, the dose ranges from greater than about 1.0x10 ⁵ to about 2.5x10 ⁸ T cells (including primary T cells and T cells differentiated from engineered induced pluripotent stem cells).

In some embodiments, the pharmaceutical composition is administered as a single dose of about 1.0x10 ⁵ to about 1.0x10 ⁷ engineered cells (such as primary cells or cells differentiated from engineered induced pluripotent stem cells) per kg body weight for a subject of 50kg or less. In some embodiments, the pharmaceutical composition is administered as a single dose of about 0.5x10 ⁵ to about 1.0x10 ⁷, about 1.0x10 ⁵ to about 1.0x10 ⁷, for a subject of 50kg or less, About 1.0x10 ⁵ to about 1.0x10 ⁷, about 5.0x10 ⁵ to about 1x10 ⁷, About 1.0x10 ⁶ to about 1x10 ⁷, about 5.0x10 ⁶ to about 1.0x10 ⁷, About 1.0x10 ⁵ to about 5.0x10 ⁶, about 1.0x10 ⁵ to about 1.0x10 ⁶, About 1.0x10 ⁵ to about 5.0x10 ⁵, about 1.0x10 ⁵ to about 5.0x10 ⁶, about 2.0x10 ⁵ to about 5.0x10 ⁶, about 3.0x10 ⁵ to about 5.0x10 ⁶, About 4.0x10 ⁵ to about 5.0x10 ⁶, about 5.0x10 ⁵ to about 5.0x10 ⁶, About 6.0x10 ⁵ to about 5.0x10 ⁶, about 7.0x10 ⁵ to about 5.0x10 ⁶, About 8.0x10 ⁵ to about 5.0x10 ⁶ or about 9.0x10 ⁵ to about 5.0x10 ⁶ cells/kg body weight. In some embodiments, the dose is about 0.2x10 ⁶ to about 5.0x10 ⁶ cells/kg body weight for a subject of 50kg or less. In many embodiments, the dose is in the range of less than about 0.2x10 ⁶ to about 5.0x10 ⁶ cells/kg body weight for a subject of 50kg or less. In many embodiments, the dose is in the range of greater than about 0.2x10 ⁶ to about 5.0x10 ⁶ cells/kg body weight for a subject of 50kg or less. In an exemplary embodiment, the volume of a single dose is about 10ml to 50ml. In some embodiments, the dose is administered intravenously.

In exemplary embodiments, the cells are administered in a single dose of about 1.0x10 ⁶ to about 5.0x10 ⁸ cells (such as primary cells and cells differentiated from engineered induced pluripotent stem cells) for subjects in excess of 50 kg. In some embodiments, the pharmaceutical composition is administered as a single dose of about 0.5x10 ⁶ to about 1.0x10 ⁹, about 1.0x10 ⁶ to about 1.0x10 ⁹, for a subject of 50kg or less, About 1.0x10 ⁶ to about 1.0x10 ⁹, about 5.0x10 ⁶ to about 1.0x10 ⁹, About 1.0x10 ⁷ to about 1.0x10 ⁹, about 5.0x10 ⁷ to about 1.0x10 ⁹, About 1.0x10 ⁶ to about 5.0x10 ⁷, about 1.0x10 ⁶ to about 1.0x10 ⁷, About 1.0x10 ⁶ to about 5.0x10 ⁷, about 1.0x10 ⁷ to about 5.0x10 ⁸, About 2.0x10 ⁷ to about 5.0x10 ⁸, about 3.0x10 ⁷ to about 5.0x10 ⁸, About 4.0x10 ⁷ to about 5.0x10 ⁸, about 5.0x10 ⁷ to about 5.0x10 ⁸, About 6.0x10 ⁷ to about 5.0x10 ⁸, about 7.0x10 ⁷ to about 5.0x10 ⁸, About 8.0x10 ⁷ to about 5.0x10 ⁸ or about 9.0x10 ⁷ to about 5.0x10 ⁸ cells/kg body weight. In many embodiments, the cells are administered in a single dose of about 1.0x10 ⁷ to about 2.5x10 ⁸ cells for subjects exceeding 50 kg. In some embodiments, the cells are administered in a single dose ranging from less than about 1.0x10 ⁷ to about 2.5x10 ⁸ cells for subjects exceeding 50 kg. In some embodiments, the cells are administered in a single dose ranging from greater than about 1.0x10 ⁷ to about 2.5x10 ⁸ cells for subjects exceeding 50 kg. In some embodiments, the dose is administered intravenously. In an exemplary embodiment, the volume of a single dose is about 10ml to 50ml. In some embodiments, the dose is administered intravenously.

In exemplary embodiments, the dose is administered intravenously at a rate of about 1 to 50ml per minute, 1 to 40ml per minute, 1 to 30ml per minute, 1 to 20ml per minute, 10 to 30ml per minute, 10 to 40ml per minute, 10 to 50ml per minute, 20 to 50ml per minute, 30 to 50ml per minute, 40 to 50ml per minute. In various embodiments, the pharmaceutical composition is stored in one or more infusion bags for intravenous administration. In some embodiments, the dose is administered entirely at no more than 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 240 minutes, or 300 minutes.

In some embodiments, a single dose of the pharmaceutical composition is present in a single infusion bag. In other embodiments, a single dose of the pharmaceutical composition is divided into 2,3, 4 or 5 separate infusion bags.

In some embodiments, the cells described herein are administered in multiple doses (such as 2, 3,4, 5, 6, or more doses). In some embodiments, each of the plurality of doses is administered to the subject in a range of 1 to 24 hours apart. In some cases, the subsequent dose is administered from about 1 hour to about 24 hours (e.g., about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or about 24 hours) after the initial dose or the previous dose. In some embodiments, each of the plurality of doses is administered to the subject in a range of about 1 to 28 days apart. In some cases, the subsequent dose is administered from about 1 day to about 28 days (e.g., about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or about 28 days) after the initial dose or the previous dose. In many embodiments, each of the plurality of doses is administered to the subject in a range of 1 week to about 6 weeks apart. In certain instances, the subsequent dose is administered from about 1 week to about 6 weeks (e.g., about 1, 2, 3,4, 5, or 6 weeks) after the initial dose or the previous dose. In several embodiments, each of the plurality of doses is administered to the subject in a range of about 1 month to about 12 months apart. In several cases, the subsequent dose is administered from about 1 month to about 12 months (e.g., about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 months) after the initial dose or the previous dose.

In some embodiments, the methods provided herein comprise administering to a subject (i) an engineered population of cells described herein, and (ii) an anticoagulant. In some embodiments, the anticoagulant is any anticoagulant described in section V.C below.

In some embodiments, the anticoagulant is administered at the time of administration of the engineered cell population, wherein heparin is administered at a dose of 70U/kg of recipient body weight. In some embodiments, the anticoagulant is provided as a separate formulation and administered concurrently with the engineered cell population. In some embodiments, the anticoagulant and the engineered cell population are provided in separate infusion bags, for example for intravenous administration. In some embodiments, the anticoagulant is heparin. In some embodiments, heparin is administered systemically. In some embodiments, heparin is administered by infusion through a mesenteric vein to the portal vein. In some embodiments, the patient is continuously infused with heparin for 12-36 hours (e.g., about 24 hours) 2,3, 4, 5, 6, 7, 8, 9, or 10 hours after surgery. In some embodiments, the heparin dosage administered during the post-operative period is lower than the initial administration dosage of heparin. In some embodiments, the operation after 200-400U/h (e.g., about 300U/h dose) heparin dosage to administer heparin. Heparin was administered by continuous infusion of 300U/h heparin at a dose of 300U/h heparin for 24 hours after surgery. In some embodiments, heparin is administered by continuous infusion at a dose of 300U/h heparin for 24 hours beginning 6 hours post-surgery.

In some embodiments, a first dosage regimen is administered to a subject at a first time point and then a second dosage regimen is administered to the subject at a second time point. In some embodiments, the first dosage regimen is the same as the second dosage regimen. In other embodiments, the first dosage regimen is different from the second dosage regimen. In some cases, the number of cells in the first dose regimen and the second dose regimen is the same. In some cases, the number of cells in the first and second dosage regimens is different. In some cases, the number of doses of the first dose regimen and the second dose regimen is the same. In some cases, the number of doses of the first dose regimen and the second dose regimen are different.

In some embodiments, the cell is an engineered T cell (e.g., a primary T cell or a T cell differentiated from an engineered induced pluripotent stem cell), and the first dose regimen comprises an engineered T cell expressing a first CAR, and the second dose regimen comprises an engineered T cell expressing a second CAR, such that the first CAR and the second CAR are different. For example, the first CAR and the second CAR bind different target antigens. In some cases, the first CAR comprises an scFv that binds an antigen, and the second CAR comprises an scFv that binds a different antigen. In some embodiments, the first dose regimen comprises engineered T cells expressing the first CAR and the second dose regimen comprises engineered T cells or primary T cells expressing the second CAR such that the first CAR and the second CAR are the same. The first dosage regimen may be administered to the subject at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1-3 months, 1-6 months, 4-6 months, 3-9 months, 3-12 months or more apart from the second dosage regimen. In some embodiments, a plurality of dosage regimens are administered to a subject during a disease (e.g., an autoimmune disease), and at least two of the dosage regimens comprise the same type of engineered T cells described herein. In other embodiments, at least two of the plurality of dosage regimens comprise different types of engineered T cells described herein.

B. Immunosuppressant

In some embodiments, the immunosuppressant and/or immunomodulatory agent is not administered to the patient prior to the first administration of the engineered cell population or composition containing the same.

In some embodiments, immunosuppressants and/or immunomodulators may be administered to patients receiving engineered cell administration. In some embodiments, the immunosuppressant and/or immunomodulator is administered prior to administration of the engineered cells. In some embodiments, the immunosuppressant and/or immunomodulator is administered prior to the administration of the first and/or second administration of the engineered cells.

Non-limiting examples of immunosuppressants and/or immunomodulators include cyclosporin, azathioprine, mycophenolic acid esters, corticosteroids such as prednisone, methotrexate, gold salts, sulfasalazine, antimalarial, buquinate, leflunomide, mizoribine, 15-deoxyspergualin, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyte globulin, thymopentin, thymosin-alpha and the like. In some embodiments, the immunosuppressant and/or immunomodulator is selected from the group of immunosuppressive antibodies consisting of: antibodies that bind to p75 of the IL-2 receptor, antibodies that bind to, for example MHC、CD2、CD3、CD4、CD7、CD28、B7、CD40、CD45、IFN-γ、TNF-α、IL-4、IL-5、IL-6R、IL-6、IGF、IGFR1、IL-7、IL-8、IL-10、CD11a or CD58, and antibody conjugates that bind to any of their ligands. In some embodiments, wherein the immunosuppressant and/or immunomodulator is administered to the patient prior to or after the first administration of the cells, the dose administered is lower than the dose required for cells having expression of one or more MHC class I molecules and/or one or more MHC class II molecules and no exogenous CD47 expression.

In one embodiment, such immunosuppressants and/or immunomodulators can be selected from soluble IL-15R, IL-10, B7 molecules (e.g., B7-1, B7-2, variants thereof, and fragments thereof), ICOS and OX40, inhibitors of negative T cell regulators (such as antibodies to CTLA-4), and the like.

In some embodiments, an immunosuppressant and/or immunomodulatory agent may be administered to the patient prior to the first administration of the engineered cell population. In some embodiments, the immunosuppressant and/or immunomodulator is administered at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more prior to the first administration of the cells. In some embodiments, the immunosuppressant and/or immunomodulator is administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more prior to the first administration of the cells.

In certain embodiments, the immunosuppressant and/or immunomodulator is not administered to the patient after the first administration of the cells, or is administered for at least 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more after the first administration of the cells. In some embodiments, the immunosuppressant and/or immunomodulator is administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more after the first administration of the cells.

In some embodiments, the immunosuppressant and/or immunomodulatory agent is not administered to the patient prior to administration of the engineered cell population. In many embodiments, the immunosuppressant and/or immunomodulatory agent is administered to the patient prior to the first and/or second administration of the engineered cell population. In some embodiments, the immunosuppressant and/or immunomodulatory agent is administered at least 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more prior to administration of the cells. In some embodiments, the immunosuppressant and/or immunomodulator is administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more prior to the first and/or second administration of the cells. In certain embodiments, the immunosuppressant and/or immunomodulatory agent is administered at least 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more after administration of the cells. In some embodiments, the immunosuppressant and/or immunomodulator is administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more after the first and/or second administration of the cells.

In some embodiments, wherein the immunosuppressant and/or immunomodulator is administered to the patient prior to or after administration of the cells, the dose administered is lower than the dose required for immunogenic cells (e.g., a population of cells of the same or similar cell type or phenotype of the engineered cells but without modification (e.g., genetic modification), e.g., having endogenous levels of CD142, expression of one or more MHC class I molecules and/or one or more MHC class II molecules, and without increased (e.g., exogenous) CD47 expression). In some embodiments, the cells are administered in a lower dose than is required for reducing immune rejection of an immunogenic cell (e.g., a population of cells of the same or similar cell type or phenotype of an engineered cell but without modification (e.g., genetic modification), e.g., having endogenous levels of CD142, MHC class I molecule(s), and/or MHC class II molecule(s) expressed without increased (e.g., exogenous) CD47 expression).

C. Anticoagulant

In some embodiments, the anticoagulant is administered to the patient during and/or after administration of the engineered cell population. In some embodiments, the anticoagulant and the engineered cell population are administered to the patient simultaneously. In some embodiments, the anticoagulant and the engineered cell population are administered sequentially.

In some embodiments, heparin is administered at the time of administration of the engineered cell population, wherein heparin is administered at a dose of 70U/kg of recipient body weight. In some embodiments, heparin is administered systemically. In some embodiments, heparin is administered by infusion through a mesenteric vein to the portal vein. In some embodiments, the patient is continuously infused with heparin for 12-36 hours (e.g., about 24 hours) 2,3, 4, 5, 6, 7, 8, 9, or 10 hours after surgery. In some embodiments, the heparin dosage administered during the post-operative period is lower than the initial administration dosage of heparin. In some embodiments, the operation after 200-400U/h (e.g., about 300U/h dose) heparin dosage to administer heparin. Heparin was administered by continuous infusion of 300U/h heparin at a dose of 300U/h heparin for 24 hours after surgery. In some embodiments, heparin is administered by continuous infusion at a dose of 300U/h heparin for 24 hours beginning 6 hours post-surgery.

In some embodiments, the anticoagulant is administered systemically. In some embodiments, the anticoagulant is administered by IV infusion.

Exemplary embodiments

1. An engineered cell comprising a modification that (I) increases expression of one or more tolerogenic factors, (II) reduces expression of CD142, and (iii) reduces expression of one or more MHC class I molecules and/or one or more MHC class II molecules, wherein the increased expression of (I) and the reduced expression of (II) and (iii) are relative to a cell of the same cell type that does not comprise the modification.

2. The engineered cell of embodiment 1, wherein one or more of the modifications in (iii) reduces expression of:

a. One or more MHC class I molecules;

b. One or more MHC class II molecules; or (b)

C. one or more MHC class I molecules and one or more MHC class II molecules.

3. The engineered cell of embodiment 1 or embodiment 2, wherein the one or more modifications reduce expression ：B2M、TAP I、NLRC5、CIITA、HLa-a、HLA-B、HLA-C、HLA-DP、HLA-DM、HLA-DOA、HLA-DOB、HLA-DQ、HLA-DR、RFX5、RFXANK、RFXAP、NFY-A、NFY-B and/or NFY-C of one or more molecules selected from the group consisting of and any combination thereof.

4. The engineered cell of any one of embodiments 1-3, wherein the engineered cell does not express one or more molecules ：B2M、TAP I、NLRC5、CIITA、HLa-a、HLA-B、HLA-C、HLA-DP、HLA-DM、HLA-DOA、HLA-DOB、HLA-DQ、HLA-DR、RFX5、RFXANK、RFXAP、NFY-A、NFY-B and/or NFY-C selected from the group consisting of and combinations thereof.

5. The engineered cell of any one of embodiments 1-4, wherein the one or more tolerogenic factors comprise one or more tolerogenic factors selected from the group consisting of: CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDOl, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8 and SERPINB9 and any combination thereof.

6. The engineered cell of embodiment 5, wherein the one or more tolerogenic factors are selected from the group consisting of: CD47, PD-L1, HLA-E or HLA-G, CCL21, FASL, SERPINB9, CD200, MFGE8, and any combination thereof.

7. The engineered cell of any one of embodiments 1-6, wherein the one or more tolerogenic factors comprise one or more tolerogenic factors selected from the group consisting of: A20/TNFAIP3, C1-inhibitor, CCL21, CCL22, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, fasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, IL-10, IL15-RF, IL-35, MANF, mfge8, PD-1, PD-L1, serpinb9, and any combination thereof.

8. The engineered cell of any one of embodiments 1-7, wherein the one or more tolerogenic factors comprise CD47.

9. The engineered cell of any one of embodiments 1-8, wherein the one or more tolerogenic factors comprise HLA-E.

10. The engineered cell of any one of embodiments 1-9, wherein the one or more tolerogenic factors comprise CD24.

11. The engineered cell of any one of embodiments 1-10, wherein the one or more tolerogenic factors comprises PDL1.

12. The engineered cell of any one of embodiments 1-11, wherein the one or more tolerogenic factors comprise CD55.

13. The engineered cell of any one of embodiments 1-12, wherein the one or more tolerogenic factors comprise CR1.

14. The engineered cell of any one of embodiments 1-13, wherein the one or more tolerogenic factors comprise MANF.

15. The engineered cell of any one of embodiments 1-14, wherein the one or more tolerogenic factors comprise a20/TNFAIP3.

16. The engineered cell of any one of embodiments 1-15, wherein the one or more tolerogenic factors comprise HLA-E and CD47.

17. The engineered cell of any one of embodiments 1-16, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of CD24, CD47, and PDL1, optionally wherein the one or more tolerogenic factors comprise CD24, CD47, and PDL1.

18. The engineered cell of any one of embodiments 1-17, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD47, and PDL1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD, CD47, and PDL1.

19. The engineered cell of any one of embodiments 1-18, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of CD46, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise CD46, CD55, CD59, and CR1.

20. The engineered cell of any one of embodiments 1-19, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD46, CD55, CD59, and CR1.

21. The engineered cell of any one of embodiments 1-20, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD47, PDL1, CD46, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD, CD47, PDL1, CD46, CD55, CD59, and CR1.

22. The engineered cell of any one of embodiments 1-21, wherein the one or more tolerogenic factors comprise HLA-E and PDL1.

23. The engineered cell of any one of embodiments 1-22, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1 and a20/TNFAIP, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1 and a20/TNFAIP.

24. The engineered cell of any one of embodiments 1-23, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1 and MANF, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1 and MANF.

25. The engineered cell of any one of embodiments 1-24, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1, a20/TNFAIP and MANF, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1, a20/TNFAIP and MANF.

26. An engineered cell comprising a modification that (i) increases expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD, CD200, and MFGE8, and (ii) decreases expression of CD142, wherein the increased expression of (i) and the decreased expression of (ii) are relative to a cell of the same cell type that does not comprise the modification.

27. The engineered cell of embodiment 26, wherein one or more of (i) increasing expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD, CD200, and MFGE8, (ii) increasing expression of CD46, and (iii) increasing expression of CD59 comprises one or more modifications that increase gene activity of an endogenous gene.

28. The engineered cell of embodiment 27, wherein the endogenous gene encodes the CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200, MFGE8, CD46, or CD59.

29. The engineered cell of embodiments 27 or 28, wherein the one or more modifications that increase the gene activity of an endogenous gene comprise the introduction of one or more modifications or heterologous promoters of the endogenous promoter or enhancer of the gene.

30. The engineered cell of embodiment 29, wherein the heterologous promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter for HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter for HIV, moloney virus promoter, epstein Barr Virus (EBV) promoter and Rous Sarcoma Virus (RSV) promoter, and UBC promoter.

31. The engineered cell of any one of embodiments 1-30, wherein the engineered cell further comprises one or more modifications that increase expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD55, wherein the increased expression of the one or more complement inhibitors is relative to a cell of the same cell type that does not comprise the modification.

32. The engineered cell of any one of embodiments 1-31, wherein the modification that increases expression comprises increased surface expression and/or the modification that decreases expression comprises decreased surface expression.

33. The engineered cell of embodiment 31 or embodiment 32, wherein the modification that increases expression of the one or more complement inhibitors comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and/or an exogenous polynucleotide encoding CD 55.

34. The engineered cell of any one of embodiments 31-33, wherein the one or more complement inhibitors are CD46 and CD59, optionally wherein the modification comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD59.

35. The engineered cell of any one of embodiments 31-34, wherein the one or more complement inhibitors are CD46, CD59, and CD55, optionally wherein the modification comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55.

36. The engineered cell of any one of embodiments 33-35, wherein the exogenous polynucleotide encoding CD46 encodes an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID No. 3.

37. The engineered cell of embodiment 36, wherein the exogenous polynucleotide encoding CD46 encodes the sequence set forth in SEQ ID No. 3.

38. The engineered cell of any one of embodiments 33-37, wherein the exogenous polynucleotide encoding CD59 encodes an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID No. 5 and exhibits complement inhibitory activity.

39. The engineered cell of embodiment 38, wherein the exogenous polynucleotide encoding CD59 encodes the sequence set forth in SEQ ID No. 5.

40. The engineered cell of any one of embodiments 33-39, wherein the exogenous polynucleotide encoding CD55 encodes an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID No. 8 and exhibits complement inhibitory activity.

41. The engineered cell of embodiment 40, wherein the exogenous polynucleotide encoding CD55 encodes the sequence set forth in SEQ ID No. 8.

42. The engineered cell of any one of embodiments 33-41, wherein the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, and/or the exogenous polynucleotide encoding CD55 are each operably linked to a promoter.

43. The engineered cell of any one of embodiments 5-42, wherein the modification that increases CD47 expression comprises an exogenous polynucleotide encoding the CD47 protein.

44. The engineered cell of embodiment 43, wherein the exogenous polynucleotide encoding CD47 encodes an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID No. 1 and reduces innate immune killing of the engineered cell.

45. The engineered cell of any one of embodiments 43-44, wherein the exogenous polynucleotide encoding CD47 encodes the sequence set forth in SEQ ID No. 1.

46. The engineered cell of any one of embodiments 43-45, wherein the exogenous polynucleotide encoding CD47 is operably linked to a promoter.

47. The engineered cell of any one of embodiments 1-46, wherein the engineered cell comprises a polycistronic vector comprising two or more exogenous polynucleotides selected from the group consisting of: one or more exogenous polynucleotides encoding the one or more tolerogenic factors, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding a CD55 polypeptide.

48. The engineered cell of embodiment 47, wherein each of said polynucleotides is isolated by an IRES or self-cleaving peptide.

49. The engineered cell of any one of embodiments 47-48, wherein each polynucleotide of the polycistronic vector is operably linked to the same promoter.

50. The engineered cell of any one of embodiments 47-49, wherein the polycistronic vector comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59.

51. The engineered cell of any one of embodiments 47-50, wherein the polycistronic vector comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55.

52. The engineered cell of embodiment 50 or embodiment 51, wherein the polycistronic vector further comprises an exogenous polynucleotide encoding CD47.

53. The engineered cell of embodiment 50 or embodiment 51, wherein the polycistronic vector is a first transgene and the engineered cell comprises a separate transgene comprising a polynucleotide encoding CD 47.

54. The engineered cell of any one of embodiments 1-53, wherein the engineered cell comprises a transgene comprising a polynucleotide encoding CD 47.

55. The engineered cell of any one of embodiments 1-46, wherein the engineered cell comprises a first transgene and a second transgene,

Wherein the first and second transgenes each comprise one or more exogenous polynucleotides selected from the group consisting of: exogenous polynucleotide encoding CD47, exogenous polynucleotide encoding CD46, exogenous polynucleotide encoding CD59, and exogenous polynucleotide encoding CD55 polypeptide, and

Wherein the first and second transgenes are monocistronic or polycistronic vectors.

56. The engineered cell of any one of embodiments 42-55, wherein the promoter is a constitutive promoter.

57. The engineered cell of any one of embodiments 42-56, wherein the promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter for HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter for HIV, moloney virus promoter, epstein Barr Virus (EBV) promoter and Rous Sarcoma Virus (RSV) promoter, and UBC promoter.

58. The engineered cell of any one of embodiments 33-57, wherein the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, and/or the exogenous polynucleotide encoding CD55 polypeptide is integrated into the genome of the engineered cell.

59. The engineered cell of any one of embodiments 43-58, wherein the exogenous polynucleotide encoding CD47 is integrated into the genome of the engineered cell.

60. The engineered cell of embodiment 58 or embodiment 59, wherein said integration is by non-targeted insertion into the genome of said engineered cell, optionally by introducing said exogenous polynucleotide into said cell using a lentiviral vector.

61. The engineered cell of embodiment 58 or embodiment 59, wherein said integration is by targeted insertion into a target genomic locus of said cell.

62. The engineered cell of embodiment 61, wherein the target genomic locus is selected from the group consisting of: MICA locus, MICB locus, B2M locus, CIITA locus, TRAC locus or TRBC locus, CD142 locus, CCR5 locus, CXCR4 locus, PPP1R12C (also known as AAVS 1) locus, albumin locus, SHS231 locus, CLYBL locus, ROSA26 locus, LRP1 locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus and KDM5D locus.

63. The engineered cell of embodiment 62, wherein the target genomic locus is a MICA locus, a MICB locus, a TAP1 locus, a B2M locus, a CIITA locus, a TRAC locus, a TRBC locus, or a safe harbor locus.

64. The engineered cell of embodiment 63, wherein the target genomic locus is selected from the group consisting of: CCR5 locus, CXCR4 locus, PPP1R12C (also known as AAVS 1) locus, albumin locus, SHS231 locus, CLYBL locus and ROSA26 locus.

65. The engineered cell of embodiment 64, wherein the safe harbor locus is selected from the group consisting of: AAVS1, ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA, and SHS231 loci.

66. The engineered cell of any one of embodiments 61-65, wherein the exogenous polynucleotide encoding CD47 is integrated into a first target genomic locus, the exogenous polynucleotide encoding CD46 is integrated into a second target genomic locus, and the polynucleotide encoding CD59 is integrated into a third target genomic locus.

67. The engineered cell of embodiment 66, wherein the exogenous polynucleotide encoding CD55 is integrated into a fourth target genomic locus.

68. The engineered cell of embodiment 66, wherein at least two of the first, second, and third target genomic loci are the same locus.

69. The engineered cell of embodiment 67 or embodiment 68, wherein at least two of the first, second, third, and fourth target genomic loci are the same locus.

70. The engineered cell of any one of embodiments 66-69, wherein the first, second, and third target genomic loci are the same locus.

71. The engineered cell of embodiments 66-70, wherein the first, second, third, and fourth target genomic loci are the same locus.

72. The engineered cell of embodiment 66 or embodiment 67, wherein each of the first, second, and third target genomic loci are different loci.

73. The engineered cell of embodiment 67, wherein the first, second, third, and fourth target genomic loci are different loci.

74. The engineered cell of any one of embodiments 1-73, wherein the modification that reduces expression of CD142 reduces CD142 protein expression.

75. The engineered cell of embodiment 74, wherein the modification eliminates CD142 gene activity.

76. The engineered cell of embodiment 74 or 75, wherein the modification comprises inactivation or disruption of both alleles of the CD142 gene.

77. The engineered cell of any one of embodiments 75-76, wherein the modification comprises inactivation or disruption of all CD142 coding sequences in the cell.

78. The engineered cell of embodiment 76 or embodiment 77, wherein said inactivation or disruption comprises an indel in said CD142 gene.

79. The engineered cell of any one of embodiments 61-65, wherein the modification is a frameshift mutation or deletion of a stretch of contiguous genomic DNA of the CD142 gene.

80. The engineered cell of any one of embodiments 75-79, wherein the CD142 gene is knocked out.

81. The engineered cell of any one of embodiments 75-80, wherein the modification is by a genomic modification protein, optionally wherein the modification is by nuclease-mediated genome editing.

82. The engineered cell of embodiment 81, wherein the nuclease-mediated genome editing is by a Zinc Finger Nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas combination targeted to the CD142 gene, optionally wherein the Cas is selected from Cas9 or Cas12.

83. The engineered cell of embodiment 82, wherein the nuclease-mediated genome editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site within the CD142 gene.

84. The engineered cell of embodiment 83, wherein the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising the gRNA and a Cas protein.

85. The engineered cell of any one of embodiments 1-25 and 27-84, wherein the modification that reduces expression of one or more MHC class I molecules reduces expression of one or more MHC class I molecule proteins.

86. The engineered cell of any one of embodiments 1-25 and 27-85, wherein the modification that reduces expression of one or more MHC class I molecules comprises reduced expression of B2M.

87. The engineered cell of any one of embodiments 74-86, wherein the modification that reduces expression of one or more MHC class I molecules comprises reduced protein expression of B2M.

88. The engineered cell of embodiment 86 or embodiment 87, wherein the modification eliminates B2M gene activity.

89. The engineered cell of any one of embodiments 86-88, wherein the modification comprises inactivation or disruption of both alleles of the B2M gene.

90. The engineered cell of any one of embodiments 86-89, wherein the modification comprises inactivation or disruption of all B2M coding sequences in the cell.

91. The engineered cell of embodiment 89 or embodiment 90, wherein said inactivation or disruption comprises an indel in said B2M gene. .

92. The engineered cell of any one of embodiments 86-91, wherein the modification is a frameshift mutation or deletion of a stretch of contiguous genomic DNA of the B2M gene.

93. The engineered cell of any one of embodiments 86-92, wherein the B2M gene is knocked out.

94. The engineered cell of any one of embodiments 85-93, wherein the modification is by a genomic modification protein, optionally wherein the modification is by nuclease-mediated gene editing.

95. The engineered cell of embodiment 94, wherein the modification by the genomic modification protein is performed using ：Cas3、Cas4、Cas5、Cas8a、Cas8b、Cas8c、Cas9、Cas10、Cas12、Cas12a(Cpf1)、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12f(C2c10)、Cas12g、Cas12h、Cas12i、Cas12k(C2c5)、Cas13、Cas13a(C2c2)、Cas13b、Cas13c、Cas13d、C2c4、C2c8、C2c9、Cmr5、Cse1、Cse2、Csf1、Csm2、Csn2、Csx10、Csx11、Csy1、Csy2、Csy3、Mad7、 zinc-finger nuclease (ZFN), transcription-activating factor-like effector nuclease (TALEN), meganuclease, CRISPR-associated transposase, or any CRISPR-Cas combination that targets the B2M gene, optionally wherein the modification is performed by nuclease-mediated gene editing using Cas9 or Cas 12.

96. The engineered cell of embodiment 95, wherein the nuclease-mediated gene editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site within the B2M gene.

97. The engineered cell of embodiment 96, wherein the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising the gRNA and a Cas protein.

98. The engineered cell of any one of embodiments 1-25 and 27-97, wherein the modification that reduces expression of one or more MHC class II molecules reduces expression of one or more MHC class II molecule proteins.

99. The engineered cell of any one of embodiments 1-25 and 27-98, wherein the modification that reduces expression of one or more MHC class II molecules comprises reduced expression of CIITA.

100. The engineered cell of embodiment 99, wherein the modification that reduces expression of one or more MHC class II molecules comprises reduced protein expression of CIITA.

101. The engineered cell of embodiment 99 or embodiment 100, wherein the modification eliminates CIITA.

102. The engineered cell of any one of embodiments 99-101, wherein the modification comprises inactivation or disruption of both alleles of the CIITA gene.

103. The engineered cell of any one of embodiments 99-102, wherein the modification comprises inactivation or disruption of all CIITA coding sequences in the cell.

104. The engineered cell of embodiment 102 or embodiment 103, wherein said inactivation or disruption comprises an indel in said CIITA gene.

105. The engineered cell of any one of embodiments 102-104, wherein the indel is a frameshift mutation or deletion of a stretch of contiguous genomic DNA of the CIITA gene.

106. The engineered cell of any one of embodiments 99-105, wherein the CIITA gene is knocked out.

107. The engineered cell of any one of embodiments 1-106, wherein the modification is by a genomic modification protein.

108. The engineered cell of embodiment 107, wherein the modification by the genomic modification protein is a modification by CRISPR-associated transposase, guided editing, or programmable addition via a site-specific targeting element (PASTE).

109. The engineered cell of embodiment 107 or 108, wherein the modification by the genomic modification protein is nuclease-mediated gene editing.

110. The engineered cell of embodiment 109, wherein the nuclease-mediated gene editing is by a Zinc Finger Nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas combination, optionally wherein the Cas is selected from Cas9 or Cas12.

111. The engineered cell of embodiment 107 or 108, wherein the modification by the genome modification protein is performed by one or more proteins selected from the group consisting of ：Cas3、Cas4、Cas5、Cas8a、Cas8b、Cas8c、Cas9、Cas10、Cas12、Cas12a(Cpf1)、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12f(C2c10)、Cas12g、Cas12h、Cas12i、Cas12k(C2c5)、Cas13、Cas13a(C2c2)、Cas13b、Cas13c、Cas13d、C2c4、C2c8、C2c9、Cmr5、Cse1、Cse2、Csf1、Csm2、Csn2、Csx10、Csx11、Csy1、Csy2、Csy3、Mad7、 Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and CRISPR-associated transposases.

112. The engineered cell of any one of embodiments 1-111, wherein the modification:

Decreasing expression of any one or more of NLRC5, TRAC, TRB, CD, 142, ABO, CD38, CD52, PCDH11Y, NLGN Y, and RHD.

113. The engineered cell of any one of embodiments 1-112, wherein one or more of (i) increasing expression of one or more tolerogenic factors, (ii) increasing expression of CD46, and (iii) the modification that increases expression of CD59 comprises one or more modifications that increase gene activity of an endogenous gene.

114. The engineered cell of embodiment 113, wherein the endogenous gene encodes the one or more tolerogenic factors CD46 or CD59.

115. The engineered cell of embodiment 113 or 1114, wherein the one or more modifications that increase the gene activity of the endogenous gene comprise the introduction of one or more modifications or heterologous promoters of an endogenous promoter or enhancer of the gene.

116. The engineered cell of embodiment 115, wherein the heterologous promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter for HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter for HIV, moloney virus promoter, epstein Barr Virus (EBV) promoter and Rous Sarcoma Virus (RSV) promoter, and UBC promoter.

117. The engineered cell of any one of embodiments 1-116, wherein the engineered cell is a human cell or an animal cell.

118. The engineered cell of embodiment 117, wherein the engineered cell is a human cell.

119. The engineered cell of any one of embodiments 1-118, wherein the cell is a blood-exposed cell type or a cell type capable of differentiating into a blood-exposed cell type.

120. The engineered cell of any one of embodiments 1-119, wherein the engineered cell is a differentiated cell derived from a pluripotent stem cell or progeny thereof.

121. The engineered cell of embodiment 120, wherein the pluripotent stem cell is an induced pluripotent stem cell.

122. The engineered cell of any one of embodiments 1-119, wherein the engineered cell is a primary cell isolated from a donor subject.

123. The engineered cell of embodiment 122, wherein the donor subject is healthy or not suspected of having a disease or disorder at the time a donor sample is obtained from the individual donor.

124. The engineered cell of any one of embodiments 1-123, wherein the engineered cell is selected from the group consisting of: islet cells, beta islet cells, pancreatic islet cells, immune cells, B cells, T cells, natural Killer (NK) cells, natural Killer T (NKT) cells, macrophages, endothelial cells, muscle cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, dopaminergic neurons, retinal pigment epithelial cells, visual cells, liver cells, thyroid cells, skin cells, glial progenitor cells, neural cells, cardiac cells, stem cells, hematopoietic stem cells, induced pluripotent stem cells (ipscs), mesenchymal Stem Cells (MSCs), embryonic Stem Cells (ESCs), pluripotent Stem Cells (PSCs), and blood cells.

125. The engineered cell of any one of embodiments 1-124, wherein the engineered cell is an endothelial cell.

126. The engineered cell of any one of embodiments 1-125, wherein the engineered cell is an epithelial cell.

127. The engineered cell of embodiment 126, wherein the engineered cell is a T cell.

128. The engineered cell of embodiment 127, wherein the engineered cell is an NK cell.

129. The engineered cell of embodiment 127 or embodiment 128, wherein the engineered cell comprises a Chimeric Antigen Receptor (CAR).

130. The engineered cell of embodiment 124, wherein the engineered cell is a stem cell.

131. The engineered cell of embodiment 124, wherein the engineered cell is a Hematopoietic Stem Cell (HSC).

132. The engineered cell of embodiment 124, wherein the engineered cell is a beta islet cell.

133. The engineered cell of embodiment 124, wherein the engineered cell is a hepatocyte.

134. The engineered cell of embodiment 124, wherein the engineered cell is a pluripotent stem cell.

135. The engineered cell of embodiment 124, wherein the engineered cell is an induced pluripotent stem cell.

136. The engineered cell of embodiment 124, wherein the engineered cell is an embryonic stem cell.

137. The engineered cell of any one of embodiments 1-136, wherein the cell is ABO blood group O.

138. The engineered cell of any one of embodiments 1-137, wherein the cell is rhesus factor negative (Rh-).

139. The engineered cell of any one of embodiments 1-136 and 138, wherein the cell comprises a functional ABO a allele and/or a functional ABO B allele.

140. The engineered cell of any one of embodiments 1-137 and 139, wherein the cell is rhesus factor positive (rh+).

141. A method of generating an engineered cell, the method comprising:

a. Reducing or eliminating expression of one or more MHC class I molecules and/or one or more MHC class II molecules in the cell;

b. reducing expression of CD142 in the cell; and

C. Increasing expression of a tolerogenic factor in said cell.

142. The method of embodiment 141, wherein the one or more tolerogenic factors are selected from the group consisting of: CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDOl, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8 and SERPINB9 and any combination thereof.

143. The method of embodiment 142, wherein the one or more tolerogenic factors are selected from the group consisting of: CD47, PD-L1, HLA-E or HLA-G, CCL21, FASL, SERPINB9, CD200, MFGE8, and any combination thereof.

144. The method of any one of embodiments 141-143, wherein the one or more tolerogenic factors comprises one or more tolerogenic factors selected from the group consisting of: A20/TNFAIP3, C1-inhibitor, CCL21, CCL22, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, fasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, IL-10, IL15-RF, IL-35, MANF, mfge8, PD-1, PD-L1, serpinb9, and any combination thereof.

145. The method of embodiment 143 or embodiment 144, wherein the one or more tolerogenic factors comprises CD47.

146. The method of any one of embodiments 141-145, wherein the one or more tolerogenic factors comprise HLA-E.

147. The method of any one of embodiments 141-146, wherein the one or more tolerogenic factors comprise CD24.

148. The method of any one of embodiments 141-147, wherein the one or more tolerogenic factors comprises PDL1.

149. The method of any one of embodiments 141-148, wherein the one or more tolerogenic factors comprise CD55.

150. The method of any one of embodiments 141-149, wherein the one or more tolerogenic factors comprise CR1.

151. The method of any of embodiments 141-150, wherein the one or more tolerogenic factors comprise MANF.

152. The method of any one of embodiments 141-151, wherein the one or more tolerogenic factors comprise a20/TNFAIP3.

153. The method of any one of embodiments 141-152, wherein the one or more tolerogenic factors comprise HLA-E and CD47.

154. The method of any one of embodiments 141-153, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of CD24, CD47, and PDL1, optionally wherein the one or more tolerogenic factors comprises CD24, CD47, and PDL1.

155. The method of any one of embodiments 141-154, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD47, and PDL1, optionally wherein the one or more tolerogenic factors comprises HLA-E, CD, CD47, and PDL1.

156. The method of any one of embodiments 141-155, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of CD46, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprises CD46, CD55, CD59, and CR1.

157. The method of any one of embodiments 141-156, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD46, CD55, CD59, and CR1.

158. The method of any one of embodiments 141-157, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of HLA-E, CD24, CD47, PDL1, CD46, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprises HLA-E, CD, CD47, PDL1, CD46, CD55, CD59, and CR1.

159. The method of any one of embodiments 141-158, wherein the one or more tolerogenic factors comprise HLA-E and PDL1.

160. The method of any one of embodiments 141-159, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1 and a20/TNFAIP, optionally wherein the one or more tolerogenic factors comprises HLA-E, PDL1 and a20/TNFAIP.

161. The method of any of embodiments 141-160, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1 and MANF, optionally wherein the one or more tolerogenic factors comprises HLA-E, PDL1 and MANF.

162. The method of any of embodiments 141-161, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1, a20/TNFAIP and MANF, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1, a20/TNFAIP and MANF.

163. The method of any one of embodiments 141-162, wherein the method comprises reducing expression of one or more MHC class I molecules and one or more MHC class II molecules.

164. The method of any one of embodiments 141-163, wherein increasing expression of the tolerogenic factors comprises increasing gene activity of an endogenous gene.

165. The method of embodiment 164, wherein the endogenous gene encodes the tolerogenic factor.

166. The method of embodiments 164 or 165 wherein increasing the gene activity of the endogenous gene comprises modifying an endogenous promoter or enhancer of the gene, or introducing a heterologous promoter.

167. The method of embodiment 166, wherein the heterologous promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter for HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter for HIV, moloney virus promoter, epstein Barr Virus (EBV) promoter and Rous Sarcoma Virus (RSV) promoter, and UBC promoter.

168. A method of generating a low-immunogenicity cell, the method comprising:

a. Increasing expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200 and MFGE8 in said cell, and

B. reducing expression of CD142 in the cell.

169. The method of any one of embodiments 141-168, further comprising increasing expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD55 in the cell.

170. The method of embodiment 168 or 169, wherein increasing the expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200, MFGE8, and/or one or more complement inhibitors comprises increasing the gene activity of an endogenous gene.

171. The method of embodiment 170, wherein the endogenous gene encodes the CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200, MFGE8, CD46, or CD59.

172. The method of embodiment 170 or embodiment 171, wherein increasing the gene activity of the endogenous gene comprises modifying an endogenous promoter or enhancer of the gene, or introducing a heterologous promoter.

173. The method of embodiment 172, wherein the heterologous promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter for HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter for HIV, moloney virus promoter, epstein Barr Virus (EBV) promoter and Rous Sarcoma Virus (RSV) promoter, and UBC promoter.

174. The method of any of embodiments 141-173, wherein the reduced expression comprises reduced surface expression and/or the increased expression comprises increased surface expression, optionally wherein the reduced surface expression comprises no detectable surface expression.

175. The method of any one of embodiments 169-174, wherein increasing expression of the one or more complement inhibitors comprises introducing an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and/or an exogenous polynucleotide encoding CD55 into the cell.

176. The method of any one of embodiments 169-175, wherein the one or more complement inhibitors are CD46 and CD59, optionally wherein increasing expression of the one or more complement inhibitors comprises introducing an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59.

177. The method of any one of embodiments 169-176, wherein the one or more complement inhibitors are CD46, CD59, and CD55, optionally wherein increasing expression of the one or more complement inhibitors comprises introducing an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55.

178. The method of any of embodiments 169-177, wherein the exogenous polynucleotide encoding CD46 encodes an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID No. 3 and exhibits complement inhibitory activity.

179. The method of embodiment 178, wherein the exogenous polynucleotide encoding CD46 encodes the sequence set forth in SEQ ID NO. 3.

180. The method of any of embodiments 169-179, wherein the exogenous polynucleotide encoding CD59 encodes an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID No. 5 and exhibits complement inhibitory activity.

181. The method of embodiment 180, wherein the exogenous polynucleotide encoding CD59 encodes the sequence set forth in SEQ ID NO. 5.

182. The method of any of embodiments 169-181, wherein said exogenous polynucleotide encoding CD55 encodes a sequence that has at least 85% identity to the amino acid sequence of SEQ ID No. 8 and exhibits complement inhibitory activity.

183. The method of embodiment 182, wherein the exogenous polynucleotide encoding CD55 encodes the sequence set forth in SEQ ID NO. 8.

184. The method of any one of embodiments 169-183, wherein each of the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, and/or the exogenous polynucleotide encoding CD55 is operably linked to a promoter.

185. The method of any one of embodiments 169-184, wherein said modification that increases CD47 expression comprises an exogenous polynucleotide encoding said CD47 protein.

186. The method of any of embodiments 169-185, wherein said exogenous polynucleotide encoding CD47 encodes a sequence that has at least 85% identity to the amino acid sequence of SEQ ID No.1 and reduces innate immune killing of said engineered cells.

187. The method of embodiment 186, wherein the exogenous polynucleotide encoding CD47 encodes the sequence set forth in SEQ ID No. 1.

188. The method of any one of embodiments 169-187, wherein the exogenous polynucleotide encoding CD47 is operably linked to a promoter.

189. The method of any one of embodiments 169-188, wherein the method comprises introducing into the cell a polycistronic vector comprising two or more exogenous polypeptides selected from the group consisting of: one or more exogenous polynucleotides encoding the one or more tolerogenic factors, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding a CD55 polypeptide.

190. The method of embodiment 189, wherein each of the polynucleotides is isolated by an IRES or self-cleaving peptide.

191. The method of embodiment 189 or embodiment 190, wherein the two or more exogenous polynucleotides are selected from the group consisting of: an exogenous polynucleotide encoding CD47, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding a CD55 polypeptide.

192. The method of any one of embodiments 189-191, wherein each polynucleotide of the polycistronic vector is operably linked to the same promoter.

193. The method of any one of embodiments 189-192, wherein the polycistronic vector comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59.

194. The method of any one of embodiments 189-193, wherein the polycistronic vector comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55.

195. The method of embodiment 193 or embodiment 194, wherein the polycistronic vector further comprises an exogenous polynucleotide encoding CD47.

196. The method of embodiment 193 or embodiment 194, wherein the engineered cell comprises a separate transgene comprising a polynucleotide encoding CD 47.

197. The method of any one of embodiments 169-196, wherein the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, and/or the exogenous polynucleotide encoding CD55 is integrated into the genome of the engineered cell.

198. The method of any one of embodiments 185-197, wherein the exogenous polynucleotide encoding CD47 is integrated into the genome of the engineered cell.

199. The method of embodiment 197 or embodiment 198, wherein the integration is by non-targeted insertion into the genome of the engineered cell.

200. The method of embodiment 199, wherein the integration is performed by introducing the exogenous polynucleotide into the cell using a lentiviral vector.

201. The method of embodiment 197 or embodiment 198, wherein the integration is by targeted insertion into a target genomic locus of the cell, optionally wherein the targeted insertion is by nuclease-mediated gene editing using homology directed repair.

202. The method of embodiment 201, wherein the target genomic locus is selected from the group consisting of: MICA locus, MICB locus, B2M locus, CIITA locus, TRAC locus or TRBC locus, CD142 locus, CCR5 locus, CXCR4 locus, PPP1R12C (also known as AAVS 1) locus, albumin locus, SHS231 locus, CLYBL locus, ROSA26 locus, LRP1 locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus and KDM5D locus.

203. The method of embodiment 201 or embodiment 202, wherein the target genomic locus is a MICA locus, a MICB locus, a TAP1 locus, a B2M locus, a CIITA locus, a TRAC locus, a TRBC locus, or a safe harbor locus.

204. The method of embodiment 201 or embodiment 202, wherein the target genomic locus is selected from the group consisting of: CCR5 locus, CXCR4 locus, PPP1R12C (also known as AAVS 1) locus, albumin locus, SHS231 locus, CLYBL locus and ROSA26 locus.

205. The method of any one of embodiments 201-204, wherein the target genomic locus is a safe harbor locus.

206. The method of any one of embodiments 201-205, wherein the nuclease-mediated gene editing is by a Zinc Finger Nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas combination targeted to the target genomic locus, optionally wherein the Cas is selected from Cas9 or Cas12.

207. The method of embodiment 206, wherein the nuclease-mediated gene editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to a target sequence of the target genomic locus and a homology directed repair template comprising the CD46 encoding exogenous polynucleotide, the CD59 encoding exogenous polynucleotide, the CD55 encoding exogenous polynucleotide, and/or the CD47 encoding exogenous polynucleotide.

208. The method of embodiment 207, wherein the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising the gRNA and a Cas protein.

209. The method of any one of embodiments 141-208, wherein reducing expression of CD142 reduces CD142 protein expression.

210. The method of embodiment 209, wherein reducing expression of CD142 comprises introducing a modification that reduces activity of a CD142 gene.

211. The method of embodiment 210, wherein the modification that reduces the activity of the CD142 gene comprises inactivation or disruption of both alleles of the CD142 gene.

212. The method of embodiment 210 or embodiment 211, wherein said modification that reduces CD142 gene activity comprises inactivation or disruption of all CD142 coding sequences in said cell.

213. The method of embodiment 211 or embodiment 212, wherein said inactivation or disruption comprises an indel in said CD142 gene or a deletion of a stretch of contiguous genomic DNA of said CD142 gene.

214. The method of embodiment 213, wherein the indel is a frameshift mutation.

215. The method of any one of embodiments 210-214, wherein the CD142 gene is knocked out.

216. The method of any of embodiments 210-215, wherein the modification that reduces CD142 gene activity is introduced by a genomic modification protein, optionally wherein the modification that reduces CD142 gene activity is introduced by nuclease-mediated gene editing.

217. The method of embodiment 216, wherein the modification by the genome modification protein is performed using ：Cas3、Cas4、Cas5、Cas8a、Cas8b、Cas8c、Cas9、Cas10、Cas12、Cas12a(Cpf1)、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12f(C2c10)、Cas12g、Cas12h、Cas12i、Cas12k(C2c5)、Cas13、Cas13a(C2c2)、Cas13b、Cas13c、Cas13d、C2c4、C2c8、C2c9、Cmr5、Cse1、Cse2、Csf1、Csm2、Csn2、Csx10、Csx11、Csy1、Csy2、Csy3、Mad7、 zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, CRISPR-associated transposase, or any CRISPR-Cas combination that targets the CD142 gene, optionally wherein the modification is performed by nuclease-mediated gene editing using Cas9 or Cas 12.

218. The method of embodiment 217, wherein the nuclease-mediated gene editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site within the CD142 gene.

219. The method of embodiment 218, wherein the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising the gRNA and a Cas protein.

220. The method of any one of embodiments 141-219, wherein reducing expression of one or more MHC class I molecules comprises introducing a modification that reduces expression of one or more MHC class I molecule proteins.

221. The method of embodiment 220, wherein the modification that reduces expression of one or more MHC class I molecule proteins comprises reduced expression of B2M.

222. The method of embodiment 220 or 221, wherein the modification that reduces expression of one or more MHC class I molecule proteins comprises reduced expression of B2M protein.

223. The method of embodiment 221 or 222, wherein the modification that reduces expression of one or more MHC class I molecule proteins reduces B2M gene activity.

224. The method of any one of embodiments 221-223, wherein the modification that reduces expression of one or more MHC class I molecules comprises inactivation or disruption of both alleles of the B2M gene.

225. The method of any one of embodiments 221-223, wherein the modification that reduces expression of one or more MHC class I molecule proteins comprises inactivation or disruption of all B2M coding sequences in the cell.

226. The method of embodiment 224 or embodiment 225, wherein said inactivation or disruption comprises an indel in said B2M gene or a deletion of a stretch of contiguous genomic DNA of said B2M gene.

227. The method of embodiment 226, wherein the indel is a frameshift mutation.

228. The method of any one of embodiments 221-227, wherein the B2M gene is knocked out.

229. The method of any one of embodiments 221-228, wherein the modification to reduce expression of one or more MHC class I molecule proteins is by a genomic modification protein, optionally wherein the modification to reduce expression of one or more MHC class I molecule proteins is by nuclease-mediated gene editing.

230. The method of embodiment 229, wherein the modification by the genome modification protein is performed using ：Cas3、Cas4、Cas5、Cas8a、Cas8b、Cas8c、Cas9、Cas10、Cas12、Cas12a(Cpf1)、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12f(C2c10)、Cas12g、Cas12h、Cas12i、Cas12k(C2c5)、Cas13、Cas13a(C2c2)、Cas13b、Cas13c、Cas13d、C2c4、C2c8、C2c9、Cmr5、Cse1、Cse2、Csf1、Csm2、Csn2、Csx10、Csx11、Csy1、Csy2、Csy3、Mad7、 zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, CRISPR-associated transposase, or any CRISPR-Cas combination that targets the B2M gene, optionally wherein the modification is performed by nuclease-mediated gene editing using Cas9 or Cas 12.

231. The method of embodiment 230, wherein the nuclease-mediated gene editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site within the B2M gene.

232. The method of embodiment 231, wherein the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising the gRNA and a Cas protein.

233. The method of any one of embodiments 141-232, wherein reducing expression of one or more MHC class II molecules comprises introducing a modification that reduces expression of one or more MHC class II molecule proteins.

234. The method of embodiment 233, wherein the modification that reduces expression of one or more MHC class II molecule proteins comprises reduced expression of CIITA.

235. The method of embodiment 233 or 234, wherein the modification that reduces expression of one or more MHC class II molecule proteins comprises reduced protein expression of CIITA.

236. The method of embodiment 233 or embodiment 234, wherein the modification that reduces expression of one or more MHC class II molecule proteins reduces CIITA gene activity.

237. The method of any one of embodiments 233-236, wherein the modification that reduces expression of one or more MHC class II molecule proteins comprises inactivation or disruption of both alleles of the CIITA gene.

238. The method of any one of embodiments 233-237, wherein the modification comprises inactivation or disruption of all CIITA coding sequences in the cell.

239. The method of embodiment 237 or embodiment 238, wherein the inactivation or disruption comprises an indel in the CIITA gene or a deletion of a stretch of contiguous genomic DNA of the CIITA gene.

240. The method of embodiment 239, wherein the indel is a frameshift mutation.

241. The method of any one of embodiments 234-240, wherein the CIITA gene is knocked out.

242. The method of any one of embodiments 141-241, wherein the cell is a human cell or an animal cell, optionally wherein the animal cell is a pig (pig/candidate) cell, a cow (cow/candidate) cell, or a sheep (shaep/ovine) cell.

243. The method of any one of embodiments 141-242, wherein the engineered cell is a human cell.

244. The method of any one of embodiments 141-243, wherein the cell is a blood-exposed cell type or a cell type capable of differentiating into a blood-exposed cell type.

245. The method of any one of embodiments 141-244, wherein the cell is a primary cell isolated from a donor subject.

246. The method of any one of embodiments 141-244, wherein the cell is a pluripotent stem cell, wherein the engineered cell is a differentiated cell derived from the pluripotent stem cell, and the method further comprises differentiating the pluripotent stem cell.

247. The method of embodiment 246, wherein the pluripotent stem cells are induced pluripotent stem cells.

248. The method of any one of embodiments 141-243, wherein the engineered cell is selected from the group consisting of: islet cells, beta islet cells, pancreatic islet cells, immune cells, B cells, T cells, natural Killer (NK) cells, natural Killer T (NKT) cells, macrophages, endothelial cells, muscle cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, dopaminergic neurons, retinal pigment epithelial cells, visual cells, liver cells, thyroid cells, skin cells, glial progenitor cells, neural cells, cardiac cells, stem cells, hematopoietic stem cells, induced pluripotent stem cells (ipscs), mesenchymal Stem Cells (MSCs), embryonic Stem Cells (ESCs), pluripotent Stem Cells (PSCs), and blood cells.

249. The method of embodiment 248, wherein the engineered cell is a beta islet cell.

250. The method of embodiment 248, wherein the engineered cell is a hepatocyte.

251. An engineered cell produced according to the method of any one of embodiments 141-250.

252. The engineered cell of any one of embodiments 1-140 and 251, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell are capable of escaping NK cell-mediated cytotoxicity upon administration to a recipient patient.

253. The engineered cell of any one of embodiments 1-140 and 251-252, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell are protected from cell lysis of mature NK cells after administration to a recipient patient.

254. The engineered cell of any one of embodiments 1-140 and 251-253, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce an immune response to the cell upon administration to a recipient patient.

255. The engineered cell of any one of embodiments 1-140 and 251-254, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce a systemic inflammatory response to the cell after administration to a recipient patient.

256. The engineered cell of any one of embodiments 1-140 and 251-255, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce a local inflammatory response to the cell after administration to a recipient patient.

257. The engineered cell of any one of embodiments 1-140 and 251-256, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce complement pathway activation upon administration to a recipient patient.

258. The engineered cell of any one of embodiments 1-140 and 251-257, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce clotting upon administration to a recipient patient.

259. The engineered cell of any one of embodiments 1-140 and 251-258, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce an immediate blood-mediated inflammatory response upon administration to a recipient patient.

260. The engineered cell of embodiments 258-259, wherein the cell is contacted with blood after administration to the recipient patient.

261. A cell population comprising a plurality of engineered cells of any one of embodiments 1-140 and 251-260.

262. The population of embodiment 261, wherein at least about 30% of the cells in the population are the engineered cells.

263. The engineered population of embodiments 261 or 262, wherein the plurality of engineered primary cells is derived from cells pooled from more than one donor subject.

264. The engineered primary cell population of embodiment 263, wherein each of the more than one donor subjects is a healthy subject or is not suspected of having a disease or disorder when the donor sample is obtained from the donor subject.

265. The population of any one of embodiments 261-264, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in said population comprise said modification.

266. The population of any one of embodiments 261-265, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise an exogenous polynucleotide encoding CD 47.

267. The population of any one of embodiments 261-266, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in said population comprise an exogenous polynucleotide encoding CD 46.

268. The population of any one of embodiments 261-267, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in said population comprise an exogenous polynucleotide encoding CD 59.

269. The population of any one of embodiments 261-268, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in said population comprise an exogenous polynucleotide encoding CD 55.

270. The population of any one of embodiments 261-269, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise one or more alterations that inactivate both alleles of a B2M gene.

271. The population of any one of embodiments 261-270, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise one or more alterations that inactivate both alleles of the CIITA gene.

272. The population of any one of embodiments 261-271, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise reduced CD142 expression relative to unchanged or unmodified wild-type cells.

273. The population of any one of embodiments 261-272, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in said population comprise one or more alterations that inactivate both alleles of the CD142 gene.

274. A composition comprising the population of any one of embodiments 261-273 or the engineered cell of any one of embodiments 1-140 and 251-260.

275. A composition comprising a population of engineered beta islet cells, wherein the engineered beta islet cells comprise: (i) a transgene comprising an exogenous polynucleotide encoding CD47, (ii) inactivation or disruption of both alleles of the CD142 gene, and (iii) inactivation or disruption of both alleles of the B2M gene.

276. The composition of embodiment 275, wherein the engineered β -cell comprises inactivation or disruption of both alleles of a CIITA gene.

277. A composition comprising an engineered population of hepatocytes, wherein the engineered hepatocytes comprise: (i) a transgene comprising an exogenous polynucleotide encoding CD47, (ii) inactivation or disruption of both alleles of the CD142 gene, and (iii) inactivation or disruption of both alleles of the B2M gene.

278. The composition of embodiment 277, wherein the engineered hepatocyte comprises inactivation or disruption of both alleles of the CIITA gene.

279. The composition of any one of embodiments 275-278, wherein the transgene is a polycistronic vector, and wherein the transgene further comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59.

280. The composition of any one of embodiments 275-278, wherein the beta islet cells or liver cells further comprise a polycistronic vector, wherein the polycistronic vector comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59.

281. The composition of any one of embodiments 275-278, wherein the transgene is introduced at a target genomic locus by nuclease-mediated gene editing using homology directed repair.

282. The composition of any one of embodiments 275-281, wherein the inactivation or disruption is by a genome modification protein, optionally wherein the inactivation or disruption is by nuclease-mediated gene editing.

283. The composition of any one of embodiments 281-282, wherein the modification by the genomic modification protein is performed using ：Cas3、Cas4、Cas5、Cas8a、Cas8b、Cas8c、Cas9、Cas10、Cas12、Cas12a(Cpf1)、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12f(C2c10)、Cas12g、Cas12h、Cas12i、Cas12k(C2c5)、Cas13、Cas13a(C2c2)、Cas13b、Cas13c、Cas13d、C2c4、C2c8、C2c9、Cmr5、Cse1、Cse2、Csf1、Csm2、Csn2、Csx10、Csx11、Csy1、Csy2、Csy3、Mad7、 Zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, CRISPR-associated transposase, or any CRISPR-Cas combination that targets the target genomic locus, optionally wherein the modification is performed by nuclease-mediated gene editing using Cas9 or Cas 12.

284. The composition of any one of embodiments 275-283, wherein the composition is a pharmaceutical composition.

285. The composition of embodiment 284, comprising a pharmaceutically acceptable excipient.

286. The composition of any one of embodiments 284-285, wherein the composition is formulated in a serum-free cryopreservation medium comprising a cryoprotectant.

287. The composition of embodiment 286, wherein the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (volume/volume).

288. The composition of embodiment 286 or 287, wherein the cryoprotectant is or is about 10% DMSO (v/v).

289. The composition of any one of embodiments 275-288, which is sterile.

290. A container comprising the composition of any one of embodiments 275-289.

291. The container of embodiment 290, which is a sterile bag.

292. The sterile bag of embodiment 291, wherein the bag is a cryopreservation compatible bag.

293. A method of treating a disease, disorder, or cell defect in a patient in need thereof, the method comprising administering to the patient an effective amount of the population of any one of embodiments 261-273 or the composition of any one of embodiments 274-289.

294. The method of embodiment 293, wherein the method further comprises administering to the patient an anticoagulant that reduces coagulation.

295. A method of treating a disease, disorder, or cell defect in a patient in need thereof, the method comprising

(A) Administering to the patient an effective amount of: a cell population comprising a plurality of engineered cells,

Wherein the engineered cell comprises the following modifications:

(i) Increasing expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD 55;

(ii) Increasing expression of one or more tolerogenic factors, and

(Iii) Reduces the expression of one or more MHC class I molecules and/or one or more MHC class II molecules,

Wherein the increased expression of (i) and (ii) and the decreased expression of (iii) are relative to a cell of the same cell type that does not comprise the modification; and

(B) Administering to the patient an anticoagulant that reduces coagulation.

296. The method of embodiment 295, wherein the one or more tolerogenic factors are selected from the group consisting of: CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDOl, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8 and SERPINB9.

297. The method of embodiment 296, wherein the one or more tolerogenic factors is CD47.

298. A method of treating a disease, disorder, or cell defect in a patient in need thereof, the method comprising

Wherein the engineered cell comprises the following modifications:

(i) Increasing expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD 55; and

(Ii) Increases the expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200 and MFGE8,

Wherein the increased expression of (i) and (ii) is relative to a cell of the same cell type that does not comprise the modification; and

(B) Administering to the patient an anticoagulant that reduces coagulation.

299. The method of embodiment 298, wherein the population is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

300. The method of any of embodiments 294-299, wherein the population and the anticoagulant are administered simultaneously or sequentially.

301. The method of any of embodiments 294-300, wherein the anticoagulant is heparin.

302. The method of embodiment 301, wherein the heparin is plain heparin.

303. The method of embodiment 301, wherein the heparin is low molecular weight heparin.

304. The method of any one of embodiments 301-303, wherein the heparin is soluble heparin.

305. The method of any one of embodiments 301-303, wherein the heparin is immobilized on the surface of the cells prior to administration of the cells to the patient.

306. The method of any of embodiments 294-305, wherein the anticoagulant is melagatran or LMW-DS.

307. The method of any of embodiments 294-305, wherein the anticoagulant is acetoacetcysteine (NAC).

308. The method of any of embodiments 294-305, wherein the anticoagulant is alpha-1 antitrypsin (AAT) and/or activated protein C.

309. The method of any one of embodiments 293-308, wherein the disorder or disease is selected from the group consisting of: diabetes, cancer, angiogenesis disorders, ocular diseases, thyroid diseases, skin diseases and liver diseases.

310. The method of any one of embodiments 293-308, wherein the cellular defect is associated with diabetes, or the disease or disorder is diabetes, optionally wherein the diabetes is type I diabetes.

311. The method of embodiment 310, wherein the cell population is a population of islet cells (including beta islet cells).

312. The method of embodiment 311, wherein the islet cells are selected from the group consisting of islet progenitor cells, immature islet cells, and mature islet cells.

313. The method of any of embodiments 293-309, wherein the cellular defect is associated with, or the disease or disorder is a vascular disorder or disease.

314. The method of embodiment 313, wherein the population of cells is a population of endothelial cells.

315. The method of any of embodiments 293-309, wherein the cellular defect is associated with autoimmune thyroiditis or the disease or disorder is autoimmune thyroiditis.

316. The method of embodiment 315, wherein the cell population is a thyroid progenitor cell population.

317. The method of any one of embodiments 293-309, wherein the cell defect is associated with a liver disease or the disease is a liver disease.

318. The method of embodiment 317, wherein the liver disease comprises liver cirrhosis.

319. The method of embodiment 317 or 318, wherein the cell population is a population of hepatocytes or hepatic progenitors.

320. The method of any of embodiments 293-309, wherein the cellular defect is associated with a corneal disease, or the disease is a corneal disease.

321. The method of embodiment 320, wherein the corneal disease is fox's dystrophy or congenital genetic endothelial dystrophy.

322. The method of embodiment 320 or 321, wherein the population of cells is a population of corneal endothelial progenitor cells or corneal endothelial cells.

323. The method of any one of embodiments 293-309, wherein the cell defect is associated with a kidney disease or the disease is a kidney disease.

324. The method of embodiment 323, wherein the cell population is a kidney precursor cell or a kidney cell population.

325. The method of any one of embodiments 293-309, wherein the cell defect is associated with cancer or the disease is cancer.

326. The method of embodiment 325, wherein the cancer is selected from the group consisting of: b-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous lymphoblastic leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

327. The method of embodiment 325 or 326, wherein the cell population is a T cell population or NK cell population or NKT cell population.

328. The method of any of embodiments 293-309, wherein the cell deficiency is associated with or the disease or disorder is a hematopoietic disease or disorder.

329. The method of embodiment 328, wherein the hematopoietic disease or disorder is myelodysplasia, aplastic anemia, fanconi anemia, paroxysmal sleep hemoglobinuria, sickle cell disease, congenital pure red cell aplastic anemia, schwann-Dai Mengde disease, ke Shiwen syndrome, chronic granulomatosis, adrenoleukodystrophy, leukocyte adhesion deficiency, hemophilia, thalassemia, β -thalassemia, leukemias such as Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), adult lymphoblastic leukemia, chronic Lymphoblastic Leukemia (CLL), B-cell chronic lymphoblastic leukemia (B-CLL), chronic Myeloblastic Leukemia (CML), juvenile Chronic Myelogenous Leukemia (CML), and juvenile myelomonocytic leukemia (JMML)), severe Combined Immunodeficiency Disease (SCID), X severe combined immunodeficiency, wegenet-aldrich syndrome (WAS), adenosine deaminase (adam), chronic lymphocytic leukemia (hodgkin's), hodgkin's disease, hodgkin's lymphoma, or non-aids.

330. The method of embodiment 328, wherein the cellular defect is associated with leukemia or myeloma, or wherein the disease or condition is leukemia or myeloma.

331. The method of any one of embodiments 293-309 and 328, wherein the cellular defect is associated with or is an autoimmune disease or disorder.

332. The method of embodiment 331, wherein the autoimmune disease or disorder is acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, anti-synthetase syndrome, atopic allergy, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, Autoimmune polyendocrine adenosis syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, balneosis, balneo-sclerosis, behcet's syndrome, primary Graves disease, bischtaff encephalitis, braun's syndrome, bullous pemphigoid, cancer, kalman's disease, celiac disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, chager-Schtriaus syndrome, cicatricial pemphigoid, crohn's syndrome, condensed collets, complement component 2 deficiency, craniomatis, CREST syndrome, crohn's disease, cushing's syndrome, cutaneous leukopenia vasculitis, degos ' disease, delkum's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, diffuse systemic sclerosis of the skin, dresler's syndrome, discoid lupus erythematosus, eczema, adnexitis-associated arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, acquired epidermolysis bullosa, erythema nodosum, primary mixed condensed globulinemia, ehrlichia syndrome, progressive fibrodysplasia ossificans, fibroalveolar inflammation, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, nephritis syndrome, gerbileve's disease, gill-barre syndrome (GBS), Bridge encephalitis, bridge thyroiditis, hemolytic anemia, allergic purpura, herpes gestation, hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, igA nephropathy, inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, kawasaki disease, lanbert-Eatonic syndrome, white blood cell disruption vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), graek's disease, lupus hepatitis, lupus erythematosus, ma Jide syndrome, meniere's disease, Microscopic polyangiitis, miller-Fisher syndrome, mixed connective tissue disease, scleroderma, acute acne-like lichen-like pityriasis, multiple sclerosis, myasthenia gravis, myositis, neuromyelitis optica, neuromuscular rigidity, ocular cicatricial pemphigoid, strabismus eye clonus syndrome, thyroiditis, recurrent rheumatism, paraneoplastic cerebellar degeneration, paroxysmal sleep hemoglobinuria (PNH), pa Luo Zeng syndrome, parsen-Tener syndrome, platycodon, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, Polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell dysgenesis, laplace Mu Sen encephalitis, raynaud's phenomenon, recurrent polychondritis, litty's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schmitt syndrome, schniter syndrome, scleritis, scleroderma, sjogren syndrome, spondyloarthropathies, still's disease, stiff person syndrome, subacute bacterial endocarditis, susak syndrome, sjogren's syndrome, sidner's disease, sympathogenic ophthalmitis, Arteritis, temporal arteritis, painful oculopathy syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue diseases, undifferentiated spondyloarthropathies, vasculitis, vitiligo or wegener's granulomatosis.

333. The method of any of embodiments 328-332, wherein the population of cells is a population comprising Hematopoietic Stem Cells (HSCs) and/or derivatives thereof.

334. The method of any one of embodiments 293-309, wherein the cellular deficit is associated with parkinson's disease, huntington's disease, multiple sclerosis, a neurodegenerative disease or disorder, attention Deficit Hyperactivity Disorder (ADHD), tourette's Syndrome (TS), schizophrenia, psychosis, depression, neuropsychiatric stroke (neuropsychiatric disorder stroke) or Amyotrophic Lateral Sclerosis (ALS), or wherein the disease or disorder is parkinson's disease, huntington's disease, multiple sclerosis, a neurodegenerative disease or disorder, attention Deficit Hyperactivity Disorder (ADHD), tourette's Syndrome (TS), schizophrenia, psychosis, depression, a stroke of neuropsychiatric disorder, or Amyotrophic Lateral Sclerosis (ALS).

335. The method of embodiment 334, wherein the population of cells is a population comprising neural cells and/or glial cells.

336. The method of any one of embodiments 293-335, wherein the cells are expanded and cryopreserved prior to administration.

337. The method of any one of embodiments 293-336, wherein administering the population comprises intravenous injection, intramuscular injection, intravascular injection, or transplanting the population.

338. The method of embodiment 337, wherein the population is transplanted via renal capsule transplantation or intramuscular injection.

339. The method of any one of embodiments 293-338, wherein the population is derived from a donor subject, wherein the HLA type of the donor does not match the HLA type of the patient.

340. The method of any one of embodiments 293-339, wherein the population is derived from a donor, wherein the blood group of the donor does not match the blood group of the patient, and the blood group of the donor is not type O.

341. The method of any one of embodiments 293-340, wherein the population is derived from a donor, wherein the blood group of the donor is rhesus factor (Rh) positive and the blood group of the patient is Rh negative.

342. The method of any one of embodiments 293-341, wherein the patient's serum comprises antibodies to Rh.

343. The method of any of embodiments 293-342, wherein the population is a population of human cells and the patient is a human patient.

344. The method of any one of embodiments 293-343, wherein the population of cells comprises a functional ABO a allele and/or a functional ABO B allele.

345. The method of embodiment 344, wherein the population of cells presents ABO type a antigens and the patient's serum comprises anti-a antibodies.

346. The method of embodiment 344, wherein the population of cells presents ABO type B antigens and the patient's serum comprises anti-B antibodies.

347. The method of embodiment 344, wherein the population of cells presents ABO type a and type B antigens and the patient's serum comprises anti-a and/or anti-B antibodies.

348. The method of any one of embodiments 293-347, wherein a population of cells expresses an Rh factor and the patient's serum comprises an anti-Rh antibody.

349. The method of any one of embodiments 293-348, further comprising administering one or more immunosuppressants to the patient.

350. The method of any one of embodiments 293-348, wherein one or more immunosuppressants have been administered to the patient.

351. The method of embodiment 349 or embodiment 350, wherein the one or more immunosuppressants are small molecules or antibodies.

352. The method of any one of embodiments 349-351, wherein the one or more immunosuppressants are selected from the group consisting of: cyclosporine, azathioprine, mycophenolic acid, mycophenolate ester, corticosteroids, prednisone, methotrexate, gold salts, sulfasalazine, antimalarial, buconazole, leflunomide, mizoribine, 15-deoxyspergualin, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyte globulin, thymopentapeptides (thymosin-alpha) and immunosuppressive antibodies.

353. The method of any one of embodiments 349-352, wherein the one or more immunosuppressants comprise cyclosporin.

354. The method of any one of embodiments 349-352, wherein the one or more immunosuppressants comprise mycophenolate mofetil.

355. The method of any one of embodiments 349-352, wherein the one or more immunosuppressants comprise a corticosteroid.

356. The method of any one of embodiments 349-352, wherein the one or more immunosuppressants comprise cyclophosphamide.

357. The method of any one of embodiments 349-352, wherein the one or more immunosuppressants comprise rapamycin.

358. The method of any one of embodiments 349-352, wherein the one or more immunosuppressants comprise tacrolimus (FK-506).

359. The method of any one of embodiments 349-352, wherein the one or more immunosuppressants comprise anti-thymocyte globulin.

360. The method of any one of embodiments 349-352, wherein the one or more immunosuppressants are one or more immunomodulators.

361. The method of embodiment 360, wherein the one or more immunomodulatory agents is a small molecule or an antibody.

362. The method of embodiment 351 or embodiment 361, wherein the antibody binds to one or more receptors or ligands selected from the group consisting of: p75、MHC、CD2、CD3、CD4、CD7、CD28、B7、CD40、CD45、IFN-γ、TNF-α、IL-4、IL-5、IL-6R、IL-6、IGF、IGFR1、IL-7、IL-8、IL-10、CD11a、CD58, of the IL-2 receptor and antibodies that bind to any of its ligands.

363. The method of any one of embodiments 349-362, wherein the one or more immunosuppressants are administered to the patient or have been administered to the patient prior to administration of the engineered cells.

364. The method of any one of embodiments 349-363, wherein the patient is administered or the one or more immunosuppressants have been administered to the patient at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to administration of the engineered cells.

365. The method of any one of embodiments 349-363, wherein at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more are administered to the patient or the one or more immunosuppressants have been administered to the patient prior to administration of the engineered cells.

366. The method of any one of embodiments 349-363, wherein the patient is administered or the one or more immunosuppressants have been administered to the patient at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after administration of the engineered cells.

367. The method of any one of embodiments 349-363, wherein at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more are administered to the patient or have been administered to the patient after administration of the engineered cells.

368. The method of any one of embodiments 349-363, wherein the one or more immunosuppressants are administered to the patient or have been administered to the patient on the same day as the first administration of the engineered cells.

369. The method of any one of embodiments 349-363, wherein the one or more immunosuppressants are administered to the patient or have been administered to the patient after administration of the engineered cells.

370. The method of any one of embodiments 349-363, wherein the one or more immunosuppressants are administered to the patient or have been administered to the patient after the administration of the engineered cells is first and/or second administration.

371. The method of any one of embodiments 349-363, wherein the one or more immunosuppressants are administered to the patient or have been administered to the patient prior to the administration of the first and/or second administration of the engineered cells.

372. The method of any one of embodiments 349-363, wherein the patient is administered or has been administered the one or more immunosuppressants at least 1,2,3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, or 14 days prior to the administration of the engineered cells for the first and/or second administration.

373. The method of any one of embodiments 349-363, wherein at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more are administered to the patient or the one or more immunosuppressants are administered to the patient prior to the administration of the first and/or second administration of the engineered cells.

374. The method of any one of embodiments 349-363, wherein the patient is administered or has been administered the one or more immunosuppressants at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the administration of the engineered cells first and/or second administration.

375. The method of any one of embodiments 349-363, wherein at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more are administered to the patient or have been administered to the patient after the administration of the engineered cells first and/or second.

376. The method of any one of embodiments 349-375, wherein the one or more immunosuppressants are administered at a lower dose to reduce immune rejection of modified immunogenic cells that do not comprise the engineered cells compared to the dose of the one or more immunosuppressants administered.

377. The method of any one of embodiments 293-376, wherein the engineered cell is capable of controlled killing of the engineered cell.

378. The method of any one of embodiments 293-377, wherein the engineered cell comprises a suicide gene or suicide switch.

379. The method of embodiment 378, wherein the suicide gene or the suicide switch induces controlled cell death in the presence of a drug or prodrug or after activation by a selective exogenous compound.

380. The method of embodiment 378 or embodiment 379, wherein said suicide gene or said suicide switch is an inducible protein capable of inducing apoptosis of said engineered cell.

381. The method of embodiment 380, wherein said inducible protein capable of inducing apoptosis in said engineered cell is a cysteine protease protein.

382. The method of embodiment 381, wherein the cysteine protease protein is cysteine protease 9.

383. The method of embodiment 380 or embodiment 381, wherein said suicide gene or suicide switch is selected from the group consisting of: cytosine deaminase (CyD), herpes virus thymidine kinase (HSV-Tk), inducible cysteine proteinase 9 (iCaspase 9) and rapamycin activated cysteine proteinase 9 (rapaCasp).

384. The method of any one of embodiments 378-383, wherein following administration of the one or more immunosuppressants to the patient, the suicide gene or the suicide switch is activated to induce controlled cell death.

385. The method of any one of embodiments 378-383, wherein the suicide gene or the suicide switch is activated to induce controlled cell death prior to administering the one or more immunosuppressants to the patient.

386. The method of any one of embodiments 378-385, wherein after administering the engineered cells to the patient, the suicide gene or the suicide switch is activated to induce controlled cell death.

387. The method of any one of embodiments 378-386, wherein the suicide gene or the suicide switch is activated to induce controlled cell death if it has cytotoxicity or other negative consequences for the patient.

388. The method of any one of embodiments 293-387, comprising administering an agent that allows depletion of engineered cells in the population of engineered cells.

389. The method of embodiment 388, wherein the agent that allows depletion of the engineered cell is an antibody that recognizes a protein expressed on the surface of the engineered cell.

390. The method of embodiment 389, wherein the antibody is selected from the group consisting of an antibody that recognizes CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR 8.

391. The method of embodiment 389 or embodiment 390, wherein the antibody is selected from the group consisting of: mo Geli bead mab, AFM13, MOR208, octuzumab, rituximab, oxcarbatuzumab, rituximab-Rllb, tobrauximab, RO5083945 (GA 201), cetuximab, hul4.18k322a, hul4.18-IL2, hul3F 8, rituximab, c.60c3-Rllc, and biological analogs thereof.

392. The method of any one of embodiments 293-391, comprising administering an agent that recognizes the one or more tolerogenic factors on the surface of the engineered cell.

393. The method of embodiment 392, wherein the engineered cell is engineered to express the one or more tolerogenic factors.

394. The method of any one of embodiments 293-393, wherein the one or more tolerogenic factors comprises one or more tolerogenic factors selected from the group consisting of: A20/TNFAIP3, C1-inhibitor, CCL21, CCL22, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, fasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, IL-10, IL15-RF, IL-35, MANF, mfge8, PD-1, PD-L1, serpinb9, and any combination thereof.

395. The method of embodiments 393 or 394, wherein the one or more tolerogenic factors comprises CD47.

396. The method of any one of embodiments 293-395, wherein the one or more tolerogenic factors comprise HLA-E.

397. The method of any one of embodiments 293-396, wherein the one or more tolerogenic factors comprise CD24.

398. The method of any one of embodiments 293-397, wherein the one or more tolerogenic factors comprises PDL1.

399. The method of any one of embodiments 293-398, wherein the one or more tolerogenic factors comprise CD55.

400. The method of any one of embodiments 293-399, wherein the one or more tolerogenic factors comprises CR1.

401. The method of any one of embodiments 293-400, wherein the one or more tolerogenic factors comprise MANF.

402. The method of any one of embodiments 293-401, wherein the one or more tolerogenic factors comprise a20/TNFAIP3.

403. The method of any one of embodiments 293-402, wherein the one or more tolerogenic factors comprise HLA-E and CD47.

404. The method of any one of embodiments 293-403, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of CD24, CD47, and PDL1, optionally wherein the one or more tolerogenic factors comprises CD24, CD47, and PDL1.

405. The method of any one of embodiments 293-404, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD47, and PDL1, optionally wherein the one or more tolerogenic factors comprises HLA-E, CD, CD47, and PDL1.

406. The method of any one of embodiments 293-405, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of CD46, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprises CD46, CD55, CD59, and CR1.

407. The method of any one of embodiments 293-406, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD46, CD55, CD59, and CR1.

408. The method of any one of embodiments 293-407, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD24, CD47, PDL1, CD46, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD, CD47, PDL1, CD46, CD55, CD59, and CR1.

409. The method of any one of embodiments 293-408, wherein the one or more tolerogenic factors comprise HLA-E and PDL1.

410. The method of any one of embodiments 293-409, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1 and a20/TNFAIP, optionally wherein the one or more tolerogenic factors comprises HLA-E, PDL1 and a20/TNFAIP.

411. The method of any one of embodiments 293-410, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1 and MANF, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1 and MANF.

412. The method of any one of embodiments 293-411, wherein said one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1, a20/TNFAIP and MANF, optionally wherein said one or more tolerogenic factors comprises HLA-E, PDL1, a20/TNFAIP and MANF.

413. The method of embodiment 393 or embodiment 394, wherein the one or more tolerogenic factors is CD47.

414. The method of any of embodiments 293-413, further comprising administering one or more additional therapeutic agents to the patient.

415. The method of any of embodiments 293-414, wherein one or more additional therapeutic agents have been administered to the patient.

416. The method of any one of embodiments 293-415, further comprising monitoring the therapeutic efficacy of the method.

417. The method of any one of embodiments 293-416, further comprising monitoring the prophylactic efficacy of the method.

418. The method of any one of embodiments 293-417, wherein the method is repeated until a desired inhibition of one or more disease symptoms occurs.

419. The engineered cell of any one of embodiments 1-140 and 251-260, wherein the engineered cell comprises an exogenous polynucleotide encoding a suicide gene or suicide switch.

420. The engineered cell of embodiment 419, wherein the suicide gene or suicide switch is selected from the group consisting of: cytosine deaminase (CyD), herpes virus thymidine kinase (HSV-Tk), inducible cysteine proteinase 9 (iCaspase 9) and rapamycin activated cysteine proteinase 9 (rapaCasp).

421. The engineered cell of embodiment 419 or embodiment 420, wherein the suicide gene or suicide switch and a gene associated with the suicide gene or the safety switch are expressed by a bicistronic cassette integrated into the genome of the engineered cell.

422. The engineered cell of any one of embodiments 419-421, wherein the suicide gene or suicide switch and the one or more tolerogenic factors are expressed by a bicistronic cassette integrated into the genome of the engineered cell.

423. The engineered cell of embodiment 419 or embodiment 420, wherein the bicistronic cassette is integrated by non-targeted insertion into the genome of the engineered cell, optionally by introducing the exogenous polynucleotide into the cell using a lentiviral vector.

424. The engineered cell of embodiment 419 or 420, wherein the bicistronic cassette is integrated by targeted insertion into a target genomic locus of the cell, optionally wherein the targeted insertion is by nuclease-mediated gene editing using homology directed repair.

425. The engineered cell of any one of embodiments 419-424, wherein the one or more tolerogenic factors is CD47.

426. The method of any one of embodiments 141-250, wherein the engineered cell comprises an exogenous polynucleotide encoding a suicide gene or suicide switch.

427. The method of embodiment 426 wherein said suicide gene is selected from the group consisting of: cytosine deaminase (CyD), herpes virus thymidine kinase (HSV-Tk), inducible cysteine proteinase 9 (iCaspase 9) and rapamycin activated cysteine proteinase 9 (rapaCasp).

428. The method of embodiment 426 or embodiment 427 wherein the suicide gene or suicide switch and the gene associated with the suicide gene or the safety switch are expressed by a bicistronic cassette integrated into the genome of the engineered cell.

429. The method of any one of embodiments 426 or 427, wherein the suicide gene or suicide switch and the one or more tolerogenic factors are expressed by a bicistronic cassette integrated into the genome of the engineered cell.

430. The method of embodiment 428 or embodiment 429, wherein the bicistronic cassette is integrated by non-targeted insertion into the genome of the engineered cell.

431. The method of embodiment 428 or embodiment 429, wherein the bicistronic cassette is integrated by targeted insertion into a target genomic locus of the engineered cell.

432. The method of any one of embodiments 426-431 wherein the one or more tolerogenic factors is CD47.

433. The composition of any one of embodiments 274-289, wherein an engineered cell in the population of engineered cells comprises an exogenous polynucleotide encoding a suicide gene or suicide switch.

434. The composition of embodiment 433, wherein the suicide gene or suicide switch is selected from the group consisting of: cytosine deaminase (CyD), herpes virus thymidine kinase (HSV-Tk), inducible cysteine proteinase 9 (iCaspase 9) and rapamycin activated cysteine proteinase 9 (rapaCasp).

435. The composition of embodiment 433 or embodiment 434, wherein the suicide gene and the gene associated with the suicide gene or the safety switch are expressed by a bicistronic cassette integrated into the genome of the engineered cells in the engineered cell population.

436. The composition of any one of embodiments 433-435, wherein the suicide gene or suicide switch and the exogenous CD47 are expressed by a bicistronic cassette integrated into the genome of the engineered cell.

437. The composition of embodiment 435 or embodiment 436, wherein the bicistronic cassette is integrated by non-targeted insertion into the genome, optionally by introducing the exogenous polynucleotide into an engineered cell in the engineered cell population using a lentiviral vector.

438. The composition of embodiment 435 or embodiment 436, wherein the bicistronic cassette is integrated by targeted insertion into a target genomic locus of an engineered cell in the engineered cell population, optionally wherein the targeted insertion is by nuclease-mediated gene editing using homology directed repair.

439. A combination comprising the engineered population of cells of any one of embodiments 261-273 or a population of cells comprising a plurality of the engineered cells of any one of embodiments 419-425, and an anticoagulant or cell coating that reduces blood coagulation.

440. A combination, the combination comprising:

(a) A cell population comprising a plurality of engineered cells,

Wherein the engineered cell comprises the following modifications:

(ii) Increase expression of CD47, and

(B) Anticoagulants.

441. The combination of embodiment 439 or embodiment 440, wherein the anticoagulant is selected from the group consisting of heparin, an activator of antithrombin, an inhibitor of factor II (fII), an inhibitor of factor VII (fVII), and an inhibitor of factor X (fX).

442. The combination of embodiment 441 wherein the anticoagulant is heparin.

443. The combination of embodiment 442, wherein the heparin is plain heparin.

444. The combination of embodiment 443, wherein the heparin is low molecular weight heparin.

445. The combination of any one of embodiments 441-444, wherein the heparin is soluble heparin.

446. The combination of embodiment 441 wherein the anticoagulant is melagatran or LMW-DS.

447. The combination of embodiment 441, wherein the anticoagulant is acetoacetylcysteine (NAC).

448. The combination of embodiment 441, wherein the anticoagulant is alpha-1 antitrypsin (AAT) and/or activated protein C.

449. The combination of embodiment 441 wherein the anticoagulant is an antibody directed against CD 142.

450. A kit comprising a combination according to any one of embodiments 441-448.

Examples

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 Effect of overexpression of CD46, CD55 and/or CD59 in human B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg cells on human ABO incompatibility-mediated CDC

This example describes a study to test the effect of overexpressing a membrane-bound complement inhibitor on CDC. CD46, CD55 and CD59 were expressed alone or in various combinations in human B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg human induced pluripotent stem cells (hiPSC) or endothelial cells differentiated from B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiPSC.

A. Method of

Transgenic expression of only CD46, CD55 and CD59 or combinations thereof in B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg low immune cells. B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion} hipscs were generated using standard CRISPR/Cas9 gene editing techniques. A transgene (tg) encoding exogenous CD47 and one or more exogenous membrane-bound complement inhibitors CD46, CD55 and CD59 is introduced into the cells using standard lentiviral vector transduction techniques with a lentiviral vector encoding an exogenous protein.

Endothelial cells were differentiated from the modified hipscs. Endothelial cells were differentiated from B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion} CD47tg low immune cells or were additionally engineered to overexpress CD46; CD55; CD59; CD46 and CD59; CD46 and CD55; CD55 and CD59; or hipscs of CD46, CD55 and CD 59. Endothelial cell differentiation was performed as described in Deuse et al "Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients."Nature biotechnology vol.37,3(2019):252-258, the contents of which are incorporated herein by reference in their entirety.

Flow cytometry. The surface expression levels of HLA-I, HLA-II, CD47, CD46, CD55 and/or CD59 on engineered cells were assessed by flow cytometry by staining the cells with antibody specific reagents. Isotype antibodies were used as controls. The engineered cells are pooled for analysis or sorted to give positive (+) surface expression of the transgene (30-60 fold expression compared to isotype), high (++) surface expression of the transgene (61-400 fold expression compared to isotype), or ultra high (++) expression of the transgene (more than 400 fold compared to isotype).

CDC assay of human cells using ABO incompatible serum. Will also be engineered to overexpress CD46; CD55; CD59; CD46 and CD59; CD46 and CD55; CD55 and CD59; or B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion} of CD46, CD55 and CD59, CD47tg hiPSC or hiEC were incubated with ABO incompatible human serum and CDC was analyzed by measuring cell lysis over incubation time on the xcelligent ^TM MP platform (ACEA Biosciences) to provide label-free monitoring of cell proliferation and cell viability. The change in impedance is reported as a Cell Index (CI) (a decrease in cell index indicates an increase in cell lysis or killing).

B. Results

The B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg cells were not protected from CDC. As shown in FIGS. 1A and 1B, B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiPSC and hiEC each did not express HLA-I or HLA-II, but rather over expressed CD47. As shown in fig. 2A and 2B, B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiPSC, and hiEC also each exhibited endogenous surface expression of CD46, CD55, and CD59 complement inhibitory receptors. Although complement inhibitory receptors were expressed, the results demonstrated that the cells were not protected from CDC. Specifically, incubation with ABO incompatible sera resulted in rapid killing of both B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiPSC (fig. 3A) and B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiEC (fig. 3B).

Overexpression of CD46 and CD59 or CD46, CD55 and CD59 protects cells from ABO incompatibility-mediated CDC. To determine whether overexpression of one or more membrane-bound complement inhibitors would protect cells from CDC, B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiPSC, or hiEC engineered to overexpress one or more of CD46, CD55, and CD59 were evaluated for resistance to CDC.

The expression levels of CD46, CD55 and/or CD59 in the untransduced cells (endogenously expressed) or in the transduced cell pool or individual clones are provided in table E1 (hiPSC) and table E2 (hiEC) below. Overexpression in the transduced cell pool and clones was classified as follows: +=30-60 fold compared to isotype control; ++ = control with isotype in contrast to the ratio of 61-400, ++ = and isoforms the control was more than 400-fold compared. Flow cytometry analysis of B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiPSC or hiEC transduced with a lentiviral vector containing a transgene encoding an individual complement inhibitor receptor showed increased surface expression of CD46, CD55 and CD59, as shown in tables E1 and E2.

Over-expression of CD46, CD55 or CD59 in B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiPSC or hiEC, respectively, did not protect cells from CDC, even for complement inhibitors having the following individuals representation of ultra-high (+++) expression the same is true of sexual clones: CD46 (FIG. 4A, hiPSC; FIG. 4B, hiEC); CD55 (FIG. 5A, hiPSC; FIG. 5B, hiEC), or CD59 (FIG. 6A, hiPSC; FIG. 6B, hiEC). Similar results were obtained in the ABO incompatibility CDC assay for other individual complement inhibitor expression clones shown in table E1 and table E2. Overexpression (even high (++) expression) of the combination of CD46 and CD55 in B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiPSC (table E1) or B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiEC (table E2) provided some protection against CDC but were not effective in preventing cell killing, as shown in fig. 7A (hiPSC) and fig. 7B (hiEC). Overexpression (even high (++) expression) of the combination of CD55 and CD59 in B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiPSC (table E1) or B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiEC (table E2) did not provide any protection against CDC, as shown in fig. 8A (hiPSC) and 8B (hiEC).

In contrast, overexpression of the combination of CD46 and CD59 in B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiPSC (table E1) or B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiEC (table E2) significantly reduced or avoided cell killing by CDC, as shown in fig. 9A (representative hiPSC clone) and fig. 9B (representative hiEC clone). In addition, high (++) expression of CD49 and CD59 (e.g., less than 100-fold compared to isotype control, as shown in tables E1 and E2) is also sufficient to prevent CDC.

Overexpression of the combination of CD46, CD55, and CD59 in B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiPSC (table E1) or B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiEC (table E2) also significantly reduced or avoided cell killing by CDC, resulting in hiPSC or hiEC survival, as shown in fig. 10A (representative hiPSC clone) and fig. 10B (representative hiEC clone).

CDC assay results for endothelial cells differentiated from hiPSC (hiEC) alone (i.e., without ABO-incompatible serum added) are provided in fig. 11. In the presence of ABO incompatible sera, no significant difference was observed between the survival of hiEC alone (control) and the survival of hiPSC or hiEC overexpressing CD46 and CD59 (fig. 9A to 9B) or CD46, CD55 and CD59 (fig. 10A to 10B), indicating that overexpression of CD46 and CD59 or CD46, CD55 and CD59 blocks CDC.

Although CDC assay results for representative clones are shown in the figures, similar results were obtained for all clones shown in tables E1 and E2 below. In particular, CDC mediated killing was observed with libraries and clones transduced with CD46, CD55 or CD59 and with libraries and clones transduced with CD46 and CD55 or CD55 and CD59, respectively. In contrast, survival (protection against CDC-mediated killing) of libraries and clones transduced with CD46 and CD59 or transduced with CD46, CD59 and CD55 was observed.

Together, these results demonstrate that cells (including hipscs and differentiated cells such as hiecs) that endogenously express CDC inhibitors (including membrane-bound complement inhibitors CD46, CD55, and CD 59) may not adequately protect the cells from CDC, even in B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion 、} CD47tg cells that do not trigger an innate or adaptive immune response. However, the data indicate that over-expression of CD46 with CD59 or CD46 with CD59 and CD55 protects these cells from CDC even in the presence of antibodies that bind to antigens on the cell surface (e.g., anti-a or anti-B antibodies in ABO-incompatible sera).

Table E1. Expression data of CD46, CD55 and/or CD59 in B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiPSC

Table E2. Expression data of CD46, CD55 and/or CD59 in B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg hiEC

EXAMPLE 2B 2M indels/indels, CIITA indels/indels, CD47tg, CD46tg, CD59tghiPSC derived beta islet cells Using heparin transplantation

B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg, CD46tg, CD59tg hipscs were generated as described in example 1. Differentiating the modified hipscs into beta islet cells (modified low-immunogenicity beta islet cells) according to established beta islet cell differentiation protocols, for example, as described in U.S. patent publication No. 2021/0207099; hogrebe et al ,"Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells,"Nat.Biotechnol.,2020,38:460-470,doi:10.1038/s41587-020-0430-6; and Hogrebe et al ,"Generation of insulin-producing pancreatic beta cells from multiple human stem cell lines,"Nat.Protoc.,2021,doi:10.1038/s41596-021-00560-y, which are incorporated herein by reference in their entirety.

Modified low-immunogenicity beta islet cells differentiated from B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg, CD46tg, CD59tg hiPSC were transplanted into type I diabetics via intramuscular or intravenous injection. 100, 500 or 2500 units/kg heparin are administered to the patient by intravenous injection.

The long-term function of transplanted beta islet cells was analyzed by measuring blood glucose levels. Blood glucose measurements were made 4 hours after fasting according to standard protocols. Blood glucose levels between 80 and 120mg/dL are classified as non-diabetic, while blood glucose levels >200mg/dL are classified as diabetic.

The B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg, CD46tg, CD59tg combination modifications improve long-term survival and implantation of transplanted hiPSC-derived beta islet cells and improve long-term function of transplanted hiPSC-derived beta islet cells (e.g., as monitored by blood glucose levels). Administration of heparin in combination with administration of modified, hypoimmunogenic beta islet cells (B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}, CD47tg, CD46tg, CD59 tg) may improve survival, implantation, and/or function of transplanted hipscs and/or may reduce coagulation pathway activation in response to hiPSC-derived beta islet cell transplantation (e.g., reduce levels of thrombin-antithrombin III complex (TAT) or C peptide after transplantation).

Example 3 transplantation of B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}、CD142^{Indel of insertion / Indel of insertion}, CD47 tghiPSC-derived beta islet cells

B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}、CD142^{Indel of insertion / Indel of insertion}, CD47tg hiPSC were generated as described in example 1 and differentiated into beta islet cells as described in example 2 above.

Modified low-immunogenicity beta islet cells differentiated from B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}、CD142^{Indel of insertion / Indel of insertion}, CD47tg hiPSC were transplanted into type 1 diabetic patients as described in example 2, except that heparin was not further administered to the patient. The engraftment, long-term survival and long-term function of patient-transplanted cells were monitored as described in example 2.

The B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}、CD142^{Indel of insertion / Indel of insertion}, CD47tg combination modification improves long-term survival and implantation of transplanted hiPSC-derived beta islet cells and improves long-term function of transplanted hiPSC-derived beta islet cells (e.g., as monitored by blood glucose levels). B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}、CD142^{Indel of insertion / Indel of insertion}, CD47tg can reduce complement-dependent cytotoxicity and reduce coagulation pathway activation (e.g., reduce levels of thrombin-antithrombin III complex (TAT) or C peptide after transplantation) in response to transplantation of hiPSC-derived beta islet cells. Thus, B2M ^{Indel of insertion / Indel of insertion}、CIITA^{Indel of insertion / Indel of insertion}、CD142^{Indel of insertion / Indel of insertion}, CD47tg modified beta islet cells can avoid activation of the blood-mediated immune response (IBMIR) pathway immediately after transplantation.

The present invention is not intended to limit the scope of the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the described compositions and methods will be apparent from the description and teachings herein. Such modifications may be implemented without departing from the true scope and spirit of the disclosure, and such modifications are intended to fall within the scope of the disclosure.

Sequence(s)

Claims

2. The engineered cell of claim 1, wherein one or more of the modifications in (iii) reduces expression of:

a. One or more MHC class I molecules;

b. One or more MHC class II molecules; or (b)

C. one or more MHC class I molecules and one or more MHC class II molecules.

3. The engineered cell of claim 1 or claim 2, wherein the one or more modifications reduce expression ：B2M、TAP I、NLRC5、CIITA、HLa-a、HLA-B、HLA-C、HLA-DP、HLA-DM、HLA-DOA、HLA-DOB、HLA-DQ、HLA-DR、RFX5、RFXANK、RFXAP、NFY-A、NFY-B and/or NFY-C of one or more molecules selected from the group consisting of and any combination thereof.

4. The engineered cell of any one of claims 1-3, wherein the engineered cell does not express one or more molecules ：B2M、TAP I、NLRC5、CIITA、HLa-a、HLA-B、HLA-C、HLA-DP、HLA-DM、HLA-DOA、HLA-DOB、HLA-DQ、HLA-DR、RFX5、RFXANK、RFXAP、NFY-A、NFY-B and/or NFY-C selected from the group consisting of and combinations thereof.

5. The engineered cell of any one of claims 1-4, wherein the one or more tolerogenic factors comprise one or more tolerogenic factors selected from the group consisting of: CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDOl, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8 and SERPINB9 and any combination thereof.

6. The engineered cell of claim 5, wherein the one or more tolerogenic factors are selected from the group consisting of: CD47, PD-L1, HLA-E or HLA-G, CCL21, FASL, SERPINB9, CD200, MFGE8, and any combination thereof.

7. The engineered cell of any one of claims 1-6, wherein the one or more tolerogenic factors comprise one or more tolerogenic factors selected from the group consisting of: A20/TNFAIP3, C1-inhibitor, CCL21, CCL22, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, fasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, IL-10, IL15-RF, IL-35, MANF, mfge8, PD-1, PD-L1, serpinb9, and any combination thereof.

8. The engineered cell of any one of claims 1-7, wherein the one or more tolerogenic factors comprise CD47.

9. The engineered cell of any one of claims 1-8, wherein the one or more tolerogenic factors comprise HLA-E.

10. The engineered cell of any one of claims 1-9, wherein the one or more tolerogenic factors comprise CD24.

11. The engineered cell of any one of claims 1-10, wherein the one or more tolerogenic factors comprises PDL1.

12. The engineered cell of any one of claims 1-11, wherein the one or more tolerogenic factors comprise CD55.

13. The engineered cell of any one of claims 1-12, wherein the one or more tolerogenic factors comprise CR1.

14. The engineered cell of any one of claims 1-13, wherein the one or more tolerogenic factors comprise MANF.

15. The engineered cell of any one of claims 1-14, wherein the one or more tolerogenic factors comprise a20/TNFAIP3.

16. The engineered cell of any one of claims 1-15, wherein the one or more tolerogenic factors comprise HLA-E and CD47.

17. The engineered cell of any one of claims 1-16, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of CD24, CD47, and PDL1, optionally wherein the one or more tolerogenic factors comprise CD24, CD47, and PDL1.

18. The engineered cell of any one of claims 1-17, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD47, and PDL1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD, CD47, and PDL1.

19. The engineered cell of any one of claims 1-18, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of CD46, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise CD46, CD55, CD59, and CR1.

20. The engineered cell of any one of claims 1-19, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD46, CD55, CD59, and CR1.

21. The engineered cell of any one of claims 1-20, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD47, PDL1, CD46, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD, CD47, PDL1, CD46, CD55, CD59, and CR1.

22. The engineered cell of any one of claims 1-21, wherein the one or more tolerogenic factors comprise HLA-E and PDL1.

23. The engineered cell of any one of claims 1-22, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1 and a20/TNFAIP, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1 and a20/TNFAIP.

24. The engineered cell of any one of claims 1-23, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1 and MANF, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1 and MANF.

25. The engineered cell of any one of claims 1-24, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1, a20/TNFAIP and MANF, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1, a20/TNFAIP and MANF.

27. The engineered cell of claim 26, wherein one or more of (i) increasing expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200, and MFGE8, (ii) increasing expression of CD46, and (iii) increasing expression of CD59 comprises one or more modifications that increase gene activity of an endogenous gene.

28. The engineered cell of claim 27, wherein the endogenous gene encodes the CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200, MFGE8, CD46, or CD59.

29. The engineered cell of claim 27 or 28, wherein the one or more modifications that increase the gene activity of an endogenous gene comprise the introduction of one or more modifications of an endogenous promoter or enhancer of the gene or a heterologous promoter.

30. The engineered cell of claim 29, wherein the heterologous promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter for HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter for HIV, moloney virus promoter, epstein Barr Virus (EBV) promoter and Rous Sarcoma Virus (RSV) promoter, and UBC promoter.

31. The engineered cell of any one of claims 1-30, wherein the engineered cell further comprises one or more modifications that increase expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD55, wherein the increased expression of the one or more complement inhibitors is relative to a cell of the same cell type that does not comprise the modification.

32. The engineered cell of any one of claims 1-31, wherein the modification that increases expression comprises increased surface expression and/or the modification that decreases expression comprises decreased surface expression.

33. The engineered cell of claim 31 or claim 32, wherein the modification that increases expression of the one or more complement inhibitors comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and/or an exogenous polynucleotide encoding CD 55.

34. The engineered cell of any one of claims 31-33, wherein the one or more complement inhibitors are CD46 and CD59, optionally wherein the modification comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD59.

35. The engineered cell of any one of claims 31-34, wherein the one or more complement inhibitors are CD46, CD59, and CD55, optionally wherein the modification comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55.

36. The engineered cell of any one of claims 33-35, wherein the exogenous polynucleotide encoding CD46 encodes an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID No. 3.

37. The engineered cell of claim 36, wherein the exogenous polynucleotide encoding CD46 encodes the sequence set forth in SEQ ID No. 3.

38. The engineered cell of any one of claims 33-37, wherein the exogenous polynucleotide encoding CD59 encodes an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID No. 5 and exhibits complement inhibitory activity.

39. The engineered cell of claim 38, wherein the exogenous polynucleotide encoding CD59 encodes the sequence set forth in SEQ ID No. 5.

40. The engineered cell of any one of claims 33-39, wherein the exogenous polynucleotide encoding CD55 encodes an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID No. 8 and exhibits complement inhibitory activity.

41. The engineered cell of claim 40, wherein the exogenous polynucleotide encoding CD55 encodes the sequence set forth in SEQ ID NO. 8.

42. The engineered cell of any one of claims 33-41, wherein the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, and/or the exogenous polynucleotide encoding CD55 are each operably linked to a promoter.

43. The engineered cell of any one of claims 5-42, wherein the modification that increases CD47 expression comprises an exogenous polynucleotide encoding the CD47 protein.

44. The engineered cell of claim 43, wherein the exogenous polynucleotide encoding CD47 encodes an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID NO. 1 and reduces innate immune killing of the engineered cell.

45. The engineered cell of any one of claims 43-44, wherein said exogenous polynucleotide encoding CD47 encodes the sequence set forth in SEQ ID No. 1.

46. The engineered cell of any one of claims 43-45, wherein the exogenous polynucleotide encoding CD47 is operably linked to a promoter.

47. The engineered cell of any one of claims 1-46, wherein the engineered cell comprises a polycistronic vector comprising two or more exogenous polynucleotides selected from the group consisting of: one or more exogenous polynucleotides encoding the one or more tolerogenic factors, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding a CD55 polypeptide.

48. The engineered cell of claim 47, wherein each of said polynucleotides is isolated by an IRES or self-cleaving peptide.

49. The engineered cell of any one of claims 47-48, wherein each polynucleotide of the polycistronic vector is operably linked to the same promoter.

50. The engineered cell of any one of claims 47-49, wherein the polycistronic vector comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59.

51. The engineered cell of any one of claims 47-50, wherein the polycistronic vector comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55.

52. The engineered cell of claim 50 or claim 51, wherein the polycistronic vector further comprises an exogenous polynucleotide encoding CD47.

53. The engineered cell of claim 50 or claim 51, wherein the polycistronic vector is a first transgene and the engineered cell comprises a separate transgene comprising a polynucleotide encoding CD 47.

54. The engineered cell of any one of claims 1-53, wherein the engineered cell comprises a transgene comprising a polynucleotide encoding CD 47.

55. The engineered cell of any one of claims 1-46, wherein the engineered cell comprises a first transgene and a second transgene,

56. The engineered cell of any one of claims 42-55, wherein the promoter is a constitutive promoter.

57. The engineered cell of any one of claims 42-56, wherein said promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter for HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter for HIV, moloney virus promoter, epstein Barr Virus (EBV) promoter and Rous Sarcoma Virus (RSV) promoter, and UBC promoter.

58. The engineered cell of any one of claims 33-57, wherein the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, and/or the exogenous polynucleotide encoding CD55 polypeptide is integrated into the genome of the engineered cell.

59. The engineered cell of any one of claims 43-58, wherein the exogenous polynucleotide encoding CD47 is integrated into the genome of the engineered cell.

60. The engineered cell of claim 58 or claim 59, wherein said integration is by non-targeted insertion into the genome of the engineered cell, optionally by introducing the exogenous polynucleotide into the cell using a lentiviral vector.

61. The engineered cell of claim 58 or claim 59, wherein said integration is by targeted insertion into a target genomic locus of the cell.

62. The engineered cell of claim 61, wherein the target genomic locus is selected from the group consisting of: MICA locus, MICB locus, B2M locus, CIITA locus, TRAC locus or TRBC locus, CD142 locus, CCR5 locus, CXCR4 locus, PPP1R12C (also known as AAVS 1) locus, albumin locus, SHS231 locus, CLYBL locus, ROSA26 locus, LRP1 locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus and KDM5D locus.

63. The engineered cell of claim 62, wherein the target genomic locus is a MICA locus, a MICB locus, a TAP1 locus, a B2M locus, a CIITA locus, a TRAC locus, a TRBC locus, or a safe harbor locus.

64. The engineered cell of claim 63, wherein the target genomic locus is selected from the group consisting of: CCR5 locus, CXCR4 locus, PPP1R12C (also known as AAVS 1) locus, albumin locus, SHS231 locus, CLYBL locus and ROSA26 locus.

65. The engineered cell of claim 64, wherein the safe harbor locus is selected from the group consisting of: AAVS1, ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA, and SHS231 loci.

66. The engineered cell of any one of claims 61-65, wherein the exogenous polynucleotide encoding CD47 is integrated into a first target genomic locus, the exogenous polynucleotide encoding CD46 is integrated into a second target genomic locus, and the polynucleotide encoding CD59 is integrated into a third target genomic locus.

67. The engineered cell of claim 66, wherein the exogenous polynucleotide encoding CD55 is integrated into a fourth target genomic locus.

68. The engineered cell of claim 66, wherein at least two of the first, second, and third target genomic loci are the same locus.

69. The engineered cell of claim 67 or claim 68, wherein at least two of the first, second, third, and fourth target genomic loci are the same locus.

70. The engineered cell of any one of claims 66-69, wherein the first, second, and third target genomic loci are the same locus.

71. The engineered cell of claims 66-70, wherein the first, second, third, and fourth target genomic loci are the same locus.

72. The engineered cell of claim 66 or claim 67, wherein each of the first, second, and third target genomic loci are different loci.

73. The engineered cell of claim 67, wherein the first, second, third, and fourth target genomic loci are different loci.

74. The engineered cell of any one of claims 1-73, wherein the modification that reduces expression of CD142 reduces CD142 protein expression.

75. The engineered cell of claim 74, wherein said modification eliminates CD142 gene activity.

76. The engineered cell of any one of claims 74 or 75, wherein the modification comprises inactivation or disruption of both alleles of the CD142 gene.

77. The engineered cell of any one of claims 75-76, wherein said modification comprises inactivation or disruption of all CD142 coding sequences in said cell.

78. The engineered cell of claim 76 or claim 77, wherein said inactivation or disruption comprises an indel in the CD142 gene.

79. The engineered cell of any one of claims 61-65, wherein the modification is a frameshift mutation or deletion of a stretch of contiguous genomic DNA of the CD142 gene.

80. The engineered cell of any one of claims 75-79, wherein CD142 gene is knocked out.

81. The engineered cell of any one of claims 75-80, wherein the modification is by a genomic modification protein, optionally wherein the modification is by nuclease-mediated genome editing.

82. The engineered cell of claim 81, wherein the nuclease-mediated genome editing is by a Zinc Finger Nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas combination targeted to the CD142 gene, optionally wherein the Cas is selected from Cas9 or Cas12.

83. The engineered cell of claim 82, wherein the nuclease-mediated genome editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site within the CD142 gene.

84. The engineered cell of claim 83, wherein the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising the gRNA and Cas protein.

85. The engineered cell of any one of claims 1-25 and 27-84, wherein the modification that reduces expression of one or more MHC class I molecules reduces expression of one or more MHC class I molecule proteins.

86. The engineered cell of any one of claims 1-25 and 27-85, wherein the modification that reduces expression of one or more MHC class I molecules comprises reduced expression of B2M.

87. The engineered cell of any one of claims 74-86, wherein the modification that reduces expression of one or more MHC class I molecules comprises reduced protein expression of B2M.

88. The engineered cell of claim 86 or claim 87, wherein the modification eliminates B2M gene activity.

89. The engineered cell of any one of claims 86-88, wherein the modification comprises inactivation or disruption of both alleles of the B2M gene.

90. The engineered cell of any one of claims 86-89, wherein the modification comprises inactivation or disruption of all B2M coding sequences in the cell.

91. The engineered cell of claim 89 or claim 90, wherein the inactivation or disruption comprises an indel in the B2M gene.

92. The engineered cell of any one of claims 86-91, wherein the modification is a frameshift mutation or deletion of a stretch of contiguous genomic DNA of the B2M gene.

93. The engineered cell of any one of claims 86-92, wherein the B2M gene is knocked out.

94. The engineered cell of any one of claims 85-93, wherein the modification is by a genomic modification protein, optionally wherein the modification is by nuclease-mediated gene editing.

95. The engineered cell of claim 94, wherein the modification by the genomic modification protein is performed using ：Cas3、Cas4、Cas5、Cas8a、Cas8b、Cas8c、Cas9、Cas10、Cas12、Cas12a(Cpf1)、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12f(C2c10)、Cas12g、Cas12h、Cas12i、Cas12k(C2c5)、Cas13、Cas13a(C2c2)、Cas13b、Cas13c、Cas13d、C2c4、C2c8、C2c9、Cmr5、Cse1、Cse2、Csf1、Csm2、Csn2、Csx10、Csx11、Csy1、Csy2、Csy3、Mad7、 zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, CRISPR-associated transposase, or any CRISPR-Cas combination that targets the B2M gene, optionally wherein the modification is performed by nuclease-mediated gene editing using Cas9 or Cas 12.

96. The engineered cell of claim 95, wherein the nuclease-mediated gene editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site within the B2M gene.

97. The engineered cell of claim 96, wherein the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising the gRNA and Cas protein.

98. The engineered cell of any one of claims 1-25 and 27-97, wherein the modification that reduces expression of one or more MHC class II molecules reduces expression of one or more MHC class II molecule proteins.

99. The engineered cell of any one of claims 1-25 and 27-98, wherein the modification that reduces expression of one or more MHC class II molecules comprises reduced expression of CIITA.

100. The engineered cell of claim 99, wherein the modification that reduces expression of one or more MHC class II molecules comprises reduced protein expression of CIITA.

101. The engineered cell of claim 99 or claim 100, wherein the modification eliminates CIITA.

102. The engineered cell of any one of claims 99-101, wherein the modification comprises inactivation or disruption of both alleles of the CIITA gene.

103. The engineered cell of any one of claims 99-102, wherein the modification comprises inactivation or disruption of all CIITA coding sequences in the cell.

104. The engineered cell of claim 102 or claim 103, wherein the inactivation or disruption comprises an indel in the CIITA gene.

105. The engineered cell of any one of claims 102-104, wherein the indel is a frameshift mutation or deletion of a stretch of contiguous genomic DNA of the CIITA gene.

106. The engineered cell of any one of claims 99-105, wherein the CIITA gene is knocked out.

107. The engineered cell of any one of claims 1-106, wherein the modification is by a genomic modification protein.

108. The engineered cell of claim 107, wherein the modification by the genomic modification protein is a modification by CRISPR-associated transposase, guided editing, or programmable addition via a site-specific targeting element (PASTE).

109. The engineered cell of claim 107 or 108, wherein the modification by the genomic modification protein is nuclease-mediated gene editing.

110. The engineered cell of claim 109, wherein the nuclease-mediated gene editing is by a Zinc Finger Nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas combination, optionally wherein the Cas is selected from Cas9 or Cas12.

111. The engineered cell of claim 107 or 108, wherein the modification by the genome modification protein is performed by one or more proteins selected from the group consisting of ：Cas3、Cas4、Cas5、Cas8a、Cas8b、Cas8c、Cas9、Cas10、Cas12、Cas12a(Cpf1)、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12f(C2c10)、Cas12g、Cas12h、Cas12i、Cas12k(C2c5)、Cas13、Cas13a(C2c2)、Cas13b、Cas13c、Cas13d、C2c4、C2c8、C2c9、Cmr5、Cse1、Cse2、Csf1、Csm2、Csn2、Csx10、Csx11、Csy1、Csy2、Csy3、Mad7、 Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and CRISPR-associated transposases.

112. The engineered cell of any one of claims 1-111, wherein the modification:

113. The engineered cell of any one of claims 1-112, wherein one or more of (i) increasing expression of one or more tolerogenic factors, (ii) increasing expression of CD46, and (iii) the modification that increases expression of CD59 comprises one or more modifications that increase gene activity of an endogenous gene.

114. The engineered cell of claim 113, wherein the endogenous gene encodes the one or more tolerogenic factors CD46 or CD59.

115. The engineered cell of claim 113 or 114, wherein the one or more modifications that increase the gene activity of the endogenous gene comprise the introduction of one or more modifications or heterologous promoters of endogenous promoters or enhancers of the gene.

116. The engineered cell of claim 115, wherein the heterologous promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter for HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter for HIV, moloney virus promoter, epstein Barr Virus (EBV) promoter and Rous Sarcoma Virus (RSV) promoter, and UBC promoter.

117. The engineered cell of any one of claims 1-116, wherein the engineered cell is a human cell or an animal cell.

118. The engineered cell of claim 117, wherein the engineered cell is a human cell.

119. The engineered cell of any one of claims 1-118, wherein the cell is a blood-exposed cell type or a cell type capable of differentiating into a blood-exposed cell type.

120. The engineered cell of any one of claims 1-119, wherein the engineered cell is a differentiated cell derived from a pluripotent stem cell or progeny thereof.

121. The engineered cell of claim 120, wherein the pluripotent stem cell is an induced pluripotent stem cell.

122. The engineered cell of claim 119, wherein the engineered cell is a primary cell isolated from a donor subject.

123. The engineered cell of claim 122, wherein the donor subject is healthy or not suspected of having a disease or disorder at the time a donor sample is obtained from the individual donor.

124. The engineered cell of any one of claims 1-123, wherein the engineered cell is selected from the group consisting of: islet cells, beta islet cells, pancreatic islet cells, immune cells, B cells, T cells, natural Killer (NK) cells, natural Killer T (NKT) cells, macrophages, endothelial cells, muscle cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, dopaminergic neurons, retinal pigment epithelial cells, visual cells, liver cells, thyroid cells, skin cells, glial progenitor cells, neural cells, cardiac cells, stem cells, hematopoietic stem cells, induced pluripotent stem cells (ipscs), mesenchymal Stem Cells (MSCs), embryonic Stem Cells (ESCs), pluripotent Stem Cells (PSCs), and blood cells.

125. The engineered cell of any one of claims 1-124, wherein the engineered cell is an endothelial cell.

126. The engineered cell of any one of claims 1-125, wherein the engineered cell is an epithelial cell.

127. The engineered cell of claim 126, wherein the engineered cell is a T cell.

128. The engineered cell of claim 127, wherein the engineered cell is an NK cell.

129. The engineered cell of claim 127 or claim 128, wherein the engineered cell comprises a Chimeric Antigen Receptor (CAR).

130. The engineered cell of claim 124, wherein the engineered cell is a stem cell.

131. The engineered cell of claim 124, wherein the engineered cell is a Hematopoietic Stem Cell (HSC).

132. The engineered cell of claim 124, wherein the engineered cell is a beta islet cell.

133. The engineered cell of claim 124, wherein the engineered cell is a hepatocyte.

134. The engineered cell of claim 124, wherein the engineered cell is a pluripotent stem cell.

135. The engineered cell of claim 124, wherein the engineered cell is an induced pluripotent stem cell.

136. The engineered cell of claim 124, wherein the engineered cell is an embryonic stem cell.

137. The engineered cell of any one of claims 1-136, wherein the cell is ABO blood group O.

138. The engineered cell of any one of claims 1-137, wherein the cell is rhesus factor negative (Rh-).

139. The engineered cell of any one of claims 1-136 and 138, wherein the cell comprises a functional ABO a allele and/or a functional ABO B allele.

140. The engineered cell of any one of claims 1-137 and 139, wherein the cell is rhesus factor positive (rh+).

141. A method of generating an engineered cell, the method comprising:

b. reducing expression of CD142 in the cell; and

C. Increasing expression of a tolerogenic factor in said cell.

142. The method of claim 141, wherein the one or more tolerogenic factors are selected from the group consisting of: CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDOl, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8 and SERPINB9 and any combination thereof.

143. The method of claim 142, wherein the one or more tolerogenic factors are selected from the group consisting of: CD47, PD-L1, HLA-E or HLA-G, CCL21, FASL, SERPINB9, CD200, MFGE8, and any combination thereof.

144. The method of any one of claims 141-143, wherein the one or more tolerogenic factors comprise one or more tolerogenic factors selected from the group consisting of: A20/TNFAIP3, C1-inhibitor, CCL21, CCL22, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, fasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, IL-10, IL15-RF, IL-35, MANF, mfge8, PD-1, PD-L1, serpinb9, and any combination thereof.

145. The method of claim 143 or claim 144, wherein the one or more tolerogenic factors comprise CD47.

146. The method of any one of claims 141-145, wherein the one or more tolerogenic factors comprise HLA-E.

147. The method of any one of claims 141-146, wherein the one or more tolerogenic factors comprise CD24.

148. The method of any one of claims 141-147, wherein the one or more tolerogenic factors comprise PDL1.

149. The method of any one of claims 141-148, wherein the one or more tolerogenic factors comprise CD55.

150. The method of any one of claims 141-149, wherein the one or more tolerogenic factors comprise CR1.

151. The method of any one of claims 141-150, wherein the one or more tolerogenic factors comprise MANF.

152. The method of any one of claims 141-151, wherein the one or more tolerogenic factors comprise a20/TNFAIP3.

153. The method of any one of claims 141-152, wherein the one or more tolerogenic factors comprise HLA-E and CD47.

154. The method of any one of claims 141-153, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of CD24, CD47, and PDL1, optionally wherein the one or more tolerogenic factors comprise CD24, CD47, and PDL1.

155. The method of any one of claims 141-154, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD47, and PDL1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD, CD47, and PDL1.

156. The method of any one of claims 141-155, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of CD46, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprises CD46, CD55, CD59, and CR1.

157. The method of any one of claims 141-156, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD46, CD55, CD59, and CR1.

158. The method of any one of claims 141-157, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD24, CD47, PDL1, CD46, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD, CD47, PDL1, CD46, CD55, CD59, and CR1.

159. The method of any one of claims 141-158, wherein the one or more tolerogenic factors comprise HLA-E and PDL1.

160. The method of any one of claims 141-159, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1 and a20/TNFAIP, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1 and a20/TNFAIP.

161. The method of any one of claims 141-160, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1 and MANF, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1 and MANF.

162. The method of any one of claims 141-161, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1, a20/TNFAIP and MANF, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1, a20/TNFAIP and MANF.

163. The method of any one of claims 141-162, wherein the method comprises reducing expression of one or more MHC class I molecules and one or more MHC class II molecules.

164. The method of any one of claims 141-163, wherein increasing expression of the tolerogenic factor comprises increasing gene activity of an endogenous gene.

165. The method of claim 164, wherein the endogenous gene encodes the tolerogenic factor.

166. The method of claim 164 or 165 wherein increasing the gene activity of the endogenous gene comprises modifying an endogenous promoter or enhancer of the gene, or introducing a heterologous promoter.

167. The method of claim 166, wherein the heterologous promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter for HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter for HIV, moloney virus promoter, epstein Barr Virus (EBV) promoter and Rous Sarcoma Virus (RSV) promoter, and UBC promoter.

168. A method of generating a low-immunogenicity cell, the method comprising:

B. reducing expression of CD142 in the cell.

169. The method of any one of claims 141-168, further comprising increasing expression of one or more complement inhibitors selected from the group consisting of CD46, CD59, and CD55 in the cell.

170. The method of claim 168 or 169, wherein increasing expression of CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200, MFGE8, and/or one or more complement inhibitors comprises increasing gene activity of an endogenous gene.

171. The method of claim 170, wherein the endogenous gene encodes the CCL21, PD-L1, FASL, SERPINB9, HLA-G, CD47, CD200, MFGE8, CD46, or CD59.

172. The method of claim 170 or claim 171, wherein increasing the gene activity of the endogenous gene comprises modifying an endogenous promoter or enhancer of the gene, or introducing a heterologous promoter.

173. The method of claim 172, wherein the heterologous promoter is selected from the group consisting of: the CAG promoter, cytomegalovirus (CMV) promoter, EF1a promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter for HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter for HIV, moloney virus promoter, epstein Barr Virus (EBV) promoter and Rous Sarcoma Virus (RSV) promoter, and UBC promoter.

174. The method of any one of claims 141-173, wherein the reduced expression comprises reduced surface expression and/or the increased expression comprises increased surface expression, optionally wherein the reduced surface expression comprises no detectable surface expression.

175. The method of any one of claims 169-174, wherein increasing expression of the one or more complement inhibitors comprises introducing an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and/or an exogenous polynucleotide encoding CD55 into the cell.

176. The method of any one of claims 169-175, wherein the one or more complement inhibitors are CD46 and CD59, optionally wherein increasing expression of the one or more complement inhibitors comprises introducing an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59.

177. The method of any one of claims 169-176, wherein the one or more complement inhibitors are CD46, CD59, and CD55, optionally wherein increasing expression of the one or more complement inhibitors comprises introducing an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55.

178. The method of any of claims 169-177, wherein the exogenous polynucleotide encoding CD46 encodes an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID No. 3 and exhibits complement inhibitory activity.

179. The method of claim 178, wherein the exogenous polynucleotide encoding CD46 encodes the sequence set forth in SEQ ID No. 3.

180. The method of any one of claims 169-179, wherein the exogenous polynucleotide encoding CD59 encodes an amino acid sequence that has at least 85% identity to the amino acid sequence of SEQ ID No. 5 and exhibits complement inhibitory activity.

181. The method of claim 180, wherein the exogenous polynucleotide encoding CD59 encodes the sequence set forth in SEQ ID No. 5.

182. The method of any one of claims 169-181, wherein said exogenous polynucleotide encoding CD55 encodes a sequence that has at least 85% identity to the amino acid sequence of SEQ ID No.8 and exhibits complement inhibitory activity.

183. The method of claim 182, wherein the exogenous polynucleotide encoding CD55 encodes the sequence set forth in SEQ ID No. 8.

184. The method of any one of claims 169-183, wherein each of the CD 46-encoding exogenous polynucleotide, the CD 59-encoding exogenous polynucleotide, and/or the CD 55-encoding exogenous polynucleotide is operably linked to a promoter.

185. The method of any one of claims 169-184, wherein said modification that increases CD47 expression comprises an exogenous polynucleotide encoding said CD47 protein.

186. The method of any one of claims 169-185, wherein the exogenous polynucleotide encoding CD47 encodes a sequence that has at least 85% identity to the amino acid sequence of SEQ ID No. 1 and reduces innate immune killing of the engineered cells.

187. The method of claim 186, wherein the exogenous polynucleotide encoding CD47 encodes the sequence set forth in SEQ ID No. 1.

188. The method of any one of claims 169-187, wherein the exogenous polynucleotide encoding CD47 is operably linked to a promoter.

189. The method of any one of claims 169-188, wherein the method comprises introducing into the cell a polycistronic vector comprising two or more exogenous polypeptides selected from the group consisting of: one or more exogenous polynucleotides encoding the one or more tolerogenic factors, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding a CD55 polypeptide.

190. The method of claim 189, wherein each of the polynucleotides is isolated by an IRES or self-cleaving peptide.

191. The method of claim 189 or claim 190, wherein the two or more exogenous polynucleotides are selected from the group consisting of: an exogenous polynucleotide encoding CD47, an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding a CD55 polypeptide.

192. The method of any one of claims 189-191, wherein each polynucleotide of the polycistronic vector is operably linked to the same promoter.

193. The method of any one of claims 189-192, wherein the polycistronic vector comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59.

194. The method of any one of claims 189-193, wherein the polycistronic vector comprises an exogenous polynucleotide encoding CD46, an exogenous polynucleotide encoding CD59, and an exogenous polynucleotide encoding CD 55.

195. The method of claim 193 or claim 194, wherein the polycistronic vector further comprises an exogenous polynucleotide encoding CD47.

196. The method of claim 193 or claim 194, wherein the engineered cells comprise separate transgenes comprising a polynucleotide encoding CD 47.

197. The method of any one of claims 169-196, wherein the exogenous polynucleotide encoding CD46, the exogenous polynucleotide encoding CD59, and/or the exogenous polynucleotide encoding CD55 is integrated into the genome of the engineered cell.

198. The method of any one of claims 185-197, wherein the exogenous polynucleotide encoding CD47 is integrated into the genome of the engineered cell.

199. The method of claim 197 or claim 198, wherein the integration is by non-targeted insertion into the genome of the engineered cell.

200. The method of claim 199, wherein the integrating is performed by introducing the exogenous polynucleotide into the cell using a lentiviral vector.

201. The method of claim 197 or claim 198, wherein the integration is by targeted insertion into a target genomic locus of the cell, optionally wherein the targeted insertion is by nuclease-mediated gene editing using homology-directed repair.

202. The method of claim 201, wherein the target genomic locus is selected from the group consisting of: MICA locus, MICB locus, B2M locus, CIITA locus, TRAC locus or TRBC locus, CD142 locus, CCR5 locus, CXCR4 locus, PPP1R12C (also known as AAVS 1) locus, albumin locus, SHS231 locus, CLYBL locus, ROSA26 locus, LRP1 locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus and KDM5D locus.

203. The method of claim 201 or claim 202, wherein the target genomic locus is a MICA locus, a MICB locus, a TAP1 locus, a B2M locus, a CIITA locus, a TRAC locus, a TRBC locus, or a harbour locus.

204. The method of claim 201 or claim 202, wherein the target genomic locus is selected from the group consisting of: CCR5 locus, CXCR4 locus, PPP1R12C (also known as AAVS 1) locus, albumin locus, SHS231 locus, CLYBL locus and ROSA26 locus.

205. The method of any one of claims 201-204, wherein the target genomic locus is a safe harbor locus.

206. The method of any one of claims 201-205, wherein the nuclease-mediated gene editing is by a Zinc Finger Nuclease (ZFN), TAL effector nuclease (TALEN), or CRISPR-Cas combination targeted to the target genomic locus, optionally wherein the Cas is selected from Cas9 or Cas12.

207. The method of claim 206, wherein the nuclease-mediated gene editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to a target sequence of the target genomic locus and a homology directed repair template comprising the CD46 encoding exogenous polynucleotide, the CD59 encoding exogenous polynucleotide, the CD55 encoding exogenous polynucleotide, and/or the CD47 encoding exogenous polynucleotide.

208. The method of claim 207, wherein the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising the gRNA and a Cas protein.

209. The method of any one of claims 141-208, wherein reducing expression of CD142 reduces CD142 protein expression.

210. The method of claim 209, wherein reducing expression of CD142 comprises introducing a modification that reduces activity of a CD142 gene.

211. The method of claim 210, wherein the modification that reduces activity of the CD142 gene comprises inactivation or disruption of both alleles of the CD142 gene.

212. The method of claim 210 or claim 211, wherein the modification that reduces CD142 gene activity comprises inactivation or disruption of all CD142 coding sequences in the cell.

213. The method of claim 211 or claim 212, wherein the inactivation or disruption comprises an indel in the CD142 gene or a deletion of a stretch of contiguous genomic DNA of the CD142 gene.

214. The method of claim 213, wherein the indel is a frameshift mutation.

215. The method of any one of claims 210-214, wherein the CD142 gene is knocked out.

216. The method of any one of claims 210-215, wherein the modification that reduces CD142 gene activity is introduced by a genomic modification protein, optionally wherein the modification that reduces CD142 gene activity is introduced by nuclease-mediated gene editing.

217. The method of claim 216, wherein the modification by the genomic modification protein is performed using ：Cas3、Cas4、Cas5、Cas8a、Cas8b、Cas8c、Cas9、Cas10、Cas12、Cas12a(Cpf1)、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12f(C2c10)、Cas12g、Cas12h、Cas12i、Cas12k(C2c5)、Cas13、Cas13a(C2c2)、Cas13b、Cas13c、Cas13d、C2c4、C2c8、C2c9、Cmr5、Cse1、Cse2、Csf1、Csm2、Csn2、Csx10、Csx11、Csy1、Csy2、Csy3、Mad7、 zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, CRISPR-associated transposase, or any CRISPR-Cas combination that targets the CD142 gene, optionally wherein the modification is performed by nuclease-mediated gene editing using Cas9 or Cas 12.

218. The method of claim 217, wherein the nuclease-mediated gene editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site within the CD142 gene.

219. The method of claim 218, wherein the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising the gRNA and a Cas protein.

220. The method of any one of claims 141-219, wherein reducing expression of one or more MHC class I molecules comprises introducing a modification that reduces expression of one or more MHC class I molecule proteins.

221. The method of claim 220, wherein the modification that reduces expression of one or more MHC class I molecule proteins comprises reduced expression of B2M.

222. The method of claim 220 or 221, wherein the modification that reduces expression of one or more MHC class I molecule proteins comprises reduced expression of B2M protein.

223. The method of claim 221 or 222, wherein the modification that reduces expression of one or more MHC class I molecule proteins reduces B2M gene activity.

224. The method of any one of claims 221-223, wherein the modification that reduces expression of one or more MHC class I molecules comprises inactivation or disruption of both alleles of the B2M gene.

225. The method of any one of claims 221-223, wherein the modification that reduces expression of one or more MHC class I molecule proteins comprises inactivation or disruption of all B2M coding sequences in the cell.

226. The method of claim 224 or claim 225 wherein the inactivation or disruption comprises an indel in the B2M gene or a deletion of a stretch of contiguous genomic DNA of the B2M gene.

227. The method of claim 226, wherein the indel is a frameshift mutation.

228. The method of any one of claims 221-227, wherein the B2M gene is knocked out.

229. The method of any one of claims 221-228, wherein the modification to reduce expression of one or more MHC class I molecule proteins is by a genomic modification protein, optionally wherein the modification to reduce expression of one or more MHC class I molecule proteins is by nuclease-mediated gene editing.

230. The method of claim 229, wherein the modification by the genome modification protein is performed using ：Cas3、Cas4、Cas5、Cas8a、Cas8b、Cas8c、Cas9、Cas10、Cas12、Cas12a(Cpf1)、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12f(C2c10)、Cas12g、Cas12h、Cas12i、Cas12k(C2c5)、Cas13、Cas13a(C2c2)、Cas13b、Cas13c、Cas13d、C2c4、C2c8、C2c9、Cmr5、Cse1、Cse2、Csf1、Csm2、Csn2、Csx10、Csx11、Csy1、Csy2、Csy3、Mad7、 zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, CRISPR-associated transposases, or any CRISPR-Cas combination that targets the B2M gene, optionally wherein the modification is performed by nuclease-mediated gene editing using Cas9 or Cas 12.

231. The method of claim 230, wherein the nuclease-mediated gene editing is by a CRISPR-Cas combination, and the CRISPR-Cas combination comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site within the B2M gene.

232. The method of claim 231, wherein the CRISPR-Cas combination is a Ribonucleoprotein (RNP) complex comprising the gRNA and a Cas protein.

233. The method of any one of claims 141-232, wherein reducing expression of one or more MHC class II molecules comprises introducing a modification that reduces expression of one or more MHC class II molecule proteins.

234. The method of claim 233, wherein the modification that reduces expression of one or more MHC class II molecule proteins comprises reduced expression of CIITA.

235. The method of claim 233 or 234, wherein the modification that reduces expression of one or more MHC class II molecule proteins comprises reduced protein expression of CIITA.

236. The method of claim 233 or claim 234, wherein the modification that reduces expression of one or more MHC class II molecule proteins reduces CIITA gene activity.

237. The method of any one of claims 233-236, wherein the modification that reduces expression of one or more MHC class II molecule proteins comprises inactivation or disruption of both alleles of the CIITA gene.

238. The method of any one of claims 233-237, wherein the modification comprises inactivation or disruption of all CIITA coding sequences in the cell.

239. The method of claim 237 or claim 238, wherein the inactivation or disruption comprises an indel in the CIITA gene or a deletion of a stretch of contiguous genomic DNA of the CIITA gene.

240. The method of claim 239, wherein the indels are frameshift mutations.

241. The method of any one of claims 234-240, wherein the CIITA gene is knocked out.

242. The method of any one of claims 141-241, wherein the cell is a human cell or an animal cell, optionally wherein the animal cell is a pig (pig/candidate) cell, a cow (cow/candidate) cell, or a sheep (shaep/ovine) cell.

243. The method of any one of claims 141-242, wherein the engineered cell is a human cell.

244. The method of any one of claims 141-243, wherein the cell is a blood-exposed cell type or a cell type capable of differentiating into a blood-exposed cell type.

245. The method of any one of claims 141-244, wherein the cell is a primary cell isolated from a donor subject.

246. The method of any one of claims 141-244, wherein the cell is a pluripotent stem cell, wherein the engineered cell is a differentiated cell derived from the pluripotent stem cell, and the method further comprises differentiating the pluripotent stem cell.

247. The method of claim 246, wherein the pluripotent stem cells are induced pluripotent stem cells.

248. The method of any one of claims 141-243, wherein the engineered cell is selected from the group consisting of: islet cells, beta islet cells, pancreatic islet cells, immune cells, B cells, T cells, natural Killer (NK) cells, natural Killer T (NKT) cells, macrophages, endothelial cells, muscle cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, dopaminergic neurons, retinal pigment epithelial cells, visual cells, liver cells, thyroid cells, skin cells, glial progenitor cells, neural cells, cardiac cells, stem cells, hematopoietic stem cells, induced pluripotent stem cells (ipscs), mesenchymal Stem Cells (MSCs), embryonic Stem Cells (ESCs), pluripotent Stem Cells (PSCs), and blood cells.

249. The method of claim 248, wherein the engineered cell is a beta islet cell.

250. The method of claim 248, wherein the engineered cell is a hepatocyte.

251. An engineered cell produced according to the method of any one of claims 141-250.

252. The engineered cell of any one of claims 1-140 and 251, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell are capable of escaping NK cell-mediated cytotoxicity upon administration to a recipient patient.

253. The engineered cell of any one of claims 1-140 and 251-252, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell are protected from cell lysis of mature NK cells after administration to a recipient patient.

254. The engineered cell of any one of claims 1-140 and 251-253, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce an immune response to the cell after administration to a recipient patient.

255. The engineered cell of any one of claims 1-140 and 251-254, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce a systemic inflammatory response to the cell after administration to a recipient patient.

256. The engineered cell of any one of claims 1-140 and 251-255, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce a local inflammatory response to the cell after administration to a recipient patient.

257. The engineered cell of any one of claims 1-140 and 251-256, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce complement pathway activation upon administration to a recipient patient.

258. The engineered cell of any one of claims 1-140 and 251-257, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce clotting upon administration to a recipient patient.

259. The engineered cell of any one of claims 1-140 and 251-258, wherein the engineered cell or progeny or differentiated cells derived from the engineered cell do not induce an immediate blood-mediated inflammatory response upon administration to a recipient patient.

260. The engineered cell of any one of claims 258-259, wherein the cell is in contact with blood after administration to the recipient patient.

261. A population of cells comprising a plurality of engineered cells of any one of claims 1-140 and 251-260.

262. The population of claim 261, wherein at least about 30% of the cells in the population are the engineered cells.

263. The engineered population of claim 261 or 262, wherein the plurality of engineered primary cells are derived from cells pooled from more than one donor subject.

264. The engineered primary cell population of claim 263, wherein each of the more than one donor subjects is a healthy subject or is not suspected of having a disease or disorder when the donor sample is obtained from the donor subject.

265. The population of any one of claims 261-264, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise the modification.

266. The population of any one of claims 261-265, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise an exogenous polynucleotide encoding CD 47.

267. The population of any one of claims 261-266, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise an exogenous polynucleotide encoding CD 46.

268. The population of any one of claims 261-267, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise an exogenous polynucleotide encoding CD 59.

269. The population of any one of claims 261-268, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in said population comprise an exogenous polynucleotide encoding CD 55.

270. The population of any one of claims 261-269, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise one or more alterations that inactivate both alleles of a B2M gene.

271. The population of any one of claims 261-270, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise one or more alterations that inactivate both alleles of the CIITA gene.

272. The population of any one of claims 261-271, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise reduced CD142 expression relative to unchanged or unmodified wild-type cells.

273. The population of any one of claims 261-272, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% of the cells in the population comprise one or more alterations that inactivate both alleles of the CD142 gene.

274. A composition comprising the population of any one of claims 261-273 or the engineered cell of any one of claims 1-140 and 251-260.

276. The composition of claim 275, wherein the engineered β -cell comprises inactivation or disruption of both alleles of a CIITA gene.

278. The composition of claim 277, wherein the engineered hepatocyte comprises inactivation or disruption of both alleles of the CIITA gene.

279. The composition of any one of claims 275-278, wherein the transgene is a polycistronic vector, and wherein the transgene further comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD 59.

280. The composition of any one of claims 275-278, wherein said beta islet or liver cell further comprises a polycistronic vector, wherein said polycistronic vector comprises an exogenous polynucleotide encoding CD46 and an exogenous polynucleotide encoding CD59.

281. The composition of any one of claims 275-278, wherein said transgene is introduced at a target genomic locus by nuclease-mediated gene editing using homology directed repair.

282. The composition of any one of claims 275-281, wherein said inactivation or disruption is by a genome modification protein, optionally wherein said inactivation or disruption is by nuclease-mediated gene editing.

283. The composition of any one of claims 281-282, wherein the modification by the genomic modification protein is performed using ：Cas3、Cas4、Cas5、Cas8a、Cas8b、Cas8c、Cas9、Cas10、Cas12、Cas12a(Cpf1)、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12f(C2c10)、Cas12g、Cas12h、Cas12i、Cas12k(C2c5)、Cas13、Cas13a(C2c2)、Cas13b、Cas13c、Cas13d、C2c4、C2c8、C2c9、Cmr5、Cse1、Cse2、Csf1、Csm2、Csn2、Csx10、Csx11、Csy1、Csy2、Csy3、Mad7、 Zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, CRISPR-associated transposase, or any CRISPR-Cas combination that targets the target genomic locus, optionally wherein the modification is performed by nuclease-mediated gene editing using Cas9 or Cas 12.

284. The composition of any one of claims 275-283, wherein the composition is a pharmaceutical composition.

285. The composition of claim 284, comprising a pharmaceutically acceptable excipient.

286. The composition of any one of claims 284-285, wherein the composition is formulated in a serum-free cryopreservation medium comprising a cryoprotectant.

287. The composition of claim 286, wherein the cryoprotectant is DMSO and the cryopreservation medium is 5% to 10% DMSO (v/v).

288. The composition of claim 286 or 287, wherein the cryoprotectant is or is about 10% dmso (v/v).

289. The composition of any one of claims 275-288, which is sterile.

290. A container comprising the composition of any one of claims 275-289.

291. The container of claim 290, being a sterile bag.

292. The sterile bag of claim 291, wherein the bag is a cryopreservation compatible bag.

293. A method of treating a disease, disorder, or cell defect in a patient in need thereof, the method comprising administering to the patient an effective amount of the population of any one of claims 261-273 or the composition of any one of claims 274-289.

294. The method of claim 293, wherein the method further comprises administering to the patient an anticoagulant that reduces coagulation.

Wherein the engineered cell comprises the following modifications:

(ii) Increasing expression of one or more tolerogenic factors, and

(B) Administering to the patient an anticoagulant that reduces coagulation.

296. The method of claim 295, wherein the one or more tolerogenic factors are selected from the group consisting of: CD47, CD27, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDOl, CTLA4-Ig, cl-inhibitor, IL-10, IL-35, FASL, CCL21, MFGE8 and SERPINB9.

297. The method of claim 296, wherein the one or more tolerogenic factors is CD47.

298. A method of treating a disease, disorder, or cell defect in a patient in need thereof, the method comprising:

Wherein the engineered cell comprises the following modifications:

(B) Administering to the patient an anticoagulant that reduces coagulation.

299. The method of claim 298, wherein the population is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

300. The method of any one of claims 294-299, wherein the population and the anticoagulant are administered simultaneously or sequentially.

301. The method of any one of claims 294-300, wherein the anticoagulant is heparin.

302. The method of claim 301, wherein the heparin is plain heparin.

303. The method of claim 301, wherein the heparin is low molecular weight heparin.

304. The method of any one of claims 301-303, wherein the heparin is soluble heparin.

305. The method of any one of claims 301-303, wherein the heparin is immobilized on the surface of the cells prior to administration of the cells to the patient.

306. The method of any one of claims 294-305, wherein the anticoagulant is melagatran or LMW-DS.

307. The method of any of claims 294-305, wherein the anticoagulant is acetoacetcysteine (NAC).

308. The method of any one of claims 294-305, wherein the anticoagulant is alpha-1 antitrypsin (AAT) and/or activated protein C.

309. The method of any one of claims 293-308, wherein the disorder or disease is selected from the group consisting of: diabetes, cancer, angiogenesis disorders, ocular diseases, thyroid diseases, skin diseases and liver diseases.

310. The method of any one of claims 293-308, wherein the cellular defect is associated with diabetes, or the disease or disorder is diabetes, optionally wherein the diabetes is type I diabetes.

311. The method of claim 310, wherein the cell population is an islet cell population, comprising a beta islet cell population.

312. The method of claim 311, wherein the islet cells are selected from the group consisting of islet progenitor cells, immature islet cells, and mature islet cells.

313. The method of any one of claims 293-309, wherein the cell defect is associated with, or the disease or disorder is, a vascular disorder or disease.

314. The method of claim 313, wherein the population of cells is a population of endothelial cells.

315. The method of any one of claims 293-309, wherein the cellular defect is associated with autoimmune thyroiditis, or the disease or condition is autoimmune thyroiditis.

316. The method of claim 315, wherein the population of cells is a population of thyroid progenitor cells.

317. The method of any one of claims 293-309, wherein the cellular defect is associated with a liver disease or the disease is a liver disease.

318. The method of claim 317, wherein the liver disease comprises cirrhosis.

319. The method of claim 317 or 318, wherein the cell population is a population of hepatocytes or hepatic progenitors.

320. The method of any one of claims 293-309, wherein the cellular defect is associated with a corneal disease, or the disease is a corneal disease.

321. The method of claim 320, wherein the corneal disease is fox's dystrophy or congenital genetic endothelial dystrophy.

322. The method of claim 320 or 321, wherein the population of cells is a population of corneal endothelial progenitor cells or corneal endothelial cells.

323. The method of any one of claims 293-309, wherein the cell defect is associated with or the disease is a kidney disease.

324. The method of claim 323, wherein the population of cells is a kidney precursor cell or population of kidney cells.

325. The method of any one of claims 293-309, wherein the cell defect is associated with cancer or the disease is cancer.

326. The method of claim 325, wherein the cancer is selected from the group consisting of: b-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous lymphoblastic leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

327. The method of claim 325 or 326, wherein the cell population is a T cell population or an NK cell population or an NKT cell population.

328. The method of any one of claims 293-309, wherein the cell deficiency is associated with or the disease or disorder is a hematopoietic disease or disorder.

329. The method of claim 328, wherein the hematopoietic disease or disorder is myelodysplasia, aplastic anemia, fanconi anemia, paroxysmal sleep hemoglobinuria, sickle cell disease, congenital pure red cell aplastic anemia, schwann-Dai Mengde disease, ke Shiwen syndrome, chronic granulomatosis, adrenoleukodystrophy, leukocyte adhesion deficiency, hemophilia, thalassemia, β -thalassemia, leukemias such as Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), adult lymphoblastic leukemia, chronic Lymphoblastic Leukemia (CLL), B-cell chronic lymphoblastic leukemia (B-CLL), chronic Myeloblastic Leukemia (CML), juvenile Chronic Myelogenous Leukemia (CML), and juvenile myelomonocytic leukemia (JMML)), severe Combined Immunodeficiency Disease (SCID), X severe combined immunodeficiency, wegenet-aldrich syndrome (WAS), adenosine deaminase (adam), chronic lymphocytic leukemia (hodgkin's), hodgkin's disease, hodgkin's lymphoma, or non-aids.

330. The method of claim 328, wherein the cellular defect is associated with leukemia or myeloma, or wherein the disease or condition is leukemia or myeloma.

331. The method of any one of claims 293-309 and 328, wherein the cellular defect is associated with or is an autoimmune disease or disorder.

332. The method of claim 331, wherein the autoimmune disease or disorder is acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, anti-synthetase syndrome, atopic allergy, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, Autoimmune polyendocrine adenosis syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, balneosis, balneo-sclerosis, behcet's syndrome, primary Graves disease, bischtaff encephalitis, braun's syndrome, bullous pemphigoid, cancer, kalman's disease, celiac disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, chager-Schtriaus syndrome, cicatricial pemphigoid, crohn's syndrome, condensed collets, complement component 2 deficiency, craniomatis, CREST syndrome, crohn's disease, cushing's syndrome, cutaneous leukopenia vasculitis, degos ' disease, delkum's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, diffuse systemic sclerosis of the skin, dresler's syndrome, discoid lupus erythematosus, eczema, adnexitis-associated arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, acquired epidermolysis bullosa, erythema nodosum, primary mixed condensed globulinemia, ehrlichia syndrome, progressive fibrodysplasia ossificans, fibroalveolar inflammation, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, nephritis syndrome, gerbileve's disease, gill-barre syndrome (GBS), Bridge encephalitis, bridge thyroiditis, hemolytic anemia, allergic purpura, herpes gestation, hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, igA nephropathy, inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, kawasaki disease, lanbert-Eatonic syndrome, white blood cell disruption vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), graek's disease, lupus hepatitis, lupus erythematosus, ma Jide syndrome, meniere's disease, Microscopic polyangiitis, miller-Fisher syndrome, mixed connective tissue disease, scleroderma, acute acne-like lichen-like pityriasis, multiple sclerosis, myasthenia gravis, myositis, neuromyelitis optica, neuromuscular rigidity, ocular cicatricial pemphigoid, strabismus eye clonus syndrome, thyroiditis, recurrent rheumatism, paraneoplastic cerebellar degeneration, paroxysmal sleep hemoglobinuria (PNH), pa Luo Zeng syndrome, parsen-Tener syndrome, platycodon, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, Polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell dysgenesis, laplace Mu Sen encephalitis, raynaud's phenomenon, recurrent polychondritis, litty's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatoid fever, sarcoidosis, schmitt syndrome, schniter syndrome, scleritis, scleroderma, sjogren syndrome, spondyloarthropathies, still's disease, stiff person syndrome, subacute bacterial endocarditis, susak syndrome, sjogren's syndrome, sidner's disease, sympathogenic ophthalmitis, Arteritis, temporal arteritis, painful oculopathy syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue diseases, undifferentiated spondyloarthropathies, vasculitis, vitiligo or wegener's granulomatosis.

333. The method of any one of claims 328-332, wherein the population of cells is a population comprising Hematopoietic Stem Cells (HSCs) and/or derivatives thereof.

334. The method of any one of claims 293-309, wherein the cellular deficit is associated with parkinson's disease, huntington's disease, multiple sclerosis, a neurodegenerative disease or disorder, attention Deficit Hyperactivity Disorder (ADHD), tourette's Syndrome (TS), schizophrenia, psychosis, depression, neuropsychiatric stroke, or Amyotrophic Lateral Sclerosis (ALS), or wherein the disease or disorder is parkinson's disease, huntington's disease, multiple sclerosis, a neurodegenerative disease or disorder, attention Deficit Hyperactivity Disorder (ADHD), tourette's Syndrome (TS), schizophrenia, psychosis, depression, neuropsychiatric stroke, or Amyotrophic Lateral Sclerosis (ALS).

335. The method of claim 334, wherein the population of cells is a population comprising neural cells and/or glial cells.

336. The method of any one of claims 293-335, wherein the cells are expanded and cryopreserved prior to administration.

337. The method of any one of claims 293-336, wherein administering the population comprises intravenous injection, intramuscular injection, intravascular injection, or transplanting the population.

338. The method of claim 337, wherein the population is transplanted via renal capsule transplantation or intramuscular injection.

339. The method of any one of claims 293-338, wherein the population is derived from a donor subject, wherein the HLA type of the donor does not match the HLA type of the patient.

340. The method of any one of claims 293-339, wherein the population is derived from a donor, wherein the blood group of the donor does not match the blood group of the patient and the blood group of the donor is not type O.

341. The method of any one of claims 293-340, wherein the population is derived from a donor, wherein the blood group of the donor is rhesus factor (Rh) positive and the blood group of the patient is Rh negative.

342. The method of any one of claims 293-341, wherein the patient's serum comprises antibodies to Rh.

343. The method of any one of claims 293-342, wherein the population is a population of human cells and the patient is a human patient.

344. The method of any one of claims 293-343, wherein the population of cells comprises a functional ABO a allele and/or a functional ABO B allele.

345. The method of claim 344, wherein the population of cells presents ABO type a antigens and the patient's serum comprises anti-a antibodies.

346. The method of claim 344, wherein the population of cells presents ABO type B antigens and the patient's serum comprises anti-B antibodies.

347. The method of claim 344, wherein the population of cells presents ABO type a and type B antigens and the patient's serum comprises anti-a and/or anti-B antibodies.

348. The method of any one of claims 293-347, wherein a population of cells expresses an Rh factor and the patient's serum comprises anti-Rh antibodies.

349. The method of any one of claims 293-348, further comprising administering one or more immunosuppressants to the patient.

350. The method of any one of claims 293-348, wherein one or more immunosuppressants have been administered to the patient.

351. The method of claim 349 or claim 350, wherein the one or more immunosuppressants are small molecules or antibodies.

352. The method of any one of claims 349-351, wherein the one or more immunosuppressants are selected from the group consisting of: cyclosporine, azathioprine, mycophenolic acid, mycophenolate ester, corticosteroids, prednisone, methotrexate, gold salts, sulfasalazine, antimalarial, buconazole, leflunomide, mizoribine, 15-deoxyspergualin, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyte globulin, thymopentapeptides (thymosin-alpha) and immunosuppressive antibodies.

353. The method of any one of claims 349-352, wherein the one or more immunosuppressants comprise cyclosporin.

354. The method of any one of claims 349-352, wherein the one or more immunosuppressants comprise mycophenolate mofetil.

355. The method of any one of claims 349-352, wherein the one or more immunosuppressives comprise a corticosteroid.

356. The method of any one of claims 349-352, wherein the one or more immunosuppressants comprise cyclophosphamide.

357. The method of any one of claims 349-352, wherein the one or more immunosuppressants comprise rapamycin.

358. The method of any one of claims 349-352, wherein the one or more immunosuppressants comprise tacrolimus (FK-506).

359. The method of any one of claims 349-352, wherein the one or more immunosuppressants comprise anti-thymocyte globulin.

360. The method of any one of claims 349-352, wherein the one or more immunosuppressants are one or more immunomodulatory agents.

361. The method of claim 360, wherein the one or more immunomodulatory agents is a small molecule or an antibody.

362. The method of claim 351 or claim 361, wherein said antibody binds to one or more receptors or ligands selected from the group consisting of: p75、MHC、CD2、CD3、CD4、CD7、CD28、B7、CD40、CD45、IFN-γ、TNF-α、IL-4、IL-5、IL-6R、IL-6、IGF、IGFR1、IL-7、IL-8、IL-10、CD11a、CD58, of the IL-2 receptor and antibodies that bind to any of its ligands.

363. The method of any one of claims 349-362, wherein the one or more immunosuppressants are administered to the patient or have been administered to the patient prior to administration of the engineered cells.

364. The method of any one of claims 349-363, wherein the patient is administered or the one or more immunosuppressants have been administered to the patient at least 1, 2, 3,4, 5,6,7, 8, 9, 10, 11, 12, 13, or 14 days prior to administration of the engineered cells.

365. The method of any one of claims 349-363, wherein the one or more immunosuppressants are administered to or have been administered to the patient at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more prior to administration of the engineered cells.

366. The method of any one of claims 349-363, wherein the patient is administered or the one or more immunosuppressants have been administered to the patient at least 1, 2, 3,4, 5,6,7, 8, 9, 10, 11, 12, 13, or 14 days after administration of the engineered cells.

367. The method of any one of claims 349-363, wherein at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more are administered to the patient or have been administered to the patient after administration of the engineered cells.

368. The method of any one of claims 349-363, wherein the one or more immunosuppressants are administered to the patient or have been administered to the patient on the same day as the first administration of the engineered cells.

369. The method of any one of claims 349-363, wherein the one or more immunosuppressants are administered to the patient or have been administered to the patient after administration of the engineered cells.

370. The method of any one of claims 349-363, wherein the one or more immunosuppressants are administered to the patient or have been administered to the patient after the administration of the engineered cells is first and/or second administration.

371. The method of any one of claims 349-363, wherein the one or more immunosuppressants are administered to the patient or have been administered to the patient prior to the administration of the first and/or second administration of the engineered cells.

372. The method of any one of claims 349-363, wherein the patient is administered or has been administered the one or more immunosuppressants at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to the administration of the engineered cells for the first and/or second administration.

373. The method of any one of claims 349-363, wherein at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more are administered to the patient or the one or more immunosuppressants are administered to the patient prior to the administration of the first and/or second administration of the engineered cells.

374. The method of any one of claims 349-363, wherein the patient is administered or has been administered the one or more immunosuppressants at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the administration of the engineered cells first and/or second administration.

375. The method of any one of claims 349-363, wherein at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more are administered to the patient or have been administered to the patient after the administration of the engineered cells first and/or second.

376. The method of any one of claims 349-375, wherein the one or more immunosuppressants are administered at a lower dose to reduce immune rejection of modified immunogenic cells that do not comprise the engineered cells compared to the dose of the one or more immunosuppressants administered.

377. The method of any one of claims 293-376, wherein the engineered cell is capable of controlled killing of the engineered cell.

378. The method of any one of claims 293-377, wherein the engineered cell comprises a suicide gene or suicide switch.

379. The method of claim 378, wherein the suicide gene or the suicide switch induces controlled cell death in the presence of a drug or prodrug or after activation by a selective exogenous compound.

380. The method of claim 378 or claim 379, wherein the suicide gene or the suicide switch is an inducible protein capable of inducing apoptosis of the engineered cell.

381. The method of claim 380, wherein the inducible protein capable of inducing apoptosis in the engineered cell is a cysteine protease protein.

382. The method of claim 381, wherein the cysteine protease protein is cysteine protease 9.

383. The method of claim 380 or claim 381, wherein the suicide gene or suicide switch is selected from the group consisting of: cytosine deaminase (CyD), herpes virus thymidine kinase (HSV-Tk), inducible cysteine proteinase 9 (iCaspase 9) and rapamycin activated cysteine proteinase 9 (rapaCasp).

384. The method of any one of claims 378-383, wherein after administration of the one or more immunosuppressants to the patient, the suicide gene or the suicide switch is activated to induce controlled cell death.

385. The method of any one of claims 378-383, wherein the suicide gene or the suicide switch is activated to induce controlled cell death prior to administration of the one or more immunosuppressants to the patient.

386. The method of any one of claims 378-385, wherein after the engineered cells are administered to the patient, the suicide gene or the suicide switch is activated to induce controlled cell death.

387. The method of any one of claims 378-386, wherein the suicide gene or the suicide switch is activated to induce controlled cell death if it has cytotoxicity or other negative outcome to the patient.

388. The method of any one of claims 293-387, comprising administering an agent that allows depletion of engineered cells in the population of engineered cells.

389. The method of claim 388, wherein the agent that allows depletion of the engineered cell is an antibody that recognizes a protein expressed on the surface of the engineered cell.

390. The method of claim 389, wherein the antibody is selected from the group consisting of an antibody that recognizes CCR4, CD16, CD19, CD20, CD30, EGFR, GD2, HER1, HER2, MUC1, PSMA, and RQR 8.

391. The method of claim 389 or claim 390, wherein the antibody is selected from the group consisting of: mo Geli bead mab, AFM13, MOR208, octuzumab, rituximab, oxcarbatuzumab, rituximab-Rllb, tobrauximab, RO5083945 (GA 201), cetuximab, hul4.18k322a, hul4.18-IL2, hul3F 8, rituximab, c.60c3-Rllc, and biological analogs thereof.

392. The method of any one of claims 293-391, comprising administering an agent that recognizes the one or more tolerogenic factors on the surface of the engineered cell.

393. The method of claim 392, wherein the engineered cells are engineered to express the one or more tolerogenic factors.

394. The method of any one of claims 293-393, wherein the one or more tolerogenic factors comprises one or more tolerogenic factors selected from the group consisting of: A20/TNFAIP3, C1-inhibitor, CCL21, CCL22, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD200, CR1, CTLA4-Ig, DUX4, fasL, H2-M3, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, IL-10, IL15-RF, IL-35, MANF, mfge8, PD-1, PD-L1, serpinb9, and any combination thereof.

395. The method of claim 393 or 394, wherein the one or more tolerogenic factors comprises CD47.

396. The method of any one of claims 293-395, wherein the one or more tolerogenic factors comprise HLA-E.

397. The method of any one of claims 293-396, wherein the one or more tolerogenic factors comprise CD24.

398. The method of any one of claims 293-397, wherein the one or more tolerogenic factors comprises PDL1.

399. The method of any one of claims 293-398, wherein the one or more tolerogenic factors comprise CD55.

400. The method of any one of claims 293-399, wherein the one or more tolerogenic factors comprises CR1.

401. The method of any one of claims 293-400, wherein the one or more tolerogenic factors comprise MANF.

402. The method of any one of claims 293-401, wherein the one or more tolerogenic factors comprise a20/TNFAIP3.

403. The method of any one of claims 293-402, wherein the one or more tolerogenic factors comprise HLA-E and CD47.

404. The method of any one of claims 293-403, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of CD24, CD47, and PDL1, optionally wherein the one or more tolerogenic factors comprises CD24, CD47, and PDL1.

405. The method of any one of claims 293-404, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD47, and PDL1, optionally wherein the one or more tolerogenic factors comprises HLA-E, CD, CD47, and PDL1.

406. The method of any one of claims 293-405, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of CD46, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprises CD46, CD55, CD59, and CR1.

407. The method of any one of claims 293-406, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD46, CD55, CD59, and CR1.

408. The method of any one of claims 293-407, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, CD24, CD47, PDL1, CD46, CD55, CD59, and CR1, optionally wherein the one or more tolerogenic factors comprise HLA-E, CD, CD47, PDL1, CD46, CD55, CD59, and CR1.

409. The method of any one of claims 293-408, wherein the one or more tolerogenic factors comprise HLA-E and PDL1.

410. The method of any one of claims 293-409, wherein the one or more tolerogenic factors comprises two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1 and a20/TNFAIP, optionally wherein the one or more tolerogenic factors comprises HLA-E, PDL1 and a20/TNFAIP.

411. The method of any one of claims 293-410, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1 and MANF, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1 and MANF.

412. The method of any one of claims 293-411, wherein the one or more tolerogenic factors comprise two or more tolerogenic factors selected from the group consisting of HLA-E, PDL1, a20/TNFAIP and MANF, optionally wherein the one or more tolerogenic factors comprise HLA-E, PDL1, a20/TNFAIP and MANF.

413. The method of claim 393 or claim 394, wherein the one or more tolerogenic factors is CD47.

414. The method of any one of claims 293-413, further comprising administering one or more additional therapeutic agents to the patient.

415. The method of any one of claims 293-414, wherein one or more additional therapeutic agents have been administered to the patient.

416. The method of any one of claims 293-415, further comprising monitoring the efficacy of treatment of the method.

417. The method of any one of claims 293-416, further comprising monitoring the prophylactic efficacy of the method.

418. The method of any one of claims 293-417, wherein the method is repeated until a desired inhibition of one or more disease symptoms occurs.

419. The engineered cell of any one of claims 1-140 and 251-260, wherein the engineered cell comprises an exogenous polynucleotide encoding a suicide gene or suicide switch.

420. The engineered cell of claim 419, wherein the suicide gene or suicide switch is selected from the group consisting of: cytosine deaminase (CyD), herpes virus thymidine kinase (HSV-Tk), inducible cysteine proteinase 9 (iCaspase 9) and rapamycin activated cysteine proteinase 9 (rapaCasp).

421. The engineered cell of claim 419 or claim 420, wherein the suicide gene or suicide switch and a gene associated with the suicide gene or the safety switch are expressed by a bicistronic cassette integrated into the genome of the engineered cell.

422. The engineered cell of any one of claims 419-421, wherein the suicide gene or suicide switch and the one or more tolerogenic factors are expressed by a bicistronic cassette integrated into the genome of the engineered cell.

423. The engineered cell of claim 419 or claim 420, wherein the bicistronic cassette is integrated by non-targeted insertion into the genome of the engineered cell, optionally by introducing the exogenous polynucleotide into the cell using a lentiviral vector.

424. The engineered cell of claim 419 or 420, wherein the bicistronic cassette is integrated by targeted insertion into a target genomic locus of the cell, optionally wherein the targeted insertion is by nuclease-mediated gene editing using homology directed repair.

425. The engineered cell of any one of claims 419-424, wherein the one or more tolerogenic factors is CD47.

426. The method of any one of claims 141-250, wherein the engineered cell comprises an exogenous polynucleotide encoding a suicide gene or suicide switch.

427. The method of claim 426 wherein the suicide gene is selected from the group consisting of: cytosine deaminase (CyD), herpes virus thymidine kinase (HSV-Tk), inducible cysteine proteinase 9 (iCaspase 9) and rapamycin activated cysteine proteinase 9 (rapaCasp).

428. The method of claim 426 or claim 427 wherein the suicide gene or suicide switch and a gene associated with the suicide gene or the safety switch are expressed by a bicistronic cassette integrated into the genome of the engineered cell.

429. The method of any one of claim 426 or claim 427 wherein the suicide gene or suicide switch and the one or more tolerogenic factors are expressed by a bicistronic cassette integrated into the genome of the engineered cell.

430. The method of claim 428 or claim 429, wherein the bicistronic cassette is integrated by non-targeted insertion into the genome of the engineered cell.

431. The method of claim 428 or claim 429, wherein the bicistronic cassette is integrated by targeted insertion into a target genomic locus of the engineered cell.

432. The method of any one of claims 426-431 wherein the one or more tolerogenic factors is CD47.

433. The composition of any one of claims 274-289, wherein an engineered cell in the population of engineered cells comprises an exogenous polynucleotide encoding a suicide gene or suicide switch.

434. The composition of claim 433, wherein said suicide gene or suicide switch is selected from the group consisting of: cytosine deaminase (CyD), herpes virus thymidine kinase (HSV-Tk), inducible cysteine proteinase 9 (iCaspase 9) and rapamycin activated cysteine proteinase 9 (rapaCasp).

435. The composition of claim 433 or claim 434, wherein said suicide gene and a gene associated with said suicide gene or said safety switch are expressed by a bicistronic cassette integrated into the genome of an engineered cell in said engineered cell population.

436. The composition of any one of claims 433-435, wherein the suicide gene or suicide switch and the exogenous CD47 are expressed by a bicistronic cassette integrated into the genome of the engineered cell.

437. The composition of claim 435 or claim 436, wherein the bicistronic cassette is integrated by non-targeted insertion into the genome, optionally by introducing the exogenous polynucleotide into an engineered cell in the engineered cell population using a lentiviral vector.

438. The composition of claim 435 or claim 436, wherein the bicistronic cassette is integrated by targeted insertion into a target genomic locus of an engineered cell in the population of engineered cells, optionally wherein the targeted insertion is by nuclease-mediated gene editing using homology directed repair.

439. A combination comprising the engineered population of cells of any one of claims 261-273 or a population of cells comprising a plurality of engineered cells of any one of claims 419-425, and an anticoagulant or cell coating that reduces blood coagulation.

440. A combination, the combination comprising:

(a) A cell population comprising a plurality of engineered cells,

Wherein the engineered cell comprises the following modifications:

(ii) Increase expression of CD47, and

(Iii) Lowering one or more MHC class I molecules and/or one or more MHC

The expression of the class II molecule is carried out,

(B) Anticoagulants.

441. The combination of claim 439 or claim 440, wherein said anticoagulant is selected from the group consisting of heparin, an activator of antithrombin, an inhibitor of factor II (fhi), an inhibitor of factor VII (fhi), and an inhibitor of factor X (fX).

442. The combination of claim 441, wherein the anticoagulant is heparin.

443. The combination of claim 442, wherein the heparin is plain heparin.

444. The combination of claim 443, wherein the heparin is low molecular weight heparin.

445. The combination of any one of claims 441-444, wherein the heparin is soluble heparin.

446. The combination of claim 441, wherein the anticoagulant is melagatran or LMW-DS.

447. The combination of claim 441, wherein the anticoagulant is acetoacetcysteine (NAC).

448. The combination of claim 441, wherein the anticoagulant is alpha-1 antitrypsin (AAT) and/or activated protein C.

449. The combination of claim 441, wherein the anticoagulant is an antibody directed against CD 142.

450. A kit comprising the combination of any one of claims 441-448.