CN118043344A

CN118043344A - Modification of expression of Y chromosome-linked antigen in hypoimmunogenic cells

Info

Publication number: CN118043344A
Application number: CN202280061779.0A
Authority: CN
Inventors: S·施雷普费尔; E·雷拜尔; D·高曼
Original assignee: Sana Biotechnology Co ltd
Current assignee: Sana Biotechnology Co ltd
Priority date: 2021-07-14
Filing date: 2022-07-12
Publication date: 2024-05-14

Abstract

Disclosed herein are engineered cells and/or low immunogenic cells, including engineered cells and/or low immunogenic stem cells, engineered cells and/or low immunogenic cells differentiated therefrom, and engineered cells and/or low immunogenic CAR-T cells (primary or differentiated from engineered and/or low immunogenic stem cells), and related methods of their use and production, comprising reduced expression of one or more Y chromosome genes and reduced expression of MHC I and/or MHC II human leukocyte antigen molecules, and overexpression of CD 47. Provided herein are cells that further exhibit reduced expression of T cell receptors.

Description

Modification of expression of Y chromosome-linked antigen in hypoimmunogenic cells

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application number 63/221,887 filed on day 7, month 14 of 2021 and U.S. provisional application number 63/255,914 filed on day 10, month 14 of 2021, the disclosure of each provisional application being incorporated herein in its entirety.

Background

Ready CAR-T cells and other therapeutic cells can provide advantages over autologous cell-based strategies, including ease of manufacture, quality control, and avoidance of malignant contamination and T cell dysfunction. However, a strong host versus graft immune response against tissue-incompatible T cells prevents expansion and persistence of allogeneic CAR-T cells and reduces the efficacy of this pathway.

There is a great deal of evidence in both animal models and human patients that low immunogenicity cell transplantation is a scientifically viable and clinically promising approach to the treatment of a variety of disorders, conditions and diseases.

There remains a need for new approaches, compositions and methods for generating cell-based therapies that avoid detection by the recipient immune system.

Disclosure of Invention

In some embodiments, provided herein is an engineered cell comprising reduced expression of one or more Y chromosome genes and Major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigen molecules relative to an unmodified wild-type or control cell and a first exogenous polynucleotide encoding CD47, wherein the engineered cell is propagated by a primary T cell or progeny thereof, or derived from an Induced Pluripotent Stem Cell (iPSC) or progeny thereof.

In some embodiments, provided herein is a low-immunogenicity T cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules relative to an unmodified wild-type or control cell and a first exogenous polynucleotide encoding CD47, wherein the low-immunogenicity T cell is propagated by a primary T cell or progeny thereof, or derived from an iPSC or progeny thereof.

In some embodiments, provided herein is a non-activated T cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified or wild-type or control cell, wherein the non-activated T cell is propagated by a primary T cell or progeny thereof, or derived from an iPSC or progeny thereof.

In some embodiments, provided herein is an islet cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules relative to an unmodified wild-type or control cell, and a first exogenous polynucleotide encoding CD47, wherein the islet cell is derived from an iPSC or a progeny thereof.

In some embodiments, provided herein is a cardiomyocyte comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified wild-type or control cell, wherein the cardiomyocyte is derived from iPSC or a progeny thereof.

In some embodiments, provided herein is a glial progenitor cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified or wild-type or control cell, wherein the cardiomyocyte is derived from iPSC or a progeny thereof.

In some embodiments, provided herein is an NK cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified wild-type or control cell, wherein the cardiomyocyte is derived from iPSC or a progeny thereof.

In some embodiments, the Y chromosome gene is a Y chromosome-linked antigen or a minor histocompatibility antigen associated with the Y chromosome.

In some embodiments, the one or more Y chromosome-linked antigens are Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

In some embodiments, the cell has reduced expression of Y-linked tropocadherin 11.

In some embodiments, the cells have reduced expression of Y-linked fibronectin 4.

In some embodiments, the cells have reduced expression of Y-linked tropocadherin 11 and reduced expression of Y-linked fibronectin 4.

In some embodiments, the cells are genetically engineered to have reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

In some embodiments, the cell does not express Y-linked tropocadherin 11.

In some embodiments, the cell does not express Y-linked fibronectin 4.

In some embodiments, the cell does not express Y-linked tropocadherin 11 and does not express Y-linked fibronectin 4.

In some embodiments, the cells are genetically engineered to not express Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

In some embodiments, the reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is caused by a knockout of PCDH11Y and/or NLGN4Y gene, respectively.

In some embodiments, the cells are derived from human cells or animal cells.

In some embodiments, the human or animal cells are from a donor subject that does not have a Y chromosome.

In some embodiments, the human or animal cell is from a donor subject having a Y chromosome, and the cell is genetically engineered to have reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

In some embodiments, the cells are genetically engineered to not express Y-linked tropocadherin 11.

In some embodiments, the cells are genetically engineered to not express Y-linked fibronectin 4.

In some embodiments, the cells are genetically engineered to not express Y-linked tropocadherin 11 and not express Y-linked fibronectin 4.

In some embodiments, the cells are propagated from or derived from a cell pool isolated from one or more donor subjects other than the patient, and the one or more donor subjects optionally include one or more subjects having a Y chromosome; one or more subjects without a Y chromosome; or a mixture of subjects with a Y chromosome and subjects without a Y chromosome.

In some embodiments, genetically engineered cells are edited using CRISPR/Cas genes to have reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

In some embodiments, CRISPR/Cas gene editing is performed using one or more guide RNAs comprising any of the sequences of tables 2-5.

In some embodiments, CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, cas12a, and Cas12 b.

In some embodiments, CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of: (a) Optionally selected from the group consisting of Cas3, cas8a, cas5, cas8b, cas8c, cas10d, cse1, cse2, csy1, csy2, csy3, and GSU 0054; (b) optionally selected from the group consisting of Cas9, csn2, and Cas 4; (c) Optionally selected from the group consisting of Cas10, csm2, cmr5, cas10, csx11, and Csx 10; (d) optionally Csf1; (e) Optionally selected from the group consisting of Cas12a, cas12b, cas12C, C2C4, C2C8, C2C5, C2C10, C2C9, casX (Cas 12 e) and CasY (Cas 12 d); and (f) optionally selected from the group consisting of Cas13, cas13a, C2, cas13b, cas13C, and Cas13 d.

In some embodiments, CRISPR/Cas gene editing is performed ex vivo from a donor subject.

In some embodiments, CRISPR/Cas gene editing is performed using lentiviral vectors.

In some embodiments, the cells comprise reduced expression of beta-2-microglobulin (B2M) and/or MHC class II transactivator (CIITA) relative to an unmodified wild-type or control cell.

In some embodiments, the cell does not express B2M and/or CIITA.

In some embodiments, the cell comprises reduced expression of RHD.

In some embodiments, the cell does not express RHD.

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

In some embodiments, the differentiated cell is selected from the group consisting of: t cells, NK cells, endothelial cells, islet cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, hepatocytes, glial progenitor cells, dopaminergic neurons, retinal pigment epithelial cells, and thyroid cells.

In some embodiments, the cell is a primary immune cell or a progeny thereof.

In some embodiments, the primary immune cell or progeny thereof is a T cell or NK cell.

In some embodiments, the cell comprises reduced expression of TCR- α and/or TCR- β.

In some embodiments, the cell does not express TCR- α and/or TCR- β.

In some embodiments, the cell further comprises a second exogenous polynucleotide encoding one or more Chimeric Antigen Receptors (CARs), and the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain that are specific for CD19, CD20, CD22, or BCMA.

In some embodiments, one or more CARs comprise a CD8 a hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

In some embodiments, one or more CARs comprise a CD8 a hinge domain having the amino acid sequence of SEQ ID No. 9.

In some embodiments, one or more CARs comprise a CD28 hinge domain having the amino acid sequence of SEQ ID No. 10 or 113.

In some embodiments, one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID No. 11 or 12.

In some embodiments, one or more CARs comprise a CD 8a transmembrane domain or a CD28 transmembrane domain.

In some embodiments, one or more CARs comprise a CD 8a transmembrane domain having the amino acid sequence of SEQ ID No. 14.

In some embodiments, one or more CARs comprise a CD28 transmembrane domain having the amino acid sequence of SEQ ID No. 15 or 114.

In some embodiments, one or more CARs comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3 zeta signaling domain.

In some embodiments, one or more CARs comprise a 4-1BB co-stimulatory domain having the amino acid sequence of SEQ ID NO. 16.

In some embodiments, one or more CARs comprise a CD28 co-stimulatory domain having the amino acid sequence of SEQ ID No. 17.

In some embodiments, one or more CARs comprise a CD3 zeta signaling domain having the amino acid sequence of SEQ ID NO:18 or 115.

In some embodiments, one or more CARs comprise an extracellular ligand binding domain comprising an scFv sequence of any one of SEQ ID NOs 19, 37, 45, 54, 63, 72, 81, or 118, or a CAR having an scFv sequence comprising a heavy and a light chain sequence of any one of SEQ ID NOs 20, 25, 38, 42, 46, 50, 64, 68, 73, 77, 119, or 123.

In some embodiments, one or more CARs have the sequence of any of SEQ ID NOs 32, 34, 36, 117 or 128.

In some embodiments, one or more CARs comprise the amino acid sequence set forth in SEQ ID No. 117 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to the amino acid sequence set forth in SEQ ID No. 117, the one or more CARs have the following components: CD8 a signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD8 a hinge domain, CD8 a transmembrane domain, 4-1BB co-stimulatory domain and CD3 zeta signaling domain.

In some embodiments, one or more CARs comprise the amino acid sequence set forth in SEQ ID No. 45 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to the amino acid sequence set forth in SEQ ID No. 45.

In some embodiments, one or more of the first and/or second exogenous polynucleotides is inserted into the first and/or second specific locus of at least one allele of the cell.

In some embodiments, the first and/or second specific loci are selected from the group consisting of: safe harbor or target loci, RHD loci, B2M loci, CIITA loci, TRAC loci, and TRB loci.

In some embodiments, the safe harbor or target locus is selected from the group consisting of: CCR5 locus, CXCR4 locus, PPP1R12C locus, ALB locus, SHS231 locus, CLYBL locus, rosa locus, F3 (CD 142) locus, MICA locus, MICB locus, LRP1 (CD 91) locus, HMGB1 locus, ABO locus, FUT1 locus and KDM5D locus.

In some embodiments, the first and/or second exogenous polynucleotide is introduced into the cell using a gene therapy vector or a transposase system selected from the group consisting of a transposase, a PiggyBac transposon, a sleeping beauty (SB 11) transposon, a Mos1 transposon, and a Tol2 transposon.

In some embodiments, the gene therapy vector is a retrovirus or a fusion (fusosome).

In some embodiments, the retrovirus is a lentiviral vector.

In some embodiments, the first and/or second exogenous polynucleotide is introduced into the cell using CRISPR/Cas gene editing.

In some embodiments, the cells or their progeny evade NK cell-mediated cytotoxicity after administration to the patient.

In some embodiments, the cells or their progeny are protected from cell lysis of mature NK cells after administration to a patient.

In some embodiments, the cells or their progeny evade phagocytosis by macrophages after administration to a patient.

In some embodiments, the cells or their progeny do not induce an immune response against the cells after administration to a patient.

In some embodiments, the cells or their progeny do not induce an antibody-based immune response against the cells after administration to a patient.

In some embodiments, the wild-type cell or the control cell is the starting material.

In some embodiments, provided herein is a pharmaceutical composition comprising an engineered cell, a low immunogenicity T cell, a non-activated T cell, an islet cell, a cardiomyocyte, a glial progenitor cell, or a population of NK cells described herein and a pharmaceutically acceptable additive, carrier, diluent, or excipient.

In some embodiments, the composition comprises one or more cell populations selected from the group consisting of a low immunogenicity T cell population, a non-activated T cell population, a low immunogenicity CD19 CAR T cell population, and a low immunogenicity CD22CAR T cell population, and a pharmaceutically acceptable additive, carrier, diluent, or excipient.

In some embodiments, provided herein is a method of treating a patient having a disease or condition that would benefit from cell-based therapy, the method comprising administering to the patient an engineered cell, a low-immunogenicity T cell, a non-activated T cell, an islet cell, a cardiomyocyte, a glial progenitor cell, or a population of NK cells described herein.

In some embodiments, the patient does not have a Y chromosome.

In some embodiments, the patient is insensitive to the Y chromosome gene.

In some embodiments, the patient is sensitive to a Y chromosome gene.

In some embodiments, the patient has previously received cell therapy derived from a donor subject having a Y chromosome or cell therapy that otherwise expresses one or more Y chromosome genes.

In some embodiments, the patient is a female patient who previously had a male child.

In some embodiments, provided herein is a method of treating cancer in a patient in need thereof, the method comprising administering to the patient a primary immune cell population disclosed herein.

In some embodiments, the primary immune cells are selected from the group consisting of T cells and NK cells.

In some embodiments, the patient does not have a Y chromosome.

In some embodiments, the patient is insensitive to the Y chromosome gene.

In some embodiments, the patient is sensitive to a Y chromosome gene.

In some embodiments, provided herein is a method of determining an appropriate cell-based therapy to be administered to a patient suffering from a disease or condition that would benefit from cell-based therapy, the method comprising: (a) Determining whether a biological sample from a patient comprises antibodies to one or more Y chromosome genes by: (i) obtaining or having obtained a biological sample from a patient; (ii) An assay is performed or has been performed to determine whether an antibody to Y-linked tropocadherin 11 is present in the biological sample; and (iii) performing or having performed an assay to determine whether an antibody to Y-linked fibronectin 4 is present in the biological sample; and (b) administering an engineered cell, a low immunogenicity T cell, a non-activated T cell, an islet cell, a cardiomyocyte, a glial progenitor cell, or a NK cell population as described herein, wherein: (i) If antibodies to Y-linked tropocadherin 11 are present in the biological sample, the population of cells comprises reduced expression of Y-linked tropocadherin 11; (ii) If antibodies to Y-linked fibronectin 4 are present in the biological sample, the population of cells comprises reduced expression of Y-linked fibronectin 4; (iii) If antibodies to Y-linked tropocadherin 11 and antibodies to Y-linked fibronectin 4 are present in the biological sample, the cell population comprises reduced expression of Y-linked tropocadherin 11 and Y-linked fibronectin 4; (iv) If there is neither an antibody to Y-linked tropocadherin 11 nor an antibody to Y-linked fibronectin 4 in the biological sample, the cell population does not comprise reduced expression of Y-linked tropocadherin 11 or Y-linked fibronectin 4.

In some embodiments, provided herein is a method of identifying a patient having a disease or condition that would benefit from a cell-based therapy comprising reduced expression of one or more Y chromosome genes, the method comprising: (a) Determining whether a biological sample from a patient comprises antibodies to one or more Y chromosome genes by: (i) obtaining or having obtained a biological sample from a patient; (ii) An assay is performed or has been performed to determine whether an antibody to Y-linked tropocadherin 11 is present in the biological sample; and (iii) performing or having performed an assay to determine whether an antibody to Y-linked fibronectin 4 is present in the biological sample; and (b) administering to the patient an engineered cell, a low immunogenicity T cell, a non-activated T cell, an islet cell, a cardiomyocyte, a glial progenitor cell, or a NK cell population as described herein, wherein: (i) If antibodies to Y-linked tropocadherin 11 are present in the biological sample, the population of cells comprises reduced expression of Y-linked tropocadherin 11; (ii) If antibodies to Y-linked fibronectin 4 are present in the biological sample, the population of cells comprises reduced expression of Y-linked fibronectin 4; (iii) If antibodies to Y-linked tropocadherin 11 and antibodies to Y-linked fibronectin 4 are present in the biological sample, the cell population comprises reduced expression of Y-linked tropocadherin 11 and Y-linked fibronectin 4; (iv) If there is neither an antibody to Y-linked tropocadherin 11 nor an antibody to Y-linked fibronectin 4 in the biological sample, the cell population does not comprise reduced expression of Y-linked tropocadherin 11 or Y-linked fibronectin 4.

In some embodiments, provided herein is a method for identifying a patient having a disease or condition that would benefit from a cell-based therapy comprising reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4, the method comprising: (a) Determining whether a biological sample from a patient comprises antibodies to Y-linked tropocadherin 11 and/or antibodies to Y-linked fibronectin 4 by: (i) obtaining or having obtained a biological sample from a patient; (ii) An assay is performed or has been performed to determine whether an antibody to Y-linked tropocadherin 11 is present in the biological sample; and (iii) performing or having performed an assay to determine whether an antibody to Y-linked fibronectin 4 is present in the biological sample; and (b) administering to the patient an engineered cell, a low immunogenicity T cell, a non-activated T cell, an islet cell, a cardiomyocyte, a glial progenitor cell, or a NK cell population as described herein, wherein: (i) If antibodies to Y-linked tropocadherin 11 are present in the biological sample, the population of cells comprises reduced expression of Y-linked tropocadherin 11; (ii) If antibodies to Y-linked fibronectin 4 are present in the biological sample, the population of cells comprises reduced expression of Y-linked fibronectin 4; (iii) If antibodies to Y-linked tropocadherin 11 and antibodies to Y-linked fibronectin 4 are present in the biological sample, the cell population comprises reduced expression of Y-linked tropocadherin 11 and Y-linked fibronectin 4; (iv) If there is neither an antibody to Y-linked tropocadherin 11 nor an antibody to Y-linked fibronectin 4 in the biological sample, the cell population does not comprise reduced expression of Y-linked tropocadherin 11 or Y-linked fibronectin 4.

In some embodiments, provided herein is a method of determining whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to NK-mediated cytotoxicity after administration to a patient, the method comprising: (a) Determining whether a biological sample from a patient comprises antibodies to Y-linked tropocadherin 11 and/or antibodies to Y-linked fibronectin 4 by: (i) obtaining or having obtained a biological sample from a patient; (ii) An assay is performed or has been performed to determine whether an antibody to Y-linked tropocadherin 11 is present in the biological sample; and (iii) performing or having performed an assay to determine whether an antibody to Y-linked fibronectin 4 is present in the biological sample; and (b) administering to the patient an engineered cell, a low immunogenicity T cell, a non-activated T cell, an islet cell, a cardiomyocyte, a glial progenitor cell, or a NK cell population as described herein, wherein: (i) If antibodies to Y-linked tropocadherin 11 are present in the biological sample, the population of cells comprises reduced expression of Y-linked tropocadherin 11; (ii) If antibodies to Y-linked fibronectin 4 are present in the biological sample, the population of cells comprises reduced expression of Y-linked fibronectin 4; (iii) If antibodies to Y-linked tropocadherin 11 and antibodies to Y-linked fibronectin 4 are present in the biological sample, the cell population comprises reduced expression of Y-linked tropocadherin 11 and Y-linked fibronectin 4; (iv) If there is neither an antibody to Y-linked tropocadherin 11 nor an antibody to Y-linked fibronectin 4 in the biological sample, the cell population does not comprise reduced expression of Y-linked tropocadherin 11 or Y-linked fibronectin 4.

In some embodiments, provided herein is a method of determining whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to lysis by mature NK cells after administration to a patient, the method comprising: (a) Determining whether a biological sample from a patient comprises antibodies to Y-linked tropocadherin 11 and/or antibodies to Y-linked fibronectin 4 by: (i) obtaining or having obtained a biological sample from a patient; (ii) An assay is performed or has been performed to determine whether an antibody to Y-linked tropocadherin 11 is present in the biological sample; and (iii) performing or having performed an assay to determine whether an antibody to Y-linked fibronectin 4 is present in the biological sample; and (b) administering to the patient an engineered cell, a low immunogenicity T cell, a non-activated T cell, an islet cell, a cardiomyocyte, a glial progenitor cell, or a NK cell population as described herein, wherein: (i) If antibodies to Y-linked tropocadherin 11 are present in the biological sample, the population of cells comprises reduced expression of Y-linked tropocadherin 11; (ii) If antibodies to Y-linked fibronectin 4 are present in the biological sample, the population of cells comprises reduced expression of Y-linked fibronectin 4; (iii) If antibodies to Y-linked tropocadherin 11 and antibodies to Y-linked fibronectin 4 are present in the biological sample, the cell population comprises reduced expression of Y-linked tropocadherin 11 and Y-linked fibronectin 4; (iv) If there is neither an antibody to Y-linked tropocadherin 11 nor an antibody to Y-linked fibronectin 4 in the biological sample, the cell population does not comprise reduced expression of Y-linked tropocadherin 11 or Y-linked fibronectin 4.

In some embodiments, provided herein is a method of determining whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to phagocytosis by macrophages after administration to a patient, the method comprising: (a) Determining whether a biological sample from a patient comprises antibodies to Y-linked tropocadherin 11 and/or antibodies to Y-linked fibronectin 4 by: (i) obtaining or having obtained a biological sample from a patient; (ii) An assay is performed or has been performed to determine whether an antibody to Y-linked tropocadherin 11 is present in the biological sample; and (iii) performing or having performed an assay to determine whether an antibody to Y-linked fibronectin 4 is present in the biological sample; and (b) administering to the patient an engineered cell, a low immunogenicity T cell, a non-activated T cell, an islet cell, a cardiomyocyte, a glial progenitor cell, or a NK cell population as described herein, wherein: (i) If antibodies to Y-linked tropocadherin 11 are present in the biological sample, the population of cells comprises reduced expression of Y-linked tropocadherin 11; (ii) If antibodies to Y-linked fibronectin 4 are present in the biological sample, the population of cells comprises reduced expression of Y-linked fibronectin 4; (iii) If antibodies to Y-linked tropocadherin 11 and antibodies to Y-linked fibronectin 4 are present in the biological sample, the cell population comprises reduced expression of Y-linked tropocadherin 11 and Y-linked fibronectin 4; (iv) If there is neither an antibody to Y-linked tropocadherin 11 nor an antibody to Y-linked fibronectin 4 in the biological sample, the cell population does not comprise reduced expression of Y-linked tropocadherin 11 or Y-linked fibronectin 4.

In some embodiments, provided herein is a method of determining whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to an induced immune response after administration to a patient, the method comprising: (a) Determining whether a biological sample from a patient comprises antibodies to Y-linked tropocadherin 11 and/or antibodies to Y-linked fibronectin 4 by: (i) obtaining or having obtained a biological sample from a patient; (ii) An assay is performed or has been performed to determine whether an antibody to Y-linked tropocadherin 11 is present in the biological sample; and (iii) performing or having performed an assay to determine whether an antibody to Y-linked fibronectin 4 is present in the biological sample; and (b) administering to the patient an engineered cell, a low immunogenicity T cell, a non-activated T cell, an islet cell, a cardiomyocyte, a glial progenitor cell, or a NK cell population as described herein, wherein: (i) If antibodies to Y-linked tropocadherin 11 are present in the biological sample, the population of cells comprises reduced expression of Y-linked tropocadherin 11; (ii) If antibodies to Y-linked fibronectin 4 are present in the biological sample, the population of cells comprises reduced expression of Y-linked fibronectin 4; (iii) If antibodies to Y-linked tropocadherin 11 and antibodies to Y-linked fibronectin 4 are present in the biological sample, the cell population comprises reduced expression of Y-linked tropocadherin 11 and Y-linked fibronectin 4; (iv) If there is neither an antibody to Y-linked tropocadherin 11 nor an antibody to Y-linked fibronectin 4 in the biological sample, the cell population does not comprise reduced expression of Y-linked tropocadherin 11 or Y-linked fibronectin 4.

In some embodiments, provided herein is a method of determining whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to an induced antibody-based immune response after administration to a patient, the method comprising: (a) Determining whether a biological sample from a patient comprises antibodies to Y-linked tropocadherin 11 and/or antibodies to Y-linked fibronectin 4 by: (i) obtaining or having obtained a biological sample from a patient; (ii) An assay is performed or has been performed to determine whether an antibody to Y-linked tropocadherin 11 is present in the biological sample; and (iii) performing or having performed an assay to determine whether an antibody to Y-linked fibronectin 4 is present in the biological sample; and (b) administering to the patient an engineered cell, a low immunogenicity T cell, a non-activated T cell, an islet cell, a cardiomyocyte, a glial progenitor cell, or a NK cell population as described herein, wherein: (i) If antibodies to Y-linked tropocadherin 11 are present in the biological sample, the population of cells comprises reduced expression of Y-linked tropocadherin 11; (ii) If antibodies to Y-linked fibronectin 4 are present in the biological sample, the population of cells comprises reduced expression of Y-linked fibronectin 4; (iii) If antibodies to Y-linked tropocadherin 11 and antibodies to Y-linked fibronectin 4 are present in the biological sample, the cell population comprises reduced expression of Y-linked tropocadherin 11 and Y-linked fibronectin 4; (iv) If there is neither an antibody to Y-linked tropocadherin 11 nor an antibody to Y-linked fibronectin 4 in the biological sample, the cell population does not comprise reduced expression of Y-linked tropocadherin 11 or Y-linked fibronectin 4.

In some embodiments, provided herein is a method of treating a patient having a disease or condition that would benefit from cell-based therapy, the method comprising: (a) Determining whether a biological sample from a patient comprises antibodies to one or more Y chromosome genes by: (i) obtaining or having obtained a biological sample from a patient; (ii) An assay is performed or has been performed to determine whether an antibody to Y-linked tropocadherin 11 is present in the biological sample; and (iii) performing or having performed an assay to determine whether an antibody to Y-linked fibronectin 4 is present in the biological sample; and (b) administering to the patient an engineered cell, a low immunogenicity T cell, a non-activated T cell, an islet cell, a cardiomyocyte, a glial progenitor cell, or a NK cell population as described herein, wherein: (i) If antibodies to Y-linked tropocadherin 11 are present in the biological sample, the population of cells comprises reduced expression of Y-linked tropocadherin 11; (ii) If antibodies to Y-linked fibronectin 4 are present in the biological sample, the population of cells comprises reduced expression of Y-linked fibronectin 4; (iii) If antibodies to Y-linked tropocadherin 11 and antibodies to Y-linked fibronectin 4 are present in the biological sample, the cell population comprises reduced expression of Y-linked tropocadherin 11 and Y-linked fibronectin 4; (iv) If there is neither an antibody to Y-linked tropocadherin 11 nor an antibody to Y-linked fibronectin 4 in the biological sample, the cell population does not comprise reduced expression of Y-linked tropocadherin 11 or Y-linked fibronectin 4.

In some embodiments, the cell does not express Y-linked tropocadherin 11.

In some embodiments, the cell does not express Y-linked fibronectin 4.

In some embodiments, the cells are derived from human cells or animal cells.

In some embodiments, the cells comprise reduced expression of B2M and/or CIITA relative to unmodified or wild-type or control cells.

In some embodiments, the cell does not express B2M and/or CIITA.

In some embodiments, the cell comprises reduced expression of RHD.

In some embodiments, the cell does not express RHD.

In some embodiments, the cell is a primary immune cell or a progeny thereof.

In some embodiments, the cell does not express TCR- α and/or TCR- β.

In some embodiments, the cell further comprises a second exogenous polynucleotide encoding one or more CARs, and the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain that are specific for CD19, CD20, CD22, or BCMA.

In some embodiments, the gene therapy vector is a retrovirus or fusion.

In some embodiments, the retrovirus is a lentiviral vector.

In some embodiments, provided herein is a use of an engineered T cell population for treating a disorder or condition in a patient, wherein the engineered T cell comprises reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules relative to an unmodified wild-type or control cell and a first exogenous polynucleotide encoding CD47, wherein the engineered T cell is propagated by a primary T cell or progeny thereof, or derived from an iPSC or progeny thereof.

In some embodiments, provided herein is a use of an engineered differentiated cell population for treating a disorder or condition in a patient, wherein the engineered differentiated cell comprises reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules relative to an unmodified wild-type or control cell and a first exogenous polynucleotide encoding CD47, wherein the engineered differentiated cell is derived from an iPSC or progeny thereof.

In some embodiments, the cell does not express Y-linked tropocadherin 11.

In some embodiments, the cell does not express Y-linked fibronectin 4.

In some embodiments, the cells are derived from human cells or animal cells.

In some embodiments, the cell does not express B2M and/or CIITA.

In some embodiments, the cell comprises reduced expression of RHD.

In some embodiments, the cell does not express RHD.

In some embodiments, the cell is a primary immune cell or a progeny thereof.

In some embodiments, the cell does not express TCR- α and/or TCR- β.

In some embodiments, the first and/or second exogenous polynucleotide is introduced into the engineered T cell using a gene therapy vector or a transposase system selected from the group consisting of a transposase, a PiggyBac transposon, a sleeping beauty (SB 11) transposon, a Mos1 transposon, and a Tol2 transposon.

In some embodiments, the gene therapy vector is a retrovirus or fusion.

In some embodiments, the retrovirus is a lentiviral vector.

In some embodiments, provided herein is a method for producing an engineered cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified wild-type or control cell, the method comprising: (a) obtaining an isolated cell; (b) Genetically modifying the cell to reduce expression of one or more Y chromosome genes in the cell; (c) Genetically modifying the cell to reduce expression of MHC class I human leukocyte antigen molecules and/or MHC class II human leukocyte antigen molecules in the cell; and (d) introducing a polynucleotide encoding CD47 into the isolated cell, thereby producing an engineered cell.

In some embodiments, provided herein is a method for producing an engineered cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified wild-type or control cell, the method comprising: (a) obtaining an isolated cell; (b) Contacting the cell with a composition comprising a lentiviral vector comprising (I) a CD4 binding agent or CD8 binding agent, (II) a polynucleotide encoding a CRISPR/Cas gene-editing component targeting one or more Y chromosome loci, (iii) a polynucleotide encoding a CRISPR/Cas gene-editing component targeting MHC class I and/or class II human leukocyte antigen loci, and (iv) a first exogenous polynucleotide encoding CD47, thereby producing an engineered cell.

A detailed description of hypoimmunogenic cells, methods of their production and methods of their use is found in WO2016183041, filed 5, 9, 2015; WO2018132783 submitted on 14 days 1 and 1 in 2018; WO2018176390 submitted on 3 and 20 days 2018; WO2020018615 submitted on 7/17/2019; WO2020018620 submitted on 7/17/2019; PCT/US2020/44635 filed on 31 th 7 th 2020; WO2021022223 filed in 7.31 in 2020; WO2021041316 submitted in month 8 and 24 of 2020; WO2021222285 filed on day 27, 4 of 2021; and WO2021222285 filed on month 4, 2021, 27, the disclosures of which including examples, sequence listings and figures are incorporated herein by reference in their entirety.

Drawings

FIG. 1A depicts flow cytometry data measuring tropocadherin-Y and fibronectin-Y levels on the cell surface of an iPSC derived from a male donor as compared to an isotype control.

Fig. 1B depicts flow cytometry data measuring tropocadherin-Y and fibronectin-Y levels on the cell surface of ipscs derived from female donors as compared to isotype controls.

FIG. 2A depicts flow cytometry data measuring tropocadherin-Y and fibronectin-Y levels on the cell surface of CD3+ T cells from three blood male donors analyzed after thawing, as compared to isotype control.

FIG. 2B depicts flow cytometry data measuring tropocadherin-Y and fibronectin-Y levels on the cell surface of CD3+ T cells from two blood male donors analyzed after thawing, as compared to isotype control.

Figure 2C depicts flow cytometry data measuring tropocadherin-Y and fibronectin-Y levels on the cell surface of cd3+ T cells from two female donors analyzed after thawing, as compared to isotype control.

Figures 3A, 3B and 3C show CDC of HIP T cells from male O-type blood donors using serum from different volunteers.

Figures 4A, 4B and 4C show ADCC (NK cells) of HIP T cells from male O-type blood donors using serum from different volunteers.

Figures 5A, 5B and 5C show CDC and ADCC (NK cells) of HIP T cells from male blood O donors using serum from different volunteers and flow analysis.

Other objects, advantages and embodiments of the present disclosure will be apparent from the following detailed description.

Detailed Description

I. Introduction to the invention

Described herein are engineered or modified immune evasion cells, including but not limited to human immune evasion cells, based in part on PCT/US21/65157 filed on 12 months 23 of WO2018132783 and 2021, each of which is incorporated herein by reference in its entirety. To overcome the problem of immune rejection of these primary and/or stem cell-derived grafts by a subject, the inventors have developed and described herein low-immunogenic cells (e.g., low-immunogenic pluripotent cells, differentiated cells derived from such low-immunogenic pluripotent cells, and primary cells) that represent a viable source of any transplantable cell type. Such cells are protected from adaptive and/or innate immune rejection following administration to a recipient subject. Advantageously, the cells disclosed herein are not rejected by the immune system of the recipient subject, regardless of the genetic constitution of the subject, as they are protected from adaptive and innate immune rejection following administration to the recipient subject. In some embodiments, the engineered and/or hypoimmunogenic cells do not express one or more Y chromosome genes and/or do not express MHC I and/or II antigen molecules and/or T cell receptors. In certain embodiments, the engineered and/or hypoimmunogenic cells do not express one or more Y chromosome genes and do not express MHC I antigens. In certain embodiments, the engineered and/or hypoimmunogenic cells do not express one or more Y chromosome genes, do not express MHC I and/or II antigen molecules and/or T cell receptors, and overexpress CD47 protein. In certain embodiments, the engineered and/or hypoimmunogenic cells do not express one or more Y chromosome genes, do not express MHC I and II antigen molecules and/or T cell receptors, and overexpress CD47 protein. In certain embodiments, the engineered and/or low immunogenic cells (such as low immunogenic T cells) do not express one or more Y chromosome genes, do not express MHC I and/or II antigen molecules and/or T cell receptors, overexpress CD47 protein, and express exogenous CARs. In certain embodiments, the engineered and/or low immunogenic cells (such as low immunogenic T cells) do not express one or more Y chromosome genes, do not express MHC I and II antigen molecules and/or T cell receptors, overexpress CD47 protein, and express exogenous CARs.

In some embodiments, the hypoimmunogenic cells outlined herein do not undergo innate immune cell rejection. In some cases, the hypoimmunogenic cells are insensitive to NK cell-mediated lysis. In some cases, the hypoimmunogenic cells are not susceptible to phagocytosis by macrophages. In some embodiments, the hypoimmunogenic cells do not induce an immune response. In some embodiments, the low immunogenicity 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 need for immunosuppressants. Such low-immunogenicity cells retain cell-specific features and characteristics after transplantation, including, for example, pluripotency, and are capable of implantation and function similarly to the corresponding native cells.

The technology disclosed herein utilizes expression of tolerogenic factors and modulation (e.g., reduction or elimination) of one or more Y chromosome genes and optionally MHC I molecules, MHC II molecules, and/or TCR expression in human cells. In some embodiments, genomic editing techniques utilizing rare-cutting endonucleases (rare-cutting endonuclease) (e.g., CRISPR/Cas, TALENs, zinc finger nucleases, meganucleases, and homing endonuclease systems) can also be used to reduce or eliminate expression of genes involved in immune responses in cells (e.g., by deleting genomic DNA of genes involved in immune responses or by inserting genomic DNA into such genes such that gene expression is affected). In some embodiments, genomic editing techniques or other gene regulation techniques are used to insert tolerance inducing (tolerogenic) factors in human cells, enabling the cells and their progeny (including any differentiated cells prepared therefrom) to evade immune recognition upon implantation into a recipient subject. Thus, the cells described herein exhibit modulated expression of one or more genes and factors that affect expression of one or more Y chromosome genes, MHC I molecules, MHC II molecules, and/or TCRs, and evade the immune system of the recipient subject.

Genome editing techniques are capable of effecting double-stranded DNA breaks at desired locus sites. These controlled double strand breaks promote homologous recombination at specific locus sites. This process focuses on targeting a specific sequence (such as a chromosome) with an endonuclease that recognizes and binds to the specific sequence of a nucleic acid molecule and induces a double strand break within the nucleic acid molecule. Double strand breaks are repaired by error-prone non-homologous end joining (NHEJ) or by Homologous Recombination (HR).

Surprisingly, ipscs and T cells from male donors were found to express the Y chromosome antigens Y-linked tropocadherin 11 and Y-linked fibronectin 4. These surprising findings indicate that the source of low-immunogenicity cells (such as low-immunogenicity donor T cells, non-activated T cells, islet cells, or heart cells) should lack the Y chromosome or should be genetically modified to reduce expression of Y chromosome antigens to avoid detection and elimination by the recipient's adaptive immune system.

Practice of the various 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: ALaboratory Manual (3 rd edition, 2001); sambrook et al, molecular Cloning: A Laboratory Manual (2 nd edition, 1989); maniatis et al, molecular Cloning: A Laboratory 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 editions, 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 editions, 1991); annual Review of Immunology; and monographs such as those on the ADVANCES IN Immunology journal.

II. Definition of

As described in the present disclosure, the following terms will be employed, and the terms are defined as follows.

As used herein, the term "antigen" refers to a molecule capable of eliciting an immune response. Antigens include, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and non-peptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, carbohydrates, viruses and viral extracts, and multicellular organisms such as parasites and allergens. The term antigen broadly includes any type of molecule recognized as foreign by the host immune system.

The term "autoimmune disease" or "autoimmune disorder" or "inflammatory disease" or "inflammatory disorder" refers to any disease or disorder in which a subject produces an immune response against its own tissues and/or cells. Autoimmune disorders can affect almost every organ system of a subject (e.g., a human), including but not limited to neurological, gastrointestinal, and endocrine system diseases, as well as skin and other connective tissue, eye, blood, and vascular diseases. Examples of autoimmune diseases include, but are not limited to, hashimoto's thyroiditis, systemic lupus erythematosus, sjogren's syndrome, graves ' disease, scleroderma, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and diabetes.

The term "cancer" as used herein is defined as the hyper-proliferation of cells whose unique characteristics (e.g., loss of normal control) result in unregulated growth, lack of differentiation, localized tissue invasion and metastasis. With respect to the methods of the invention, the cancer may be any cancer, including any of the following: acute lymphocytic cancer, acute myelogenous leukemia, acinar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal canal cancer or rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gall bladder cancer or pleural cancer, nasal cancer or middle ear cancer, oral cancer, vulva cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, hodgkin lymphoma (Hodgkin lymphoma), hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, large omentum and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer, ureter cancer and urinary bladder cancer. As used herein, unless explicitly stated otherwise, the term "tumor" refers to abnormal growth of malignant type of cells or tissue, and excludes benign type of tissue.

The term "cell" refers to any human or animal cell. In some embodiments, the cell is a human or animal cell from a donor subject having a Y chromosome. In some embodiments, the cell is a human or animal cell from a donor subject that does not have a Y chromosome.

The term "chronic infectious disease" refers to a disease caused by an infectious agent in which infection persists. Such diseases may include hepatitis (type A, type B or type C), herpes viruses (e.g., VZV, HSV-1, HSV-6, HSV-II, CMV and EBV) and HIV/AIDS. Non-viral examples may include chronic fungal diseases such as aspergillosis, candidiasis, coccidioidomycosis and cryptococcus-related diseases, and histoplasmosis. Non-limiting examples of chronic bacterial infectious agents may be chlamydia pneumoniae (CHLAMYDIA PNEUMONIAE), listeria monocytogenes (Listeria monocytogene) and mycobacterium tuberculosis (Mycobacterium tuberculosis). In some embodiments, the disorder is a Human Immunodeficiency Virus (HIV) infection. In some embodiments, the disorder is acquired immunodeficiency syndrome (AIDS).

As used herein, "clinically effective amount" refers to an amount sufficient to provide a clinical benefit in the treatment and/or management of a disease, disorder, or condition. In some embodiments, a clinically effective amount is an amount that has been shown to produce at least one improved clinical endpoint for the standard of care of a disease, disorder, or condition. In some embodiments, a clinically effective amount is an amount that has proven sufficient to provide a statistically significant and meaningful effectiveness for treating a disease, disorder, or condition, e.g., in a clinical trial. In some embodiments, the clinically effective amount is also a therapeutically effective amount. In some embodiments, the clinically effective amount is not a therapeutically effective amount.

In some embodiments, the alterations or modifications described herein (including, for example, genetic alterations or modifications) 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.

In additional or alternative embodiments, the present disclosure contemplates altering the target polynucleotide sequence in any manner available to the skilled artisan, for example, using a TALEN system or RNA-guided transposase. It should be appreciated that although examples of methods utilizing CRISPR/Cas (e.g., cas9 and Cas12 a) and TALENs are described in detail herein, the present disclosure is not limited to the use of these methods/systems. Other methods of targeting, such as B2M, known to the skilled artisan to reduce or eliminate expression in target cells may be utilized herein.

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%. In some embodiments, the cells are engineered to have reduced expression of one or more targets relative to an unaltered or unmodified wild-type or control cell.

In some embodiments, the engineered and/or hypoimmunogenic cells are derived from ipscs or progeny thereof. As used herein, the term "derived from an iPSC or its progeny" encompasses the initial iPSC generated and any subsequent progeny thereof. As used herein, the term "offspring" encompasses, for example, first generation offspring, i.e., offspring that are directly derived, obtained, obtainable or derived from the original iPSC by, for example, traditional breeding methods. The term "progeny" also encompasses further generations, such as second, third, fourth, fifth, sixth, seventh or more generations, i.e. cell generations derived, obtained, obtainable or derivable from the previous generation, e.g. by conventional propagation methods. The term "offspring" also encompasses modified cells resulting from modification or alteration of the original iPSC or its progeny.

The term "donor subject" refers to an animal, e.g., a human from which cells can be obtained. "non-human animal" and "non-human mammal" as used interchangeably herein include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "donor subject" also encompasses any vertebrate, including but not limited to mammals, reptiles, amphibians, and fish. Advantageously, however, the donor subject is a mammal, such as a human or other mammal (such as a domestic mammal, e.g., a dog, cat, horse, etc., or a production mammal, e.g., a cow, sheep, pig, etc.). A "donor subject" may also refer to more than one donor, such as one or more human or non-human animals or non-human mammals.

The term "endogenous" refers to a reference molecule or polypeptide that naturally occurs in a cell. Similarly, when used in reference to expression of a coding nucleic acid, the term refers to expression of the coding nucleic acid that is naturally contained within a cell and is not exogenously introduced. Similarly, when used in reference to a promoter sequence, the term refers to a promoter sequence that is naturally contained within a cell and is not exogenously introduced.

The term "engineered cell" as used herein refers to a cell that has been altered in at least some way by human intervention, including, for example, by genetic alteration or modification, such that the engineered cell differs from a wild-type cell.

As used herein, the term "exogenous" in the context of an expressed polynucleotide or polypeptide is intended to mean that the referenced molecule or referenced polypeptide is introduced into a cell of interest. The polypeptide may be introduced, for example, by introducing the encoding 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.

For the purposes of this disclosure, "gene" includes DNA regions encoding a gene product, as well as all DNA regions that regulate the production of a gene product, whether such regulatory sequences are adjacent to coding and/or transcribed 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/or locus control regions.

"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 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/or glycosylation.

The term "genetic modification" and grammatical equivalents thereof as used herein may refer to one or more alterations of a nucleic acid (e.g., a nucleic acid within the genome of an organism). For example, genetic modification may refer to alterations, additions and/or deletions of a gene or gene portion or other nucleic acid sequence. Genetically modified cells may also refer to cells having added, deleted and/or altered genes or gene portions. Genetically modified cells may also refer to cells to which nucleic acid sequences other than genes or parts of genes have been added. Genetic modifications include, for example, transient knock-in or knock-down mechanisms, as well as mechanisms that result in permanent knock-in, knock-down or knock-out of a target gene or gene portion or nucleic acid sequence. Genetic modifications include, for example, transient knockins, as well as mechanisms that result in permanent knockins of nucleic acid sequences. Genetic modifications also include, for example, reduced or increased transcription, reduced or increased mRNA stability, reduced or increased translation, and reduced or increased protein stability.

As used herein, the terms "engraftment," "Administration (ADMINISTERING)", "introduction (introducing)", "implantation (implanting)", and "transplantation (TRANSPLANTING)" and grammatical variants thereof are used interchangeably in the context of placing cells (e.g., the cells described herein) into a subject by a method or route that results in the introduction of cells to be localized or at least partially localized at a desired site or introduced systemically (e.g., into the circulation). 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, in which location at least a portion of the implanted cells or cell components remain viable. The lifetime of the cells after administration to a subject can be as short as a few hours (e.g., twenty-four hours) to days, even as long as several years. In some embodiments, the cells may also be administered (e.g., injected) in capsule form, for example, to a location other than the desired site, such as in the brain or subcutaneously, to maintain the implanted cells at the implantation site and avoid migration of the implanted cells.

An "HLA" or "human leukocyte antigen" or "HLA molecule" or "human leukocyte antigen molecule" complex is a complex of genes encoding MHC proteins in humans. These cell surface proteins constituting the HLA complex are responsible for regulating immune responses to antigens. In humans, there are two classes of MHC, class I and class II molecules, "HLA-I" and "HLA-II", or "HLA-I molecule" and "HLA-II molecule". HLA-I includes three proteins, HLA-A, HLA-B and HLA-C, which present peptides from within cells, and antigen presented by HLA-I complexes attracts killer T cells (also known as CD8+ T cells or cytotoxic T cells). HLA-I proteins are associated with beta-2 microglobulin (B2M). HLA-II includes five proteins HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR, which present antigens from outside cells to T lymphocytes. This stimulates cd4+ cells (also known as helper T cells). It will be appreciated that the use of "MHC" or "HLA" is not meant to be limiting, as it depends on whether the gene is from a Human (HLA) or from a Murine (MHC). Thus, these terms are used interchangeably herein as it relates to mammalian cells.

As used herein to characterize a cell, the term "hypoimmunogenic" generally means that such a cell is less susceptible to innate or adaptive immune rejection by the subject into which such a cell is transplanted, e.g., the cell is less susceptible to allograft rejection by the subject into which such a cell is 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 innate or adaptive immune rejection of a subject into which such a cell is transplanted relative to a cell of the same cell type that does not comprise the modification. In some embodiments, genome editing techniques are used to modulate the expression of MHC I and MHC II genes, thereby promoting the production of cells of low immunogenicity. In some embodiments, the hypoimmunogenic cells evade immune rejection in MHC mismatched allogeneic recipients. In some cases, differentiated cells generated by the low-immunogenicity stem cells outlined herein evade immune rejection when administered (e.g., transplanted or implanted) to 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. A detailed description of hypoimmunogenic cells, methods of their production and methods of their use is found in WO2016183041, filed 5, 9, 2015; WO2018132783 submitted on 14 days 1 and 1 in 2018; WO2018176390 submitted on 3 and 20 days 2018; WO2020018615 submitted on 7/17/2019; WO2020018620 submitted on 7/17/2019; PCT/US2020/44635 filed on 31 th 7 th 2020; WO2021022223 filed in 7.31 in 2020; WO2021041316 submitted in month 8 and 24 of 2020; WO2021222285 filed on day 27, 4 of 2021; and WO2021222285 filed on month 4, 2021, 27, the disclosures of which including examples, sequence listings and figures are incorporated herein by reference in their entirety.

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 or avoid eliciting an adaptive and innate immune response). Such immune responses may be measured using assays recognized by those skilled in the art. In some embodiments, the immune response assay measures the effect of a low immunogenicity cell on T cell proliferation, T cell activation, T cell killing, donor specific antibody production, NK cell proliferation, NK cell activation, and macrophage activity. In some cases, the hypoimmunogenic cells and derivatives thereof undergo a decrease in killing by T cells and/or NK cells after administration to a subject. In some cases, the cells and derivatives thereof exhibit reduced phagocytosis by macrophages compared to unmodified or wild-type cells. In some embodiments, the low-immunogenicity cells elicit a reduced or attenuated immune response in the recipient subject as compared to corresponding unmodified wild-type cells. In some embodiments, the hypoimmunogenic cells are non-immunogenic or incapable of eliciting an immune response in a recipient subject.

The term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to a specified percentage of the two or more sequences or subsequences that have the same nucleotide or amino acid residue when compared and aligned to obtain a maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to a skilled artisan), or by visual inspection. Depending on the application, a percentage "identity" may be present in the region of the sequences to be compared, for example in the functional domain, or in the full length of the two sequences to be compared. For sequence comparison, typically one sequence serves as a reference sequence for comparison to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, subsequence coordinates are designated (if necessary), and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

The optimal alignment of sequences can be made, for example, by the following algorithm to achieve comparison: the local homology algorithm of Smith and Waterman, adv.appl.Math.2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J.mol.biol.48:443 (1970), the similarity search method of Pearson and Lipman, proc.Nat' l.Acad.Sci.USA 85:2444 (1988), the computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin genetic software package (Genetics Computer Group,575Science Dr., madison, wis.) or visual inspection (see generally Ausubel et al, infra).

One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, described in Altschul et al, J.mol. Biol.215:403-410 (1990). Software for performing BLAST analysis is publicly available through the national center for biotechnology information (National Center for Biotechnology Information).

As used herein, "immune signaling factor" refers in some cases to molecules, proteins, peptides, etc. that activate immune signaling pathways.

As used herein, "immunosuppressive factor" or "immunomodulator" or "tolerogenic factor" includes low immune factors (hypoimmunity factor), complement inhibitors, and other 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. These may be combined with additional genetic modifications.

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, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including 100% increase or any increase between 10% -100% or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold or at least about 10-fold increase from a reference level, or any increase between 2-fold and 10-fold, or more. In some embodiments, the reference level, also referred to as the base level, is 0.

In some embodiments, the alteration is an indel. As used herein, "indels" refers to mutations resulting from insertions, deletions, or combinations thereof. 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. In some embodiments, the alteration is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one nucleotide. The gene editing (e.g., 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, "knockdown" refers to a decrease in expression of a target mRNA or corresponding target protein. Knockdown is typically reported relative to the level present following administration or expression of a non-control molecule (e.g., a non-targeted control shRNA, siRNA or miRNA) that does not mediate a reduction in RNA expression level. In some embodiments, the knockdown of the target gene is achieved by conditional or inducible shRNA, conditional or inducible siRNA, conditional or inducible miRNA, or conditional or inducible CRISPR interference (CRISPRi). In some embodiments, the knockdown of the target gene is achieved by protein-based methods, such as the conditional or inducible degradation solution stator (degron) methods. In some embodiments, the knockdown of the target gene is achieved by genetic modification (including shRNA, siRNA, miRNA) or using a gene editing system (e.g., CRISPR/Cas).

Knock-down is typically assessed by measuring mRNA levels using quantitative polymerase chain reaction (qPCR) amplification or by measuring protein levels using western blot or enzyme-linked immunosorbent assay (ELISA). Analysis of protein levels provides an assessment of mRNA cleavage and translational inhibition. Additional techniques for measuring knockdown include RNA solution hybridization, nuclease protection, northern hybridization, monitoring gene expression with microarrays, antibody binding, radioimmunoassay, and fluorescence-activated cell analysis. Based on the details described herein, one of skill in the art will readily understand how to knock out a target polynucleotide sequence or portion thereof using the gene editing system (e.g., CRISPR/Cas) of the present disclosure.

"Knock in" or "knock-in" herein refers to a genetic modification caused by insertion of a DNA sequence into a chromosomal locus of a host cell. This results in an increase in the initiation or level of expression of the knockin gene, gene portion, or nucleic acid sequence insertion product, e.g., an increase in the level of RNA transcripts and/or the level of encoded protein. As will be appreciated by those skilled in the art, this may be accomplished in several ways, including inserting or adding one or more additional copies of the gene or portion thereof into a host cell, or altering regulatory components of an endogenous gene to increase expression of the protein, or inserting a designated nucleic acid sequence for which expression is desired. This can be achieved by modifying the promoter, adding different promoters, adding enhancers, adding other regulatory elements or modifying other gene expression sequences.

As used herein, "knockout" or "knockout-out" includes deleting all or part of a target polynucleotide sequence in a manner that interferes with translation or function of the target polynucleotide sequence. For example, knockout can be achieved by altering a target polynucleotide sequence by inducing insertions or deletions ("indels") in the target polynucleotide sequence, including in the functional domain (e.g., 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 knock out a target polynucleotide sequence or portion thereof using the gene editing system of the present disclosure (e.g., CRISPR/Cas).

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

"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.

In additional or alternative aspects, the present disclosure contemplates altering the target polynucleotide sequence in any manner available to the skilled artisan, for example, using a nuclease system, such as a TAL effector nuclease (TALEN) or Zinc Finger Nuclease (ZFN) system. It should be appreciated that although examples of methods utilizing CRISPR/Cas (e.g., cas9 and Cas12 a) and TALENs are described in detail herein, the present disclosure is not limited to the use of these methods/systems. Other targeting methods known to the skilled artisan to reduce or eliminate expression in target cells may be utilized herein. The methods provided herein can be used to alter a target polynucleotide sequence in a cell. The present disclosure contemplates altering a target polynucleotide sequence in a cell for any purpose. In some embodiments, the target polynucleotide sequence in the cell is altered to produce a mutant cell. As used herein, "mutant cell" refers to a cell having a resulting genotype that is different from its original genotype. In some cases, a "mutant cell" exhibits a mutant phenotype, for example, when a gene of normal function is altered using the gene expression system of the present disclosure (e.g., CRISPR/Cas system). In other cases, the "mutant cells" exhibit a wild-type phenotype, such as when the mutant genotype is corrected using the gene expression systems of the present disclosure (e.g., CRISPR/Cas systems). In some embodiments, the target polynucleotide sequence in the cell is altered to correct or repair the genetic mutation (e.g., to restore the normal phenotype of the cell). In some embodiments, the target polynucleotide sequence in the cell is altered to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).

"Y-linked fibronectin 4", "fibronectin 4-Y" and "NLGN4Y" and variants thereof refer to the Y chromosome-linked antigen encoded by the NLGN4Y gene.

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.

The term "patient" or "recipient patient" refers to an animal, e.g., a human, to whom treatment (including prophylactic treatment) with cells as described herein is provided. For the treatment of those infections, conditions or disease states that are specific to a particular animal (such as a human patient), the term patient refers to that particular animal. The term "patient" also encompasses any vertebrate, including but not limited to mammals, reptiles, amphibians, and fish. Advantageously, however, the patient is a mammal, such as a human or other mammal (such as a domestic mammal, e.g., a dog, cat, horse, etc., or a production mammal, e.g., a cow, sheep, pig, etc.).

As used herein, a "pluripotent stem cell" has the potential to differentiate into any of three germ layers: endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, genitourinary tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue). The term "pluripotent stem cell" as used herein also encompasses "induced pluripotent stem cell" or "iPSC", or a class of pluripotent stem cells derived from non-pluripotent cells. In some embodiments, the pluripotent stem cells are produced or generated by cells that are not pluripotent cells. In other words, pluripotent stem cells may be direct or indirect progeny of non-pluripotent cells. Examples of parent cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such "iPS" or "iPSC" cells may be produced by inducing the expression of certain regulatory genes or by exogenous application of certain proteins. Methods for inducing iPS cells are known in the art and are described further below. (see, e.g., zhou et al STEM CELLS (11): 2667-74 (2009); huangfu et al Nature Biotechnol.26 (7): 795 (2008); woltjen et al Nature 458 (7239): 766-770 (2009); and Zhou et al CELL STEM CELL 8:381-384 (2009); each of which is incorporated herein by reference in its entirety) Induced Pluripotent Stem Cell (iPSC) production is summarized below. As used herein, "hipscs" are human induced pluripotent stem cells. In some embodiments, as used herein, "pluripotent stem cells" also encompass Mesenchymal Stem Cells (MSCs) and/or Embryonic Stem Cells (ESCs).

As used herein, "promoter," "promoter sequence," or "promoter region" refers to a DNA regulatory region/sequence capable of binding RNA polymerase and involved in initiating transcription of a downstream coding or non-coding sequence. In some examples, the promoter sequence includes a transcription initiation site and extends upstream to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. In some embodiments, the promoter sequence includes a transcription initiation site, and a protein binding domain responsible for RNA polymerase binding. Eukaryotic promoters will often, but not always, contain a "TATA" box and a "CAT" box.

In some embodiments, the engineered and/or hypoimmunogenic cells are propagated from primary T cells or progeny thereof. As used herein, the term "propagated from primary T cells or their progeny" encompasses the primary T cells isolated from a donor subject and any subsequent progeny thereof. As used herein, the term "progeny" encompasses, for example, first generation progeny, i.e., progeny that are directly derived, obtained, obtainable, or derived from the original primary T cell, e.g., by conventional propagation methods. The term "progeny" also encompasses further generations, such as second, third, fourth, fifth, sixth, seventh or more generations, i.e. cell generations derived, obtained, obtainable or derivable from the previous generation, e.g. by conventional propagation methods. The term "progeny" also encompasses modified cells resulting from modification or alteration of the original primary T cell or its progeny.

"Y-linked tropocadherin 11", "tropocadherin 11-Y" and "PCDH11Y" and variants thereof refer to the Y chromosome-linked antigen encoded by the PCDH11Y gene.

As used herein, the terms "regulatory sequence," "regulatory element," and "control element" are interchangeable and refer to a polynucleotide sequence upstream (5 'non-coding sequence), internal, or downstream (3' non-translated sequence) of a polynucleotide target to be expressed. Regulatory sequences affect, for example, but not limited to, the time of transcription, the amount or level of transcription, RNA processing or stability, and/or translation of the relevant structural nucleotide sequence. Regulatory sequences may include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor binding sequences, stem-loop structures, translation initiation sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites, and the like. It is recognized that nucleotide sequences of different lengths may have identical regulatory or promoter activity, since in most cases the exact boundaries of the regulatory sequences are not yet fully defined.

"Safe harbor locus" as used herein refers to a locus that allows expression of a transgene or foreign gene in a manner that enables the newly inserted genetic element to function predictably, and also does not cause alterations in the host genome in a manner that would risk to the host cell. Exemplary "safe harbor" loci include, but are not limited to, CCR5 genes, PPP1R12C (also known as AAVS 1) genes, CLYBL genes, and/or Rosa genes (e.g., rosa 26).

As used herein, a "target locus" refers to a locus that allows for expression of a transgene or exogenous gene. Exemplary "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, and/or KDM5D genes (also known as HY). Exogenous polynucleotides encoding exogenous genes can be inserted into the CDs region of B2M, CIITA, TRAC, TRBC, CCR, F3 (i.e., CD 142), MICA, MICB, LRP1, HMGB1, ABO, RHD, FUT1, KDM5D (i.e., HY), PDGFRa, OLIG2, and/or GFAP. An exogenous polynucleotide encoding an exogenous gene may be inserted into intron 1 or 2 of PPP1R12C (i.e., AAVS 1) or CCR 5. An exogenous polynucleotide encoding an exogenous gene may be inserted into exon 1 or 2 or 3 of CCR 5. An exogenous polynucleotide encoding an exogenous gene may be inserted into intron 2 of CLYBL. An exogenous polynucleotide encoding an exogenous gene may be inserted into a 500bp window in Ch-4:58,976,613 (i.e., SHS 231). The exogenous polynucleotide encoding the exogenous gene may be inserted into any suitable region mentioned above that allows expression of the exogenous safe harbor or target locus, including, for example, an intron, exon, or coding sequence region in the safe harbor or target locus.

The term "sensitization" as used in connection with a patient refers to a patient having antibodies that react to foreign cells. In some embodiments, the present disclosure contemplates treatment of a sensitized subject. For example, it is contemplated that the subject of the present treatment methods is susceptible to one or more alloantigens comprising a Y chromosome-linked antigen. In some embodiments, the patient is sensitized by prior pregnancy or prior allografts (including, for example, but not limited to, allogeneic cell transplantation, allogeneic blood transfusion, allogeneic tissue transplantation, and allogeneic organ transplantation). In some embodiments, the patient exhibits memory B cells and/or memory T cells that are reactive to one or more alloantigens.

In some embodiments, the present disclosure contemplates treatment of a non-sensitized subject. For example, it is contemplated that the subject of the present treatment methods is not susceptible to one or more alloantigens comprising a Y chromosome-linked antigen. In some embodiments, the patient is not sensitized by prior pregnancy or prior allografts (including, for example, but not limited to, allogeneic cell transplantation, allogeneic blood transfusion, allogeneic tissue transplantation, and allogeneic organ transplantation). In some embodiments, the patient does not exhibit memory B cells and/or memory T cells that are reactive to one or more alloantigens.

As used herein, a "target" may refer to a gene, gene portion, genome portion, or protein that is subjected to regulatable reduced expression by the methods described herein.

As used herein, "therapeutically effective amount" refers to an amount sufficient to provide a therapeutic benefit in the treatment and/or management of a disease, disorder, or condition. In some embodiments, a therapeutically effective amount is an amount sufficient to improve, alleviate, stabilize, reverse, slow, attenuate or delay the progression of a disease, disorder or condition or the progression of symptoms or side effects of a disease, disorder or condition. In some embodiments, the therapeutically effective amount is also a clinically effective amount. In other embodiments, the therapeutically effective amount is not a clinically effective amount.

As used herein, the terms "treating" and "treatment" include administering to a subject a therapeutically or clinically 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 therapeutic or clinical result. For the purposes of this technique, beneficial or desired therapeutic or 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 a condition, disease, or disorder, one or more symptoms of the condition, 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 therapeutic or 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 vehicles 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.

In some embodiments, the cells are engineered to have reduced or increased expression of one or more targets relative to an unaltered or unmodified wild type or control cell. In the context of cells, "wild-type" or "wt" or "control" means any naturally occurring cell. Examples of wild-type or control cells include naturally occurring primary cells and T cells. However, for example, in the context of engineered and/or low-immunogenicity T cells, as used herein, "wild-type" or "control" may also mean engineered and/or low-immunogenicity T cells that may contain nucleic acid changes that result in reduced expression of one or more MHC class I molecules and/or class II molecules and/or T cell receptors, and/or CD47 protein, but have not undergone the genetic editing procedures of the present disclosure that achieve reduced expression of one or more Y chromosome genes. For example, as used herein, "wild-type" or "control" means an engineered cell comprising reduced or knocked out B2M, CIITA and/or TRAC expression. Furthermore, as used herein, "wild-type" or "control" means an engineered cell comprising reduced or knocked out B2M, CIITA, TRAC and/or TRBC expression. As used herein, "wild-type" or "control" also means an engineered cell that may contain a nucleic acid change that results in overexpression of CD47 protein, but has not undergone a gene editing program that results in a reduction in expression of one or more MHC class I molecules and/or class II molecules and/or T cell receptors. In the context of an iPSC or its offspring, "wild-type" or "control" also means an iPSC or its offspring that may contain nucleic acid changes that result in pluripotency, but has not undergone the gene editing procedures of the present disclosure that achieve reduced expression of one or more MHC class I molecules and/or class II molecules and/or T cell receptors and/or one or more Y chromosome genes and/or overexpression of CD47 protein. For example, as used herein, "wild-type" or "control" is meant to encompass ipscs or progeny thereof that reduce or knock out B2M, CIITA and/or TRAC expression. Furthermore, as used herein, "wild-type" or "control" is meant to encompass ipscs or progeny thereof that reduce or knock out B2M, CIITA, TRAC and/or TRBC expression. In the context of primary T cells or their progeny, "wild-type" or "control" also means primary T cells or their progeny that may contain nucleic acid changes that result in reduced expression of one or more MHC class I molecules and/or class II molecules and/or T cell receptors, but without undergoing the gene editing procedures of the present disclosure that effect reduced expression of one or more Y chromosome genes. For example, as used herein, "wild-type" or "control" means a gene editing program comprising primary T cells or progeny thereof that reduce or knock-out B2M, CIITA and/or TRAC expression, but which has not been subjected to a reduction in expression of one or more Y chromosome genes. Furthermore, as used herein, "wild-type" or "control" means a gene editing program comprising primary T cells or progeny thereof that reduce or knock-out B2M, CIITA, TRAC and/or TRBC expression, but not subjected to reduced expression of one or more Y chromosome genes. Furthermore, in the context of primary T cells or their progeny, "wild-type" or "control" also means primary T cells or their progeny that may contain a nucleic acid change that results in overexpression of the CD47 protein, but have not undergone a gene editing procedure that achieves reduced expression of one or more Y chromosome genes. In some embodiments, the wild-type or control cell is the starting material. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes. In some embodiments, "wild-type" or "control" refers to cells having the Y chromosome.

As used herein, "Y chromosome-linked antigen", "Y chromosome antigen", "histocompatibility Y chromosome-linked antigen" or "male-specific antigen" and variants thereof refer to peptides encoded by genes on the Y chromosome that are capable of eliciting an immune response. In particular, the peptides are capable of eliciting an immune response when present in the context of MHC molecules and/or when antibodies to the peptides are present. The Y chromosome-linked antigen includes an antigen that is an antigenic portion of a gene on the Y chromosome or an antigen of an intact protein encoded by a gene on the Y chromosome. Examples of Y chromosome-linked antigens include, but are not limited to, Y-linked tropocadherin 11 (PCDH 11Y), Y-linked fibronectin 4 (NLGN 4Y), H-Y antigen, and the like.

It should be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "unique," "only," and the like, or use of a "negative" limitation when referring to claim elements. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that are readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method may be performed in the order of recited events or in any other order that is logically possible. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, representative illustrative methods and materials are now described.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. Where a range of values is provided, it is understood that each intervening value, INTERVENING VALUE, between the upper and lower limit of that range (up to one tenth of the unit of the lower limit unless the context clearly dictates otherwise) and any other stated value or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the ranges include one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. Certain ranges are presented herein with numerical values beginning with the term "about". The term "about" is used herein to provide literal support for the exact numbers that follow, as well as numbers that approximate or approximate the numbers that follow the term. In determining whether a number is close or approximates a specifically enumerated number, the close or approximated non-enumerated number may be a number that provides substantial equivalence to the specifically enumerated number in the present context. The term about is used herein to mean plus or minus ten percent (10%) of the value. For example, "about 100" refers to any number between 90 and 110.

All publications, patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated by reference herein to disclose and describe the subject matter associated with the cited publication. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present technology described herein is not entitled to antedate such publication by virtue of prior art. Furthermore, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

Before the present technology is further described, it is to be understood that this technology is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. It should also be understood that the headings used herein are not limiting and are intended only to guide the reader, but that the subject matter generally applies to the techniques disclosed herein.

III, detailed description of the invention

A. low immunogenicity cells

In some embodiments, the disclosure provides engineered (e.g., modified and genetically modified) cells comprising reduced expression of one or more Y chromosome genes and MHC class I and/or MHC class II human leukocyte antigen molecules relative to unmodified wild-type or control cells and a first exogenous polynucleotide encoding CD47, wherein the engineered cells are propagated by primary T cells or their progeny, induced pluripotent stem cells (ipscs), or their progeny. In some embodiments, the cells are capable of evading an activated NK cell-mediated and/or antibody-based immune response.

In some embodiments, the cells are induced pluripotent stem cells, any type of differentiated cells thereof, primary immune cells, and other primary cells of any tissue. In some embodiments, the differentiated cells are cardiac cells and subpopulations thereof, neural cells and subpopulations thereof, brain endothelial cells and subpopulations thereof, dopaminergic neurons and subpopulations thereof, glial progenitor cells and subpopulations thereof, endothelial cells and subpopulations thereof, thyroid cells and subpopulations thereof, hepatic cells and subpopulations thereof, islet cells and subpopulations thereof, or retinal pigment epithelial cells and subpopulations thereof. In some embodiments, the differentiated cells are T cells and subpopulations thereof, NK cells and subpopulations thereof, and endothelial cells and subpopulations thereof. In some embodiments, the primary immune cells are T cells and subpopulations thereof and NK cells and subpopulations thereof. In some embodiments, the primary tissue cells include primary endothelial cells and subpopulations thereof.

In some embodiments, the cells described herein comprise reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules relative to an unmodified or wild-type or control cell. In some embodiments, the cells described herein comprise reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 and MHC class I and/or class II human leukocyte antigen molecules relative to unmodified wild-type or control cells. In some embodiments, the cells described herein comprise reduced expression of Y-linked tropocadherin 11 and MHC class I and/or class II human leukocyte antigen molecules relative to unmodified or wild-type or control cells. In some embodiments, the cells described herein comprise reduced expression of Y-linked fibronectin 4 and MHC class I and/or class II human leukocyte antigen molecules relative to unmodified or wild-type or control cells. In some embodiments, the cells described herein comprise reduced expression of Y-linked tropocadherin 11 and Y-linked fibronectin 4, as well as MHC class I and/or class II human leukocyte antigen molecules relative to unmodified wild-type or control cells. In some embodiments, the cells described herein comprise a first exogenous polynucleotide encoding CD 47. In some embodiments, the cells described herein comprise a second exogenous polynucleotide encoding a CAR.

In some embodiments, the disclosure relates to pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (ipscs)), differentiated cells derived from such pluripotent stem cells (such as, but not limited to, T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells), and primary cells (such as, but not limited to, primary T cells and primary NK cells). In some embodiments, pluripotent stem cells, differentiated cells derived therefrom (such as T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells), and primary cells (such as primary T cells and primary NK cells) are engineered to achieve reduced expression or lack of expression of one or more Y chromosome genes and MHC class I and/or MHC class II human leukocyte antigen molecules, and in some cases, reduced expression or lack of expression of a T Cell Receptor (TCR) complex. In some embodiments, the low immune (HIP) T cells and primary T cells overexpress CD47 and Chimeric Antigen Receptor (CAR) in addition to or in the absence of reduced expression of one or more Y chromosome genes and MHC class I and/or MHC class II human leukocyte antigen molecules, and have reduced expression or lack of expression of the T Cell Receptor (TCR) complex. In some embodiments, the CAR comprises an antigen binding domain that binds to any one selected from the group consisting of CD19, CD20, CD22, CD38, CD123, CD138, and BCMA. In some embodiments, the CAR is a CD19 specific CAR. In some embodiments, the CAR is a CD 20-specific CAR. In some embodiments, the CAR is a CD 22-specific CAR. In some cases, the CAR is a CD38 specific CAR. In some embodiments, the CAR is a CD 123-specific CAR. In some embodiments, the CAR is a CD 138-specific CAR. In some cases, the CAR is a BCMA-specific CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the bispecific CAR is a CD19/CD20 bispecific CAR. In some embodiments, the bispecific CAR is a CD19/CD22 bispecific CAR. In some embodiments, the bispecific CAR is a BCMA/CD38 bispecific CAR. In some embodiments, the cell expresses a CD 19-specific CAR and a different CAR, such as, but not limited to, a CD 20-specific CAR, a CD 22-specific CAR, a CD 38-specific CAR, a CD 123-specific CAR, a CD 138-specific CAR, and a BCMA-specific CAR. In some embodiments, the cell expresses a CD 20-specific CAR and a different CAR, such as, but not limited to, a CD 22-specific CAR, a CD 38-specific CAR, a CD 123-specific CAR, a CD 138-specific CAR, a CD 19-specific CAR, and a BCMA-specific CAR. In some embodiments, the cell expresses a CD 22-specific CAR and a different CAR, such as, but not limited to, a CD 19-specific CAR, a CD 20-specific CAR, a CD 38-specific CAR, a CD 123-specific CAR, a CD 138-specific CAR, and a BCMA-specific CAR. In some embodiments, the cell expresses a CD 38-specific CAR and a different CAR, such as, but not limited to, a CD 20-specific CAR, a CD 22-specific CAR, a CD 18-specific CAR, a CD 123-specific CAR, a CD 138-specific CAR, and a BCMA-specific CAR. In some embodiments, the cell expresses a CD 123-specific CAR and a different CAR, such as, but not limited to, a CD 20-specific CAR, a CD 22-specific CAR, a CD 38-specific CAR, a CD 19-specific CAR, a CD 138-specific CAR, and a BCMA-specific CAR. In some embodiments, the cell expresses a CD 138-specific CAR and a different CAR, such as, but not limited to, a CD 20-specific CAR, a CD 22-specific CAR, a CD 38-specific CAR, a CD 123-specific CAR, a CD 19-specific CAR, and a BCMA-specific CAR. In some embodiments, the cell expresses a BCMA-specific CAR and a different CAR, such as, but not limited to, a CD 20-specific CAR, a CD 22-specific CAR, a CD 38-specific CAR, a CD 123-specific CAR, a CD 138-specific CAR, and a CD 19-specific CAR.

In some embodiments, low immune cells derived from ipscs, such as but not limited to T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells, overexpress CD47, and include genomic modifications or knockouts or knockdown of the PCDH11Y gene. In some embodiments, low immune cells derived from ipscs, such as but not limited to T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells overexpress CD47, and include genomic modifications or knockouts or knockdown of the NLGN4Y gene. In some embodiments, low immune cells derived from ipscs, such as but not limited to T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells, overexpress CD47, and include genomic modifications or knockouts or knockdown of the B2M gene. In some embodiments, low immune cells derived from ipscs, such as but not limited to T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells overexpress CD47, and include genomic modifications or knockouts or knockdown of the CIITA gene. In some embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、CIITA^-/-, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down, CD47tg cell. In some embodiments, the low immune cells derived from ipscs are produced by differentiation-induced pluripotent stem cells (such as low immunogenicity-induced pluripotent stem cells). In some embodiments, the cells are modified or engineered as compared to wild-type or control cells (including unmodified or unmodified wild-type cells or control cells). In some embodiments, the wild-type cell or the control cell is the starting material. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to produce an engineered cell.

In some embodiments, the low immune cells derived from ESC, such as, but not limited to, T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells overexpress CD47, and include genomic modification or knockdown of the PCDH11Y gene. In some embodiments, the low immune cells derived from ESC, such as but not limited to T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells overexpress CD47, and include genomic modifications or knockouts or knockdown of the NLGN4Y gene. In some embodiments, the low immune cells derived from ESC, such as but not limited to T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells overexpress CD47, and include genomic modifications or knockouts or knockdown of the B2M gene. In some embodiments, the low immune cells derived from ESC, such as but not limited to T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells overexpress CD47, and include genomic modifications or knockouts or knockdown of the CIITA gene. In some embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、CIITA^-/-, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down, CD47tg cell. In some embodiments, the low immune cells derived from ipscs are produced by differentiating pluripotent stem cells (such as low immunogenic embryonic stem cells). In some embodiments, the cells are modified or engineered as compared to wild-type or control cells (including unmodified or unmodified wild-type cells or control cells). In some embodiments, the wild-type cell or the control cell is the starting material. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to produce an engineered cell.

In some embodiments, low immune (HIP) T cells derived from ipscs and primary T cells overexpress CD47 and Chimeric Antigen Receptor (CAR), and include genomic modifications or knockouts or knockdowns of the PCDH11Y gene. In some embodiments, low immune (HIP) T cells derived from ipscs and primary T cells overexpress CD47 and Chimeric Antigen Receptor (CAR), and include genomic modifications or knockouts or knockdowns of the NLGN4Y gene. In some embodiments, low immune (HIP) T cells derived from ipscs and primary T cells overexpress CD47 and Chimeric Antigen Receptor (CAR), and include genomic modifications or knockouts or knockdowns of the B2M gene. In some embodiments, low immune (HIP) T cells derived from ipscs and primary T cells overexpress CD47 and include genomic modifications or knockouts or knockdowns of the CIITA gene. In some embodiments, low immune (HIP) T cells derived from ipscs and primary T cells overexpress CD47 and CAR, and include genomic modifications or knockouts or knockdowns of the TRAC gene. In some embodiments, low immune (HIP) T cells derived from ipscs and primary T cells overexpress CD47 and CAR, and include genomic modifications or knockouts or knockdowns of the TRB gene. In some embodiments, low immune (HIP) T cells derived from ipscs and primary T cells overexpress CD47 and CAR and include one or more genomic modifications or knockouts or knockdowns selected from the group consisting of PCDH11Y, NLGN4Y, B2M, CIITA, TRAC and TRB genes. In some embodiments, low immune (HIP) T cells derived from ipscs and primary T cells overexpress CD47 and CAR, and include genomic modifications or knockouts or knockdowns of PCDH11Y, NLGN4Y, B2M, CIITA, TRAC and TRB genes. In some embodiments, the cell is a B2M ^-/-、CIITA^-/-、TRAC^-/-, CD47tg cell that also expresses a CAR. In some embodiments, the cell is a B2M ^-/-、TRAC^-/-, CD47tg cell that also expresses a CAR. In some embodiments, the low immunity (HIP) T cells are produced by differentiation-induced pluripotent stem cells, such as low immunogenicity-induced pluripotent stem cells. In some embodiments, the cells are modified or engineered as compared to wild-type or control cells (including unmodified or unmodified wild-type cells or control cells). In some embodiments, the wild-type cell or the control cell is the starting material. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to produce an engineered cell.

In some embodiments, the low immune (HIP) T cells derived from ipscs and primary T cells are PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、CIITA^-/-、TRB^-/-, CD47tg cells that also express a CAR. In some embodiments, the low immune (HIP) T cells derived from ipscs and primary T cells are PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、TRB^-/-, CD47tg cells that also express a CAR. In some embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、CIITA^-/-、TRAC^-/-、TRB^-/-, CD47tg cell that also expresses a CAR. In some embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、TRAC^-/-、TRB^-/-, CD47tg cell that also expresses a CAR. In certain embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion}, CD47tg cell that also expresses a CAR. In certain embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion}, CD47tg cell that also expresses a CAR. In certain embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion}, CD47tg cell that also expresses a CAR. In certain embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion}, CD47tg cell that also expresses a CAR. In certain embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion}, CD47tg cell that also expresses a CAR. In certain embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion}, CD47tg cell that also expresses a CAR. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down、TRAC^Knock-down, CD47tg cell that also expresses a CAR. In certain embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down、TRB^Knock-down, CD47tg cell that also expresses a CAR. In certain embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down、TRAC^Knock-down、TRB^Knock-down, CD47tg cell that also expresses a CAR. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、TRAC^Knock-down, CD47tg cell that also expresses a CAR. In certain embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、TRB^Knock-down, CD47tg cell that also expresses a CAR. In certain embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、TRAC^Knock-down、TRB^Knock-down, CD47tg cell that also expresses a CAR. In some embodiments, the cells are modified or engineered as compared to wild-type or control cells (including unmodified or unmodified wild-type cells or control cells). In some embodiments, the wild-type cell or the control cell is the starting material. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to produce an engineered cell.

In some embodiments, the engineered or modified cell is a pluripotent stem cell, an induced pluripotent stem cell, an NK cell differentiated from such pluripotent stem cell and induced pluripotent stem cell, a T cell differentiated from such pluripotent stem cell and induced pluripotent stem cell, or a primary T cell. 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, congenital memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cells. In some embodiments, the primary T cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a memory T cell, a regulatory T cell, a tumor infiltrating lymphocyte, and combinations thereof. Non-limiting examples of NK cells and primary NK cells include immature NK cells and mature NK cells. In some embodiments, the cells are modified or engineered as compared to wild-type or control cells (including unmodified or unmodified wild-type cells or control cells). In some embodiments, the wild-type cell or the control cell is the starting material. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to produce an engineered cell.

In some embodiments, the primary T cells are from a primary T 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 T 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 T 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, primary T cells are harvested from one or more individuals, and in some cases, the primary T cells or primary T cell banks are cultured in vitro. In some embodiments, the primary T cells or primary T cell bank are engineered to exogenously express CD47 and cultured in vitro.

In certain embodiments, the primary T cells or primary T cell pool are engineered to express a Chimeric Antigen Receptor (CAR). The CAR may be any known to those skilled in the art. Useful CARs include those that bind to an antigen selected from the group consisting of CD19, CD20, CD22, CD38, CD123, CD138, and BCMA. In some cases, the CAR is the same as or equivalent to CARs used in FDA-approved CAR-T cell therapies, such as, but not limited to, those used in temozolomide (tisagenlecleucel) and alemtuzole (axicabtagene ciloleucel), or other CARs being studied in a clinical trial.

In some embodiments, the primary T cell or primary T cell pool is engineered to exhibit reduced expression of endogenous T cell receptors as compared to unmodified primary T cells. In certain embodiments, the primary T cells or primary T cell repertoires are engineered to exhibit reduced expression of CTLA-4, PD-1, or both CTLA-4 and PD-1 as compared to unmodified primary T cells. Methods for the genetic modification of cells including T cells are described in detail in, for example, WO2020/018620 and WO2016/183041, the disclosures of which are incorporated herein by reference in their entirety, including tables, appendices, sequence listings and figures.

In some embodiments, the CAR-T cell comprises a CAR selected from the group consisting of: (a) A first generation CAR comprising an antigen binding domain, a transmembrane domain, and a signaling domain; (b) A second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains; (c) A third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains; (d) A fourth generation CAR comprising 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 CAR-T comprises a CAR comprising an antigen binding domain, a transmembrane domain, and one or more signaling domains. In some embodiments, the CAR further comprises a linker. In some embodiments, the CAR comprises a CD19 antigen binding domain. In some embodiments, the CAR comprises a CD20 antigen binding domain. In some embodiments, the CAR comprises a CD22 binding domain. In some embodiments, the CAR comprises a BCMA binding domain. In some embodiments, the CAR comprises a CD28 or CD 8a transmembrane domain. In some embodiments, the CAR comprises a CD 8a signal peptide. In some embodiments, the CAR comprises a Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 15). In some embodiments, the antigen binding domain of the CAR is selected from the group including, but not limited to: (a) An antigen binding domain that targets an antigen characteristic of a tumor cell; (b) An antigen binding domain that targets an antigen characteristic of T cells; (c) An antigen binding domain that targets an antigen that is characteristic of an autoimmune disease/disorder and/or inflammatory disease/disorder; (d) An antigen binding domain that targets an antigen characteristic of senescent cells; (e) An antigen binding domain that targets an antigen characteristic of an infectious disease; and (f) an antigen binding domain that binds to a cell surface antigen of a cell.

In some embodiments, the CAR further comprises one or more linkers. The scFv is typically in the form of two variable domains joined by a flexible peptide sequence or "linker" oriented VH-linker-VL or VL-linker-VH. Any suitable linker known to those skilled in the art may be used for the CAR according to the specification. Examples of suitable linkers include, but are not limited to, GS-based linker sequences and Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 15). In some embodiments, the linker is a GS or gly-ser linker. Exemplary Gly-Ser polypeptide linkers comprise amino acid sequences Ser (Gly ₄Ser)_n, and (Gly ₄Ser)_n and/or (Gly ₄Ser₃)_n) in some embodiments, n=1, in some embodiments, n=2, in some embodiments, n=3, i.e., ser (Gly ₄Ser)₃) in some embodiments, n=4, i.e., ser (Gly ₄Ser)₄) in some embodiments, n=5, in some embodiments, n=6, in some embodiments, n=7, in some embodiments, n=8, in some embodiments, n=9, in some embodiments, n=10, another exemplary Gly-Ser polypeptide linker comprises amino acid sequences Ser (Gly ₄Ser)_n, in some embodiments, n=1, in some embodiments, n=2, in some embodiments, n=3, in another embodiment, n=4, in some embodiments, n=5, n=6, another polypeptide linker in some embodiments, n=6, n=4, in some embodiments, n=2, n=1, n=4, in some embodiments, n=4, n=2, in some embodiments, n=4, n=1, n=4, in some embodiments, n=4, n=3. In some embodiments, n=4. In another embodiment, n=5. In yet another embodiment, n=6. Another exemplary Gly-ser polypeptide linker comprises (Gly ₄Ser₃)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary Gly-ser polypeptide linker comprises (Gly ₃Ser)_n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In another embodiment, n=5. In yet another embodiment, n=6).

In some embodiments, the antigen binding domain is selected from the group consisting of: antibodies, antigen binding portions or fragments thereof, scFv, and Fab. In some embodiments, the antigen binding domain binds to CD19, CD20, CD22, CD38, CD123, CD138, or BCMA. In some embodiments, the antigen binding domain is an anti-CD 19 scFv, such as but not limited to FMC63. In some embodiments, the antigen binding domain is an anti-CD 20 scFv. In some embodiments, the antigen binding domain is an anti-CD 22 scFv. In some embodiments, the antigen binding domain is an anti-BCMA scFv.

In some embodiments, the transmembrane domain comprises a transmembrane domain ：TCRα、TCRβ、TCRζ、CD3ε、CD3γ、CD3δ、CD3ζ、CD4、CD5、CD8α、CD8β、CD9、CD16、CD28、CD45、CD22、CD33、CD34、CD37、CD40、CD40L/CD154、CD45、CD64、CD80、CD86、OX40/CD134、4-1BB/CD137、CD154、FcεRIγ、VEGFR2、FAS、FGFR2B selected from the group of transmembrane regions comprising.

In some embodiments, the signaling domain of the CAR comprises a co-stimulatory domain. For example, the signaling domain may contain a co-stimulatory domain. Or the signaling domain may contain one or more co-stimulatory domains. In certain embodiments, the signaling domain comprises a co-stimulatory domain. In other embodiments, the signaling domain comprises a plurality of co-stimulatory domains. In some cases, when the CAR comprises two or more co-stimulatory domains, the two co-stimulatory domains are not identical. In some embodiments, the co-stimulatory domain comprises two different co-stimulatory domains. In some embodiments, the one co-stimulatory domain enhances cytokine production, CAR-T cell proliferation, and/or CAR-T cell persistence during T cell activation. In some embodiments, the plurality of co-stimulatory domains enhances cytokine production, CAR-T cell proliferation, and/or CAR-T cell persistence during T cell activation.

As described herein, a fourth generation CAR can contain 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 cases, the cytokine gene is an endogenous or exogenous cytokine gene of the engineered cell and/or the hypoimmunogenic cell. In some cases, the cytokine gene encodes a proinflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is selected from the group consisting of IL-1, IL-2, IL-9, IL-12, IL-18, TNF, IFN-gamma, and functional fragments thereof. In some embodiments, the domain that induces cytokine gene expression upon successful signaling of the CAR comprises a transcription factor or a functional domain or fragment thereof.

In some embodiments, the CAR comprises a CD3 zeta (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 other 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 certain 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. In some embodiments, the CAR comprises (i) an anti-CD 19 scFv; (ii) a CD 8a hinge and a transmembrane domain or functional variant thereof; (iii) a 4-1BB co-stimulatory domain or a functional variant thereof; and (iv) a CD3 zeta signaling domain or a functional variant thereof.

Methods for introducing CAR constructs or producing CAR-T cells are well known to those of skill in the art. Details can be found, for example, in Vormittag et al, curr Opin Biotechnol,2018,53,162-181; and Eyquem et al, nature,2017,543,113-117.

In some embodiments, cells derived from primary T cells comprise reduced expression of an endogenous T cell receptor, for example, by disrupting an endogenous T cell receptor gene (e.g., T cell receptor alpha constant region (TRAC) or T cell receptor beta constant region (TRB)). In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the disrupted T cell receptor gene. In some embodiments, the exogenous nucleic acid encoding the polypeptide is inserted at the TRAC or TRB locus.

In some embodiments, the cells derived from primary T cells comprise reduced expression of cytotoxic T lymphocyte-associated protein 4 (CTLA 4) and/or programmed cell death (PD 1). Methods of reducing or eliminating CTLA4, PD1, and expression of both CTLA4 and PD1 can include any methods recognized by those skilled in the art, such as, but not limited to, genetic modification techniques using rare-cutting endonucleases and RNA silencing or RNA interference techniques. Non-limiting examples of rare-cutting endonucleases include any Cas protein, TALEN, zinc finger nuclease, meganuclease, and/or homing endonuclease. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the CTLA4 and/or PD1 locus.

In some embodiments, the CD47 transgene is inserted into a preselected locus in a cell. In some embodiments, the transgene encoding the CAR is inserted into a preselected locus of the cell. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into preselected loci of the cell. The preselected locus may be a safe harbor or a target locus. Non-limiting examples of safe harbor or target loci include, but are not limited to, CCR5 loci, PPP1R12C (also known as AAVS 1) loci, CLYBL loci, and Rosa loci (e.g., rosa26 loci). Non-limiting examples of target loci include, but are not limited to, the CXCR4 locus, the albumin locus, the SHS231 locus, the F3 locus (also known as CD 142), the MICA locus, the MICB locus, the LRP1 locus (also known as CD91 locus), the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus. The CD47 transgene may be inserted into intron 1 or 2 of PPP1R12C (i.e., AAVS 1) or CCR 5. The CD47 transgene may be inserted into exon 1 or 2 or 3 of CCR 5. The CD47 transgene may be inserted into intron 2 of CLYBL. The CD47 transgene may be inserted into a 500bp window in Ch-4:58,976,613 (i.e., SHS 231). The CD47 transgene may be inserted into any suitable region mentioned above that allows for expression of an exogenous safe harbor or target locus, including, for example, an intron, exon, or coding sequence region in the safe harbor or target locus. In some embodiments, the preselected locus is selected from the group consisting of a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In some embodiments, the preselected locus is a B2M locus. In some embodiments, the preselected locus is a CIITA locus. In some embodiments, the preselected locus is a TRAC locus. In some embodiments, the preselected locus is a TRB locus.

In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the same locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into different loci. In many cases, the CD47 transgene is inserted into a safe harbor or target locus. In many cases, the transgene encoding the CAR is inserted into a safe harbor or target locus. In some cases, the CD47 transgene is inserted into the B2M locus. In some cases, the transgene encoding the CAR is inserted into the B2M locus. In some cases, the CD47 transgene is inserted into the CIITA locus. In some cases, the transgene encoding the CAR is inserted into the CIITA locus. In certain instances, the CD47 transgene is inserted into the TRAC locus. In certain cases, the transgene encoding the CAR is inserted into the TRAC locus. In many other cases, the CD47 transgene is inserted into the TRB locus. In many other cases, the transgene encoding the CAR is inserted into the TRB locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into a safe harbor or target locus (e.g., CCR5 locus, CXCR4 locus, PPP1R12C locus, albumin locus, SHS231 locus, CLYBL locus, rosa locus, F3 (CD 142) locus, MICA locus, MICB locus, LRP1 (CD 91) locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus, and KDM5D locus).

In certain embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into a safe harbor or target locus. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are under the control of a single promoter and are inserted into a safe harbor or target locus. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are under the control of their own promoters and are inserted into a safe harbor or target locus. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the TRAC locus. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are under the control of a single promoter and are inserted into the TRAC locus. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are under the control of their own promoters and are inserted into the TRAC locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the TRB locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are under the control of a single promoter and are inserted into the TRB locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are under the control of their own promoters and are inserted into the TRB locus. In other embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the B2M locus. In other embodiments, the CD47 transgene and the transgene encoding the CAR are under the control of a single promoter and are inserted into the B2M locus. In other embodiments, the CD47 transgene and the transgene encoding the CAR are under the control of their own promoters and are inserted into the B2M locus. In various embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the CIITA locus. In various embodiments, the CD47 transgene and the transgene encoding the CAR are under the control of a single promoter and are inserted into the CIITA locus. In various embodiments, the CD47 transgene and the transgene encoding the CAR are under the control of their own promoters and are inserted into the CIITA locus.

In some cases, the promoter that controls the expression of any of the transgenes is a constitutive promoter. In other cases, the promoter of any transgene is an inducible promoter. In some embodiments, the promoter is an EF 1a promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, both the CD47 transgene and the transgene encoding the CAR are under the control of a constitutive promoter. In some embodiments, both the CD47 transgene and the transgene encoding the CAR are under the control of an inducible promoter. In some embodiments, the CD47 transgene is under the control of a constitutive promoter, and the transgene encoding the CAR is under the control of an inducible promoter. In some embodiments, the CD47 transgene is under the control of an inducible promoter, and the transgene encoding the CAR is under the control of a constitutive promoter. In various embodiments, the CD47 transgene is under the control of an EF 1a promoter, and the transgene encoding the CAR is under the control of an EF 1a promoter. In some embodiments, the CD47 transgene is under the control of a CAG promoter, and the transgene encoding the CAR is under the control of a CAG promoter. In some embodiments, the CD47 transgene is under the control of a CAG promoter, and the transgene encoding the CAR is under the control of an EF 1a promoter. In some embodiments, the CD47 transgene is under the control of the EF 1a promoter, and the transgene encoding the CAR is under the control of the CAG promoter. In some embodiments, expression of the CD47 transgene and the transgene encoding the CAR is under the control of a single EF 1a promoter. In some embodiments, expression of the CD47 transgene and the transgene encoding the CAR is under the control of a single CAG promoter.

In another embodiment, the disclosure disclosed herein relates to pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (ipscs)), differentiated cells derived from such pluripotent stem cells (e.g., low immune (HIP) T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells), and primary T cells that overexpress CD47 (such as exogenously expressed CD47 protein), have reduced expression or lack expression of MHC class I and/or MHC class II human leukocyte antigen molecules, and have reduced expression or lack expression of T Cell Receptor (TCR) complexes. In some embodiments, low immune (HIP) T cells and primary T cells overexpress CD47 (such as exogenously expressed CD47 protein), have reduced expression or lack of expression of one or more MHC class I and/or MHC class II human leukocyte antigen molecules, and have reduced expression or lack of expression of a T Cell Receptor (TCR) complex.

In some embodiments, pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (ipscs)), differentiated cells derived from such pluripotent stem cells (e.g., low immune (HIP) T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells), and primary T cells overexpress CD47 and include genomic modifications of the B2M gene. In some embodiments, pluripotent stem cells, differentiated cells derived from such pluripotent stem cells, and primary T cells overexpress CD47, and include genomic modifications of the CIITA gene. In some embodiments, the pluripotent stem cells, differentiated cells derived from such pluripotent stem cells (such as, but not limited to, T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells) are PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、CIITA^-/-, CD47tg cells. In some embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down, CD47tg cell. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47, and include genomic modifications of the PCDH11Y gene. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47, and include genomic modifications of the NLGN4Y gene. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47, and include genomic modifications of the TRAC gene. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47, and include genomic modifications of the TRB gene. In some embodiments, the pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47, and comprise one or more genomic modifications selected from the group consisting of PCDH11Y, NLGN4Y, B2M, CIITA, TRAC and TRB genes. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47, and include genomic modifications of PCDH11Y, NLGN4Y, B2M, CIITA and TRAC genes. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47, and include genomic modifications of PCDH11Y, NLGN4Y, B2M, CIITA and TRB genes. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47, and include genomic modifications of PCDH11Y, NLGN4Y, B2M, CIITA, TRAC and TRB genes. In certain embodiments, the pluripotent stem cells, differentiated cells derived from such pluripotent stem cells, and primary T cells are PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、CIITA^-/-、TRAC^-/-, CD47tg cells. In certain embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、TRAC^-/-, CD47tg cell. In certain embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、CIITA^-/-、TRB^-/-, CD47tg cell. In certain embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、TRB^-/-, CD47tg cell. In certain embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、CIITA^-/-、TRAC^-/-、TRB^-/-, CD47tg cell. In certain embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、TRAC^-/-、TRB^-/-, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion}, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion}, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion}, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion}, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion}, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion}, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down、TRAC^Knock-down, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down、TRB^Knock-down, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down、TRAC^Knock-down、TRB^Knock-down, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、TRAC^Knock-down, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、TRB^Knock-down, CD47tg cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、TRAC^Knock-down、TRB^Knock-down, CD47tg cell. In some embodiments, the engineered or modified cell is a pluripotent stem cell (e.g., an embryonic stem cell or an induced pluripotent stem cell), a T cell differentiated from such pluripotent stem cell, or a primary T cell. 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, congenital memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cells. In some embodiments, the cells are modified or engineered as compared to wild-type or control cells (including unmodified or unmodified wild-type cells or control cells). In some embodiments, the wild-type cell or the control cell is the starting material. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to produce an engineered cell.

In some embodiments, the CD47 transgene is inserted into a preselected locus in a cell. The preselected locus may be a safe harbor or a target locus. Non-limiting examples of safe harbor or target loci include CCR5 loci, CXCR4 loci, PPP1R12C loci, albumin loci, SHS231 loci, CLYBL loci, rosa loci, F3 (CD 142) loci, MICA loci, MICB loci, LRP1 (CD 91) loci, HMGB1 loci, ABO loci, RHD loci, FUT1 loci, and KDM5D loci. In some embodiments, the preselected locus is a TRAC locus. In some embodiments, the CD47 transgene is inserted into a safe harbor or target locus (e.g., CCR5 locus, CXCR4 locus, PPP1R12C locus, albumin locus, SHS231 locus, CLYBL locus, rosa locus, F3 (CD 142) locus, MICA locus, MICB locus, LRP1 (CD 91) locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus, and KDM5D locus). In certain embodiments, the CD47 transgene is inserted into the B2M locus. In certain embodiments, the CD47 transgene is inserted into the B2M locus. In certain embodiments, the CD47 transgene is inserted into the TRAC locus. In certain embodiments, the CD47 transgene is inserted into the TRB locus.

In some cases, expression of the CD47 transgene is under the control of a constitutive promoter. In other cases, expression of the CD47 transgene is under the control of an inducible promoter. In some embodiments, the promoter is an EF1 alpha (EF 1 a) promoter. In some embodiments, the promoter is a CAG promoter.

In yet another embodiment, the disclosure disclosed herein relates to pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (ipscs)), T cells derived from such pluripotent stem cells (e.g., low immune (HIP) T cells), and primary T cells having reduced expression or lack of expression of one or more Y chromosome genes and MHC class I and/or MHC class II human leukocyte antigen molecules, and having reduced expression or lack of expression of a T Cell Receptor (TCR) complex. In some embodiments, the cells have reduced or lack expression of one or more Y chromosome genes and MHC class I antigen molecules, MHC class II antigen molecules, and TCR complexes.

In some embodiments, pluripotent stem cells (e.g., ipscs), differentiated cells derived from such pluripotent stem cells (e.g., T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells) and primary T cells include genomic modification or knockdown of the PCDH11Y gene. In some embodiments, pluripotent stem cells (e.g., ipscs), differentiated cells derived from such pluripotent stem cells (e.g., T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells) and primary T cells include genomic modification or knockdown of the NLGN4Y gene. In some embodiments, pluripotent stem cells (e.g., ipscs), differentiated cells derived from such pluripotent stem cells (e.g., T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells) and primary T cells include genomic modification or knockdown of B2M genes. In some embodiments, pluripotent stem cells (e.g., ipscs), differentiated cells derived from such pluripotent stem cells (e.g., T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells) and primary T cells include genomic modification or knockdown of CIITA genes. In some embodiments, the cells comprising ipscs and differentiated cells derived from such pluripotent stem cells (such as, but not limited to, T cells, NK cells, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, hepatocytes, islet cells, and retinal pigment epithelial cells) are PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、CIITA^-/- cells. In some embodiments, the cell is a PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/- cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion} cell. In some embodiments, the cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion} cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down cell. In some embodiments, the cell is a PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down cell. In some embodiments, pluripotent stem cells (e.g., ESC or iPSC), T cells differentiated from such pluripotent stem cells, and primary T cells include genomic modification or knockdown of the PCDH11Y gene. In some embodiments, pluripotent stem cells (e.g., ESC or iPSC), T cells differentiated from such pluripotent stem cells, and primary T cells include genomic modification or knockdown of NLGN4Y gene. In some embodiments, pluripotent stem cells (e.g., ESC or iPSC), T cells differentiated from such pluripotent stem cells, and primary T cells include genomic modification or knockdown of the TRAC gene. In some embodiments, pluripotent stem cells (e.g., ipscs), T cells differentiated from such pluripotent stem cells, and primary T cells include genomic modifications or knockdown of the TRB gene. In some embodiments, pluripotent stem cells (e.g., ipscs), T cells differentiated from such pluripotent stem cells, and primary T cells include one or more genomic modifications or knockdown selected from the group consisting of B2M, CIITA and TRAC genes. In some embodiments, pluripotent stem cells (e.g., ipscs), T cells differentiated from such pluripotent stem cells, and primary T cells include one or more genomic modifications or knockdown selected from the group consisting of PCDH11Y, NLGN4Y, B2M, CIITA and TRB genes. In some embodiments, pluripotent stem cells (e.g., ipscs), T cells differentiated from such pluripotent stem cells, and primary T cells include one or more genomic modifications or knockdown selected from the group consisting of PCDH11Y, NLGN4Y, B2M, CIITA, TRAC and TRB genes. In certain embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、CIITA^-/-、TRAC^-/- cells. In certain embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、TRAC^-/- cells. In certain embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、CIITA^-/-、TRB^-/- cells. In certain embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、TRB^-/- cells. In certain embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、CIITA^-/-、TRAC^-/-、TRB^-/- cells. In certain embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^-/-、NLGN4Y^-/-、B2M^-/-、TRAC^-/-、TRB^-/- cells. In some embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion} cells. In some embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion} cells. In some embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cells. In some embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cells. In some embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cells. In some embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cells. In some embodiments, the cells comprising ESCs, iPSCs, differentiated T cells from such ESCs, iPSCs, and primary T cells are PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down、TRAC^Knock-down cells. In some embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down、TRB^Knock-down cells. In some embodiments, the cells comprising ESCs, iPSCs, differentiated T cells from such ESCs, iPSCs, and primary T cells are PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、CIITA^Knock-down、TRAC^Knock-down、TRB^Knock-down cells. In some embodiments, the cells comprising ESCs, iPSCs, differentiated T cells from such ESCs, iPSCs, and primary T cells are PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、TRAC^Knock-down cells. In some embodiments, the cells comprising ipscs, T cells differentiated from such ipscs, and primary T cells are PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、TRB^Knock-down cells. In some embodiments, the cells comprising ESCs, iPSCs, differentiated T cells from such ESCs, iPSCs, and primary T cells are PCDH11Y ^Knock-down、NLGN4Y^Knock-down、B2M^Knock-down、TRAC^Knock-down、TRB^Knock-down cells. In some embodiments, the modified cell is a pluripotent stem cell, an induced pluripotent stem cell, a T cell or a primary T cell differentiated from such pluripotent stem cell and induced pluripotent stem cell. 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, congenital memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cells.

In some embodiments, the cells are modified or engineered as compared to wild-type or control cells (including unmodified or unmodified wild-type cells or control cells). In some embodiments, the wild-type cell or the control cell is the starting material. In some embodiments, the starting material is otherwise modified or engineered to have reduced expression or lack thereof of one or more Y chromosome genes (including, but not limited to, PCDH11Y and/or NLGN 4Y). Reduction of PCDH11Y and/or NLGN4Y expression can be achieved, for example, by direct targeting of PCDH11Y and NLGN4Y genes; and/or by targeting components critical to their transcription, translation or protein stability.

The cells of the present disclosure exhibit reduced or lack expression of MHC class I antigen molecules, MHC class II antigen molecules, and/or TCR complexes. Lowering the expression of one or more MHC class I and/or class II HLA molecules can 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 MHC-II genes; (2) Removal of B2M, which will prevent surface transport of all MHC-I molecules; (3) Removal of CIITA, which will prevent surface transport of all MHC-II molecules; and/or (4) deletion of MHC enhancer components critical for HLA expression, such as LRC5, RFX5, RFXANK, RFXAP, IRFl, NF-Y (including NFY-A, NFY-B, NFY-C) and CIITA.

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

In some embodiments, the cells disclosed herein (including, but not limited to, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from such stem cells, and primary T cells) do not express one or more human leukocyte antigen molecules (e.g., HLA-A, HLa-B, HLA-C, HLA-DP, HLa-DQ, and/or HLa-DR) corresponding to MHC-I molecules and/or MHC-II molecules, and are therefore characterized as being hypoimmunogenic. For example, in certain embodiments, the disclosed pluripotent stem cells and induced pluripotent stem cells have been modified such that the stem cells or differentiated stem cells prepared therefrom do not express or exhibit reduced expression of one or more of the following MHC-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 knockdown HLA-A genes, HLA-B genes, and/or HLA-C genes may exhibit reduced or eliminated expression of each knockdown gene.

In some embodiments, the guide RNA, shRNA, siRNA or miRNA that allows simultaneous deletion of all MHC class I alleles by targeting conserved regions in HLA genes is identified as HLA razors. In some embodiments, the gRNA is part of a CRISPR system. In an alternative embodiment, the gRNA is part of a TALEN system. In some embodiments, HLA razors targeting conserved regions identified in HLA are described in WO 2016183041. In some embodiments, a plurality of HLA razors targeting the identified conserved regions are utilized. It is generally understood that any guide, siRNA, shRNA or miRNA that targets a conserved region in HLA can act as an HLA Razor.

The provided methods can be used to inactivate or eliminate MHC class I molecule expression and/or MHC class II molecule expression in cells such as, but not limited to, pluripotent stem cells, differentiated cells, and primary T cells. 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 genes involved in immune responses in cells (e.g., by deleting genomic DNA of genes involved in immune responses or by inserting genomic DNA into such genes such that gene expression is affected). In certain embodiments, genome editing techniques or other gene regulation techniques are used to insert tolerance-inducing factors in human cells so that they and differentiated cells prepared therefrom are hypoimmunogenic cells. Thus, engineered and/or hypoimmunogenic cells have reduced or eliminated MHC I molecule expression and/or MHC II molecule expression. In some embodiments, the cells are non-immunogenic (e.g., do not induce an innate and/or adaptive immune response) in the recipient subject.

In some embodiments, the cell comprises a modification to increase expression of CD47 and one or more factors selected from the group consisting of: 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 cell comprises genomic modifications of one or more target polynucleotide sequences that regulate expression of MHC class I molecules, MHC class II molecules, or MHC class I and MHC class II molecules. In some embodiments, the gene editing system is used to modify one or more target polynucleotide sequences. In some embodiments, the RNAi system is used to knock down the expression of one or more target polynucleotide sequences. In some embodiments, the targeting polynucleotide sequence is one or more selected from the group consisting of B2M, CIITA and NLRC 5. In some embodiments, the cell comprises a genetic editing modification to the B2M gene. In some embodiments, the cell comprises a genetic editing modification to the CIITA gene. In some embodiments, the cell comprises a genetic editing modification to the NLRC5 gene. In some embodiments, the cells comprise genetic editing modifications to B2M and CIITA genes. 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 various embodiments, the cells comprise genetic editing modifications to the B2M, CIITA and NLRC5 genes. In certain embodiments, the genome of the cell has been altered to reduce or delete key components of HLA expression. In some embodiments, the cells are modified or engineered as compared to wild-type or control cells (including unmodified or unmodified wild-type cells or control cells). In some embodiments, the wild-type cell or the control cell is the starting material. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to produce an engineered cell.

In some embodiments, the disclosure provides a cell (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell (such as a cardiac cell, a neural cell, a brain endothelial cell, a dopaminergic neuron, a glial progenitor cell, an endothelial cell, a thyroid cell, a hepatic cell, an islet cell, or a retinal pigment epithelial cell), a hematopoietic stem cell, a primary NK cell, a CAR-NK cell, a primary T cell, or a CAR-T cell), or a population thereof, comprising a genome in which a gene has been edited to delete a contiguous segment of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof. In certain embodiments, the disclosure provides a cell (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell (such as a cardiac cell, a neural cell, a brain endothelial cell, a dopaminergic neuron, a glial progenitor cell, an endothelial cell, a thyroid cell, a hepatic cell, an islet cell, or a retinal pigment epithelial cell), a hematopoietic stem cell, a primary NK cell, a CAR-NK cell, a primary T cell, or a CAR-T cell), or a population thereof, comprising a genome in which a gene has been edited to delete a contiguous segment of genomic DNA, thereby reducing or eliminating surface expression of MHC class II molecules in the cell or population thereof. In various embodiments, the disclosure provides a cell (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell (such as a cardiac cell, a neural cell, a brain endothelial cell, a dopaminergic neuron, a glial progenitor cell, an endothelial cell, a thyroid cell, a hepatic cell, an islet cell, or a retinal pigment epithelial cell), a hematopoietic stem cell, a primary NK cell, a CAR-NK cell, a primary T cell, or a CAR-T cell), or a population thereof, comprising a genome in which one or more genes have been edited to delete a contiguous segment of genomic DNA, thereby reducing or eliminating surface expression of MHC class II molecules in the cell or population thereof.

In certain embodiments, expression of one or more MHC class I molecules and/or MHC class II molecules (including one or more MHC class I and/or class II HLA 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, described herein are genetically edited cells (e.g., modified human cells) comprising an exogenous CD47 protein and an inactivated or modified CIITA gene sequence, and in some cases, additional genetic modifications that inactivate or modify a B2M gene sequence. In some embodiments, described herein are genetically edited cells comprising an exogenous CD47 protein and an inactivated or modified CIITA gene sequence, and in some cases, additional genetic modifications that inactivate or modify an NLRC5 gene sequence. In some embodiments, described herein are genetically edited cells comprising an exogenous CD47 protein and an inactivated or modified B2M gene sequence, and in some cases, additional genetic modifications that inactivate or modify an NLRC5 gene sequence. In some embodiments, described herein are genetically edited cells comprising an exogenous CD47 protein and an inactivated or modified B2M gene sequence, and in some cases, additional genetic modifications that inactivate or modify the CIITA gene sequence and the NLRC5 gene sequence.

Provided herein are cells that exhibit modifications of one or more targeting polynucleotide sequences that regulate expression of any one of the following: (a) MHC I antigen molecules, (b) MHC II antigen molecules, (c) TCR complexes, (d) both MHC I and II antigen molecules, and (e) MHC I and II antigen molecules and TCR complexes. In certain embodiments, the modification comprises increasing expression of CD 47. In some embodiments, the cell comprises an exogenous or recombinant CD47 polypeptide. In certain embodiments, the modification comprises expression of a chimeric antigen receptor. In some embodiments, the cell comprises an exogenous or recombinant chimeric antigen receptor polypeptide.

In some embodiments, the cell comprises genomic modifications of one or more targeting polynucleotide sequences that regulate expression of one or more MHC I antigen molecules, MHC II antigen molecules, and/or TCR complexes. In some embodiments, the gene editing system is used to modify one or more targeting polynucleotide sequences. In some embodiments, the polynucleotide sequence targets one or more genes selected from the group consisting of B2M, CIITA, TRAC and TRB. In certain embodiments, the genomes of T cells (e.g., T cells differentiated from low immunogenicity iPSCs and primary T cells) have been altered to reduce or delete key components of HLA and TCR expression, such as HLA-A antigen, HLA-B antigen, HLA-C antigen, HLA-DP antigen, HLA-DQ antigen, HLA-DR antigen, TCR-alpha and TCR-beta.

In some embodiments, the disclosure provides a cell or population thereof comprising a genome in which a gene has been edited to delete a contiguous piece of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof. In certain embodiments, the present disclosure provides a cell or population thereof comprising a genome in which a gene has been edited to delete a contiguous piece of genomic DNA, thereby reducing or eliminating surface expression of MHC class II molecules in the cell or population thereof. In certain embodiments, the present disclosure provides a cell or population thereof comprising a genome in which a gene has been edited to delete a contiguous piece of genomic DNA, thereby reducing or eliminating surface expression of a TCR molecule in the cell or population thereof. In various embodiments, the present disclosure provides a cell or population thereof comprising a genome in which one or more genes have been edited to delete a continuous piece of genomic DNA, thereby reducing or eliminating surface expression of one or more MHC class I and class II molecules and TCR complex molecules in the cell or population thereof.

In some embodiments, the cells and methods described herein include genome editing human cells to cleave CIITA gene sequences and editing the genome of such cells to alter one or more additional target polynucleotide sequences, such as, but not limited to PCDH11Y, NLGN4Y, B2M, TRAC and TRB. In some embodiments, the cells and methods described herein include genome editing human cells to cleave B2M gene sequences and editing the genome of such cells to alter one or more additional target polynucleotide sequences, such as, but not limited to PCDH11Y, NLGN4Y, CIITA, TRAC and TRB. In some embodiments, the cells and methods described herein include genome editing human cells to cleave the TRAC gene sequence and editing the genome of such cells to alter one or more additional target polynucleotide sequences, such as, but not limited to PCDH11Y, NLGN4Y, B2M, CIITA and TRB. In some embodiments, the cells and methods described herein include genome editing human cells to cleave TRB gene sequences and editing the genome of such cells to alter one or more additional target polynucleotide sequences, such as, but not limited to PCDH11Y, NLGN4Y, B2M, CIITA and TRAC.

Provided herein are low-immunogenic stem cells comprising reduced expression of PCDH11Y and/or NLGN4Y, and HLA-A, HLa-B, HLA-C, CIITA, TCR-a, and TCR- β relative to wild-type stem cells, the low-immunogenic stem cells further comprising a set of exogenous polynucleotides comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR), wherein the first and/or second exogenous polynucleotides are inserted into a particular locus of at least one allele of the cells. Also provided herein are low-immunogenicity primary T cells (including any subtype of primary T cells) comprising reduced expression of PCDH11Y and/or NLGN4Y and HLA-A, HLa-B, HLA-C, CIITA, TCR-a, and TCR- β relative to wild-type primary T cells, the low-immunogenicity stem cells further comprising a set of exogenous polynucleotides comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR), wherein the first and/or second exogenous polynucleotides are inserted into specific loci of at least one allele of the cells. Also provided herein are low-immunogenic T cells differentiated from low-immunogenicity induced pluripotent stem cells comprising reduced expression of PCDH11Y and/or NLGN4Y, and HLA-A, HLa-B, HLA-C, CIITA, TCR-a, and TCR- β relative to wild-type primary T cells, the low-immunogenic stem cells further comprising a set of exogenous polynucleotides comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR), wherein the first and/or second exogenous polynucleotides are inserted into a particular locus of at least one allele of the cells.

In some embodiments, the engineered cell population, upon administration to a patient, evades NK cell-mediated cytotoxicity. In some embodiments, the engineered cell population evades NK cell-mediated cytotoxicity through one or more subsets of NK cells. In some embodiments, the engineered cell population is protected from cell lysis of NK cells (including immature and/or mature NK cells) after administration to a patient. In some embodiments, the engineered cell population evades phagocytosis by macrophages after administration to a patient. In some embodiments, the engineered cell population does not induce an innate and/or adaptive immune response to the cells after administration to the patient.

In some embodiments, the cells described herein comprise a safety switch. The term "safety switch" as used herein refers to a system for controlling the expression of a gene or protein of interest which, when down-regulated or up-regulated, results in the clearance or death of a cell, for example, by recognition by the host immune system. The safety switch may be designed to be triggered by an exogenous molecule upon occurrence of an adverse clinical event. Safety switches can be designed by regulating the expression of DNA, RNA and protein levels. The safety switch includes a protein or molecule that allows control of cellular activity in response to an adverse event. In one embodiment, the safety switch is a "kill switch" that is expressed in an inactive state and is fatal to the cell expressing the safety switch when the switch is activated by a selective externally provided agent. In one embodiment, the safety switch gene is cis-acting relative to the gene of interest in the construct. Activation of the safety switch causes the cell to kill itself alone or to kill itself and neighboring cells by apoptosis or necrosis. In some embodiments, the cells described herein (e.g., stem cells, induced pluripotent stem cells, hematopoietic stem cells, primary cells, or differentiated cells, including but not limited to T cells, CAR-T cells, NK cells, and/or CAR-NK cells) comprise a safety switch.

In some embodiments, the safety switch comprises a therapeutic agent that inhibits or blocks the interaction of CD47 with sirpa. In some aspects, the CD 47-sirpa blocker is an agent that neutralizes, blocks, antagonizes, or interferes with the cell surface expression of CD47, sirpa, or both. In some embodiments, the CD 47-sirpa blocker inhibits or blocks the interaction of CD47, sirpa, or both. In some embodiments, the CD 47-sirpa blocker (e.g., CD 47-sirpa blocker, inhibitor, reducing agent, antagonist, neutralizing agent, or interfering agent) comprises an agent selected from the group consisting of: antibodies or fragments thereof that bind CD47, bispecific antibodies that bind CD47, immunocytokine fusion proteins that bind CD47, fusion proteins that contain CD47, antibodies or fragments thereof that bind sirpa, bispecific antibodies that bind sirpa, immunocytokine fusion proteins that bind sirpa, fusion proteins that contain sirpa, and combinations thereof.

In some embodiments, the cells described herein comprise a "suicide gene" (or "suicide switch"). Suicide genes can cause death of hypoimmunogenic cells if they grow and divide in an undesirable manner. Suicide gene ablation pathways include suicide genes in gene transfer vectors that 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. In some embodiments, the cells described herein (e.g., stem cells, induced pluripotent stem cells, hematopoietic stem cells, primary cells, or differentiated cells, including but not limited to T cells, CAR-T cells, NK cells, and/or CAR-NK cells) comprise a suicide gene.

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.

B.CIITA

In some embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of MHC class II genes by targeting and modulating (e.g., reducing or eliminating) expression of a class II transactivator (CIITA). In some embodiments, modulation is performed using a gene editing system (e.g., CRISPR/Cas system).

CIITA is a member of the LR or Nucleotide Binding Domain (NBD) Leucine Rich Repeat (LRR) protein family and regulates MHC II transcription by association with MHC enhancers.

In some embodiments, the target polynucleotide sequences of the present disclosure are variants 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 cells described herein comprise a genetic modification at a locus encoding a CIITA protein. In other words, the cell comprises a genetic modification at the CIITA locus. In some cases, the nucleotide sequences encoding the CIITA proteins are listed in refseq. No. nm_000246.4 and NCBI Genbank No. u 18259. In some cases, the CIITA locus is described in NCBI Gene ID No. 4261. In some cases, the amino acid sequence of CIITA is depicted as NCBI GenBank No. aaa88861.1. Additional descriptions of CIITA proteins and loci can be found in Uniprot No. p33076, HGNC ref No.7067 and OMIM ref No.600005.

In some embodiments, the engineered and/or hypoimmunogenic cells outlined herein comprise genetic modifications that target the CIITA gene. In some embodiments, the genetic modification to target the CIITA gene by the rare-cutting endonuclease comprises 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 for specifically targeting the CIITA gene is selected from the group consisting of SEQ ID NOS 5184-36352 of Table 12 of WO2016183041, which is incorporated herein by reference. In some embodiments, the cells have a reduced ability to induce an innate and/or adaptive immune response in the recipient subject. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the CIITA gene.

In some embodiments, the engineered and/or hypoimmunogenic cells outlined herein comprise a knockout of CIITA expression such that the cells are CIITA ^-/-. In some embodiments, the engineered and/or hypoimmunogenic cells outlined herein introduce an insertion deletion into the CIITA locus such that the cells are CIITA ^{indel of insertion / indel of insertion}. In some embodiments, the engineered and/or hypoimmunogenic cells outlined herein comprise a knock down of CIITA expression such that the cells are CIITA ^Knock-down.

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

C.B2M

In some embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) expression of the helper strand B2M. In some embodiments, modulation is performed using a gene editing system (e.g., CRISPR/Cas system).

By modulating (e.g., reducing or deleting) the expression of B2M, surface trafficking of MHC-I molecules is blocked and cells are rendered hypoimmunogenic. In some embodiments, the cells have a reduced ability to induce an innate and/or adaptive immune response in the recipient subject.

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

In some embodiments, the cells described herein comprise a genetic modification at a locus encoding a B2M protein. In other words, the cell comprises a genetic modification at the B2M locus. In some cases, the nucleotide sequences encoding the B2M protein are listed in refseq.no. nm_004048.4 and Genbank No. ab 021288.1. In some cases, the B2M locus is described in NCBI Gene ID No. 567. In some cases, the amino acid sequence of B2M is depicted as NCBI GenBank No. baa 35182.1. Additional description of B2M proteins and loci can be found in Uniprot No. p61769, HGNC ref No.914 and OMIM ref No.109700.

In some embodiments, the engineered and/or hypoimmunogenic cells outlined herein comprise genetic modifications that target the B2M gene. In some embodiments, the genetic modification to target the B2M gene by the rare-cutting endonuclease comprises 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 for specifically targeting the B2M gene is selected from the group consisting of SEQ ID NOs 81240-85644 of table 15 of WO2016183041 (which is incorporated herein by reference). In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the B2M gene.

Assays to test whether the B2M gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the B2M gene and reduction in HLA-I expression by PCR can be determined by FACS analysis. In another embodiment, western blot of cell lysates detected with antibodies against B2M protein is used to detect B2M protein expression. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating genetic modifications.

In some embodiments, the engineered and/or hypoimmunogenic cells outlined herein comprise a knockout of B2M expression such that the cells are B2M ^-/-. In some embodiments, the engineered and/or low immunogenicity cells outlined herein introduce an insertion deletion into the B2M locus such that the cells are B2M ^{indel of insertion / indel of insertion}. In some embodiments, the engineered and/or hypoimmunogenic cells outlined herein comprise a knock down of B2M expression such that the cells are B2M ^Knock-down.

D.NLRC5

In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) expression of an NLR family CARD domain (NLRC 5) containing 5/NOD27/CLR 16.1. In some embodiments, modulation is performed using a gene editing system (e.g., CRISPR/Cas system).

NLRC5 is a key regulator of MHC-I mediated immune responses, and like CIITA, NLRC5 is highly inducible by IFN-gamma and translocatable into the nucleus. NLRC5 activates the promoter of the MHC-I gene and induces transcription of MHC-I and related genes involved in MHC-I antigen presentation.

In some embodiments, the target polynucleotide sequence is a variant of NLRC 5. In some embodiments, the target polynucleotide sequence is a homolog of NLRC 5. In some embodiments, the target polynucleotide sequence is an ortholog of NLRC 5.

In some embodiments, the reduced or eliminated expression of NLRC5 reduces or eliminates expression of one or more of the following MHC I molecules: HLA-A, HLA-B and HLA-C.

In some embodiments, the cells outlined herein comprise a genetic modification that targets the NLRC5 gene. In some embodiments, the genetic modification to target the NLRC5 gene by a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the NLRC5 gene. In some embodiments, at least one guide ribonucleic acid sequence for specifically targeting the NLRC5 gene is selected from the group consisting of appendix 3 of WO2016183041 or SEQ ID NOS: 36353-81239 of Table 14, the disclosure of which is incorporated herein by reference in its entirety.

Assays to test whether the NLRC5 gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the NLRC5 gene and reduction in HLA-I expression by PCR can be determined by FACS analysis. In another embodiment, western blot of cell lysates detected with antibodies to NLRC5 protein is used to detect NLRC5 protein expression. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating genetic modifications.

In some embodiments, the engineered and/or low immunogenicity cells outlined herein comprise a knockout of NLRC5 expression such that the cells are NLRC5 ^-/-. In some embodiments, the engineered and/or low immunogenicity cells outlined herein introduce an insertion deletion into the NLRC5 locus such that the cell is NLRC5 ^{indel of insertion / indel of insertion}. In some embodiments, the engineered and/or low immunogenicity cells outlined herein comprise knockdown of NLRC5 expression such that the cells are NLRC5 ^Knock-down.

E.TRAC

In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) the expression of TCR genes (including TRAC genes) by targeting and modulating (e.g., reducing or eliminating) the expression of T cell receptor alpha chain constant regions. In some embodiments, modulation is performed using a gene editing system (e.g., CRISPR/Cas system).

By modulating (e.g., reducing or deleting) expression of TRAC, surface transport of TCR molecules is blocked. In some embodiments, the ability of the cell to induce an innate and/or adaptive immune response in the recipient subject is also reduced.

In some embodiments, the target polynucleotide sequences of the present disclosure are variants of TRAC. In some embodiments, the target polynucleotide sequence is a homolog of TRAC. In some embodiments, the target polynucleotide sequence is an ortholog of TRAC.

In some embodiments, reduced or eliminated expression of TRAC reduces or eliminates TCR surface expression.

In some embodiments, the cells (such as, but not limited to, pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells) comprise a genetic modification at a locus encoding a TRAC protein. In other words, the cell comprises a genetic modification at the TRAC locus. In some cases, the nucleotide sequence encoding the TRAC protein is set forth in Genbank No. X02592.1. In some cases, the TRAC locus is described in RefSeq.No. NG_001332.3 and NCBI Gene IDNo. 28755. In some cases, the amino acid sequence of TRAC is depicted as Uniprot No. P01848. Additional descriptions of TRAC proteins and loci can be found in UniprotNo. P01848, HGNC Ref.No.12029, and OMIM Ref.No.186880.

In some embodiments, the engineered and/or hypoimmunogenic cells outlined herein comprise a genetic modification that targets the TRAC gene. In some embodiments, the genetic modification to target the TRAC gene by a rare-cutting endonuclease comprises 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 for specifically targeting the TRAC gene is selected from the group consisting of SEQ ID NOS 532-609 and 9102-9797 of US20160348073 (which is incorporated herein by reference).

Assays to test whether the TRAC gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the TRAC gene and reduction in TCR expression by PCR can be determined by FACS analysis. In another embodiment, the expression of TRAC protein is detected using Western blotting of cell lysates detected with antibodies directed against TRAC protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating genetic modifications.

In some embodiments, the engineered and/or low immunogenicity cells outlined herein comprise a knockout of TRAC expression such that the cells are TRAC ^-/-. In some embodiments, the engineered and/or low immunogenicity cells outlined herein introduce an insertion deletion into the TRAC locus such that the cells are TRAC ^{indel of insertion / indel of insertion}. In some embodiments, the engineered and/or low immunogenicity cells outlined herein comprise knockdown of TRAC expression such that the cells are TRAC ^Knock-down.

F.TRB

In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of TCR genes, including genes encoding T cell antigen receptor beta chains (e.g., TRB, TRBC, or TCRB genes), by targeting and modulating (e.g., reducing or eliminating) expression of T cell receptor beta chain constant regions. In some embodiments, modulation is performed using a gene editing system (e.g., CRISPR/Cas system).

By modulating (e.g., reducing or deleting) the expression of TRB, surface transport of TCR molecules is blocked. In some embodiments, the ability of the cell to induce an innate and/or adaptive immune response in the recipient subject is also reduced.

In some embodiments, the target polynucleotide sequences of the present disclosure are variants of TRB. In some embodiments, the target polynucleotide sequence is a homolog of TRB. In some embodiments, the target polynucleotide sequence is an ortholog of TRB.

In some embodiments, reduced or eliminated expression of TRB reduces or eliminates TCR surface expression.

In some embodiments, the cells (such as, but not limited to, pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells) comprise a genetic modification at a locus encoding a TRB protein. In other words, the cell comprises a genetic modification at the TRB locus. In some cases, the nucleotide sequence encoding the TRB protein is listed in UniProt No. p0dse 2. In some cases, the TRB locus is described in refseq.no. ng_001333.2 and NCBI Gene ID No. 6957. In some cases, the amino acid sequence of TRB is depicted as Uniprot No. p01848. Additional descriptions of TRB proteins and loci can be found in GenBank No. l36092.2, uniprot No. p0dse2, and HGNC ref No.12155.

In some embodiments, the engineered and/or hypoimmunogenic cells outlined herein comprise a genetic modification that targets a TRB gene. In some embodiments, the genetic modification to target the TRB gene by the rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the TRB gene. In some embodiments, at least one guide ribonucleic acid sequence for specifically targeting the TRB gene is selected from the group consisting of SEQ ID NOS 610-765 and 9798-10532 of US20160348073, which is incorporated herein by reference.

Assays to test whether the TRB gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the TRB gene and reduction in TCR expression by PCR can be determined by FACS analysis. In another embodiment, western blot of cell lysates detected with antibodies to TRB proteins is used to detect TRB protein expression. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating genetic modifications.

In some embodiments, the engineered and/or low immunogenicity cells outlined herein comprise a knockout of TRB expression such that the cells are TRBs ^-/-. In some embodiments, the engineered and/or low immunogenicity cells outlined herein introduce an insertion deletion into the TRB locus such that the cells are TRBs ^{indel of insertion / indel of insertion}. In some embodiments, the engineered and/or low immunogenicity cells outlined herein comprise knockdown of TRB expression such that the cells are TRBs ^Knock-down.

G.CD142

In certain embodiments, 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 gene editing system (e.g., 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 a genetic modification that targets the CD142 gene. In some embodiments, the genetic modification to target the CD142 gene by a 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 gRNA sequence targeting CD142 are described below.

Assays for testing whether the CD142 gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the CD142 gene and 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 detected with antibodies to CD142 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating genetic modifications.

Useful 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.

H.RHD

In some embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of a RHD antigen by targeting and modulating (e.g., reducing or eliminating) expression of the RHD gene. In some embodiments, modulation is performed using a gene editing system (e.g., CRISPR/Cas system). In some embodiments, the cells have a reduced ability to induce an innate and/or adaptive immune response in the recipient subject.

In some embodiments, the target polynucleotide sequences of the present disclosure are variants of the RHD gene. In some embodiments, the target polynucleotide sequence is a homolog of the RHD gene. In some embodiments, the target polynucleotide sequence is a ortholog of the RHD gene.

In some embodiments, the cells described herein comprise a genetic modification at a locus encoding a RhD antigen protein. In other words, the cell comprises a genetic modification at the RHD locus. In some cases, the nucleotide sequence encoding a RhD antigen protein is set forth in refseq.no. nm_001127691.2, nm_001282868.1, nm_001282869.1, nm_001282871.1, or nm_016124.4, or in Genbank No. l 08429. In some cases, the RHD locus is described in NCBI Gene ID No. 6007. In some cases, the amino acid sequence of the RhD antigen protein is depicted as NCBI GenBank No. aaa02679.1. Additional descriptions of RhD proteins and loci can be found in Uniprot No. Q02161, HGNC Ref.No.10009 and OMIM Ref.No.111680.

In some embodiments, the cells outlined herein comprise a genetic modification that targets the RHD gene. In some embodiments, the genetic modification of the targeted RHD gene is generated by gene editing of the RHD gene using a gene editing tool such as, but not limited to, CRISPR/Cas, TALE-nucleases, zinc finger nucleases, other virus-based gene editing systems, or RNA interference. In some embodiments, the gene editing targets the coding sequence of the RHD gene. In some cases, the cell does not produce a functional RHD gene product. In the absence of RHD gene product, the cells were completely devoid of Rh blood group antigens.

In some embodiments, the genetic modification to target the RHD 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 RHD gene. Methods useful for identifying gRNA sequences that target RHD are described below.

Assays for testing whether the RHD gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the RHD gene and reduction in RHD expression by PCR can be determined by FACS analysis. In another embodiment, western blotting of cell lysates, which are probed with antibodies against RhD proteins, is used to detect RhD protein expression. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating genetic modifications.

I.CTLA-4

In some embodiments, the target polynucleotide sequence is CTLA-4 or a variant of CTLA-4. In some embodiments, the target polynucleotide sequence is a homolog of CTLA-4. In some embodiments, the target polynucleotide sequence is an ortholog of CTLA-4.

In some embodiments, the cells outlined herein comprise a genetic modification that targets a CTLA-4 gene. In certain embodiments, the primary T cells comprise a genetic modification that targets a CTLA-4 gene. Genetic modification can reduce expression of CTLA-4 polynucleotides and CTLA-4 polypeptides in T cells (including primary T cells and CAR-T cells). In some embodiments, the genetic modification to target the CTLA-4 gene by a 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 CTLA-4 gene. Methods useful for recognizing CTLA-4-targeted gRNA sequences are described below.

Assays for testing whether CTLA-4 genes have been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the CTLA-4 gene and reduction in CTLA-4 expression by PCR can be determined by FACS analysis. In another embodiment, western blotting of cell lysates detected with antibodies to CTLA-4 protein is used to detect CTLA-4 protein expression. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating genetic modifications.

Useful genomic, polynucleotide and polypeptide information about human CTLA-4 is provided, for example, in GeneCard identifiers GC02P203867, HGNC No.2505, NCBI Gene ID 1493, NCBI RefSeq No. NM-005214.4, NM-001037631.2, NP-001032720.1 and NP-005205.2, uniProt No. P16410, and the like.

J.PD-1

In some embodiments, the target polynucleotide sequence is PD-1 or a variant of PD-1. In some embodiments, the target polynucleotide sequence is a homolog of PD-1. In some embodiments, the target polynucleotide sequence is an ortholog of PD-1.

In some embodiments, the cells outlined herein comprise a genetic modification that targets a gene encoding a programmed cell death protein 1 (PD-1) protein or targets a PDCD1 gene. In certain embodiments, the primary T cells comprise a genetic modification that targets the PDCD1 gene. Genetic modification can reduce expression of PD-1 polynucleotides and PD-1 polypeptides in T cells (including primary T cells and CAR-T cells). In some embodiments, the genetic modification to target the PDCD1 gene by a 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 PDCD1 gene. Methods useful for identifying a gRNA sequence that targets PD-1 are described below.

Assays to test whether the PDCD1 gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the PDCD1 gene and reduction in PD-1 expression by PCR can be determined by FACS analysis. In another embodiment, the PD-1 protein expression is detected using western blotting of cell lysates detected with antibodies directed against the PD-1 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating genetic modifications.

Useful genomic, polynucleotide and polypeptide information about human PD-1 (including PDCD1 Gene) is provided, for example, in GeneCard identifiers GC02M241849, HGNC No.8760, NCBI Gene ID 5133, uniprot No. q15116, and NCBI RefSeq nos. nm_005018.2 and np_005009.2.

K.CD47

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) CD47. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express CD47. In some embodiments, the stem cells express exogenous CD47. In some cases, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD47 polypeptide. In some embodiments, the cells are genetically modified to comprise an integrated exogenous polynucleotide encoding CD47 using homology-directed repair. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of a safe harbor or target locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of the AAVS1 locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of a safe harbor or target locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of the CCR5 locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide, wherein the nucleotide sequence is inserted into at least one allele of the AAVS1 locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide, wherein the nucleotide sequence is inserted into at least one allele of the CCR5 locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide, wherein the nucleotide sequence is inserted into at least one allele of a safe harbor or target locus, such as, but not limited to, a CCR5 locus, a CXCR4 locus, a PPP1R12C locus, an albumin locus, a SHS231 locus, a CLYBL locus, a Rosa locus, an F3 (CD 142) locus, a MICA locus, a MICB locus, an LRP1 (CD 91) locus, an HMGB1 locus, an ABO locus, an RHD locus, an FUT1 locus, and a KDM5D locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide, wherein the nucleotide sequence is inserted into at least one allele of the TRAC locus.

CD47 is a leukocyte surface antigen and plays a role in cell adhesion and integrin regulation. It is expressed on the cell surface and signals circulating macrophages that they are not to phagocytic.

In some embodiments, the cells outlined herein comprise nucleotide sequences encoding CD47 polypeptides having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequences listed in NCBI ref. Sequence nos. np_001768.1 and np_ 942088.1. In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequences listed in NCBI ref.sequence No. np_001768.1 and np_ 942088.1. In some embodiments, the cell comprises a CD47 nucleotide sequence 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 the CD47 nucleotide sequences listed in NCBI ref. Sequence nos. nm_001777.3 and nm_ 198793.2. In some embodiments, the nucleotide sequence encoding the CD47 polynucleotide is a codon optimized sequence. In some embodiments, the nucleotide sequence encoding the CD47 polynucleotide is a human codon optimized sequence.

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

Exemplary amino acid sequences of human CD47 with and without signal sequences are provided in table 1.

TABLE 1 amino acid sequence of human CD47

In some embodiments, the cell comprises a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence of SEQ ID No. 97. In some embodiments, the cell comprises a CD47 polypeptide having the amino acid sequence of SEQ ID NO. 97. In some embodiments, the cell comprises a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence of SEQ ID NO. 98. In some embodiments, the cell comprises a CD47 polypeptide having the amino acid sequence of SEQ ID NO. 98.

In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence of SEQ ID No. 97. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence of SEQ ID No. 97. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence of SEQ ID No. 98. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence of SEQ ID NO. 98. In some embodiments, the nucleotide sequence is codon optimized for expression in a particular cell.

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 low-immunogenicity cell. In some cases, the polynucleotide encoding CD47 is inserted into a safe harbor or target locus, such as, but not limited to, AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD 142), MICA, MICB, LRP1 (CD 91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding CD47 is inserted into the B2M locus, CIITA locus, TRAC locus, or TRB locus. In some embodiments, a polynucleotide encoding CD47 is inserted into any one of the loci depicted in table 21 provided herein. In certain embodiments, the polynucleotide encoding CD47 is operably linked to a promoter.

In another embodiment, western blot of cell lysates detected with antibodies to CD47 protein is used to detect CD47 protein expression. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous CD47 mRNA.

L.CD24

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) CD24. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express CD24. In some embodiments, the stem cells express exogenous CD24. In some cases, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD24 polypeptide.

CD24, also known as a thermostable antigen or small cell lung cancer cluster 4 antigen, is a glycosylated glycosyl phosphatidyl inositol anchored surface protein (Pirruccello et al, J Immunol,1986,136,3779-3784; chen et al, glycobiology,2017,57,800-806). It binds to Siglec-10 on innate immune cells. CD24 via Siglec-10 has recently been demonstrated to act as an innate immune checkpoint (Barkal et al, nature,2019,572,392-396).

In some embodiments, the cells outlined herein comprise nucleotide sequences encoding CD24 polypeptides having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequences listed in NCBI ref. Sequence No. np_001278666.1, np_001278667.1, np_001278668.1, and np_ 037362.1. In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD24 polypeptide having the amino acid sequences listed in NCBI ref.sequence No. np_001278666.1, np_001278667.1, np_001278668.1, and np_ 037362.1.

In some embodiments, the cell comprises a nucleotide sequence having 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 set forth in NCBI ref.no. nm_00129737.1, nm_00129738.1, nm_001291739.1, and nm_ 013230.3. In some embodiments, the cell comprises the nucleotide sequences listed in NCBI ref.sequence No. nm_00129737.1, nm_00129738.1, nm_001291739.1, and nm_ 013230.3.

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 CD24 into a genomic locus of a low-immunogenicity cell. In some cases, a polynucleotide encoding CD24 is inserted into a safe harbor or target locus, such as, but not limited to, AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD 142), MICA, MICB, LRP1 (CD 91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding CD24 is inserted into a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus. In some embodiments, a polynucleotide encoding CD24 is inserted into any one of the loci depicted in table 20 provided herein. In certain embodiments, the polynucleotide encoding CD24 is operably linked to a promoter.

In another embodiment, CD24 protein expression is detected using western blotting of cell lysates that are probed with antibodies to CD24 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous CD24 mRNA.

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 CD24 into a genomic locus of a low-immunogenicity cell. In some cases, a polynucleotide encoding CD24 is inserted into a safe harbor or target 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 encoding CD24 is inserted into a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus. In some embodiments, a polynucleotide encoding CD24 is inserted into any one of the loci depicted in table 20 provided herein. In certain embodiments, the polynucleotide encoding CD24 is operably linked to a promoter.

M.DUX4

In some embodiments, the disclosure provides a cell (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, a hematopoietic stem cell, a primary T cell, or a CAR-T cell) or population thereof comprising a genome modified to increase expression of a tolerogenic or immunosuppressive factor (such as DUX 4). In some embodiments, the present disclosure provides a method for altering the genome of a cell to provide increased expression of DUX 4. In some embodiments, the present disclosure provides a cell or population thereof comprising an exogenously expressed DUX4 protein. In some embodiments, increased DUX4 expression inhibits, reduces or eliminates expression of one or more of the following MHC I molecules: HLA-A, HLA-B and HLA-C.

DUX4 is a transcription factor that is active in embryonic tissue and induced pluripotent stem cells and silences in normal, healthy somatic tissue (Feng et al, 2015,ELife4;De Iaco et al, 2017,Nat Genet,49,941-945; hendrickson et al, 2017,Nat Genet,49,925-934; snider et al, 2010,PLoS Genet,e1001181;Whiddon et al, 2017, nat Genet). DUX4 expression blocks IFN-gamma mediated induction of MHC class I gene expression (e.g., B2M, HLA-A, HLA-B, and HLA-C expression). DUX4 expression is associated with antigen presentation inhibited by MHC class I (Chew et al Developmental Cell,2019,50,1-14). DUX4 acts as a transcription factor in the cleavage stage gene expression (transcription) program. Target genes include, but are not limited to, coding genes, non-coding genes, and repeat elements.

DUX4 has at least two subtypes, the longest subtype including the DUX 4C-terminal transcriptional activation domain. Subtypes are produced by alternative splicing. See, e.g., geng et al, 2012,Dev Cell,22,38-51; snider et al 2010,PLoS Genet,e1001181. The active subtype of DUX4 includes its N-terminal DNA binding domain and its C-terminal activation domain. See, e.g., choi et al, 2016,Nucleic Acid Res,44,5161-5173.

It has been demonstrated that reducing the number of CpG motifs of DUX4 reduces silencing of the DUX4 transgene (Jagannathan et al Human Molecular Genetics,2016,25 (20): 4419-4431). The nucleic acid sequence provided in Jagannathan et al, supra, represents a DUX4 codon change sequence comprising one or more base substitutions to reduce the total number of CpG sites while preserving the DUX4 protein sequence. Nucleic acid sequences are commercially available from adedge catalog number 99281.

In many embodiments, at least one or more polynucleotides can be utilized to promote exogenous expression of DUX4 by a cell (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, a hematopoietic stem cell, a primary T cell, or a CAR-T cell).

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 DUX4 into a genomic locus of a low-immunogenicity cell. In some cases, a polynucleotide encoding DUX4 is inserted into a safe harbor or target locus, such as, but not limited to, AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD 142), MICA, MICB, LRP1 (CD 91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding DUX4 is inserted into a B2M locus, CIITA locus, TRAC locus, or TRB locus. In some embodiments, a polynucleotide encoding DUX4 is inserted into any one of the loci depicted in table 20 provided herein. In certain embodiments, the polynucleotide encoding DUX4 is operably linked to a promoter.

In some embodiments, the polynucleotide sequence encoding DUX4 comprises a polynucleotide sequence comprising a codon-altered DUX4 nucleotide sequence comprising one or more base substitutions to reduce the total number of CpG sites while preserving the DUX4 protein sequence. In some embodiments, the polynucleotide sequence encoding DUX4 comprises one or more base substitutions to reduce the total number of CpG sites that have at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with SEQ ID No.1 of PCT/US2020/44635 submitted on 31 th 7 th 2020. In some embodiments, the polynucleotide sequence encoding DUX4 is SEQ ID NO:1 of PCT/US 2020/44635.

In some embodiments, the polynucleotide sequence encoding DUX4 is a nucleotide sequence encoding a polypeptide sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence selected from the group consisting of seq id nos: SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ IDNO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28 and SEQ ID NO:29 as provided in PCT/US 2020/44635. In some embodiments, the polynucleotide sequence encoding DUX4 is nucleotide sequence ：SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28 and SEQ ID NO 29 encoding a polypeptide sequence selected from the group consisting of. The amino acid sequences shown as SEQ ID NOS.2-29 are shown in FIGS. 1A through 1G of PCT/US 2020/44635.

In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ACN62209.1, or comprises an amino acid sequence set forth in GenBank accession No. ACN 62209.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in NCBI RefSeq No. np_001280727.1, or comprises an amino acid sequence set forth in NCBI RefSeq No. np_ 001280727.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ACP30489.1, or comprises an amino acid sequence set forth in GenBank accession No. ACP 30489.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in UniProt No. p0cj85.1, or comprises an amino acid sequence set forth in UniProt No. p0cj85.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession number AUA60622.1, or comprises an amino acid sequence set forth in GenBank accession number AUA 60622.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24683.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24683.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ACN62210.1, or comprises an amino acid sequence set forth in GenBank accession No. ACN 62210.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24706.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24706.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24685.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24685.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ACP30488.1, or comprises an amino acid sequence set forth in GenBank accession No. ACP 30488.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24687.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24687.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ACP30487.1, or comprises an amino acid sequence set forth in GenBank accession No. ACP 30487.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24717.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24717.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24690.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24690.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24689.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24689.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24692.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24692.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24693.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24693.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24712.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24712.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24691.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24691.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in UniProt No. p0cj87.1, or comprises an amino acid sequence set forth in UniProt No. p0cj87.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24714.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24714.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24684.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24684.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24695.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24695.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24699.1, or comprises an amino acid sequence set forth in GenBank accession No. ADK 24699.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in NCBI RefSeq No. np_001768.1, or comprises an amino acid sequence set forth in NCBI RefSeq No. np_001768. In some cases, the DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence identity to a sequence set forth in NCBI RefSeq No. np_942088.1, or comprises an amino acid sequence set forth in NCBI RefSeq No. np_ 942088.1. In some cases, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO. 28 provided in PCT/US2020/44635, or comprises an amino acid sequence of SEQ ID NO. 28 provided in PCT/US 2020/44635. In some cases, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO. 29 provided in PCT/US2020/44635 or comprises an amino acid sequence of SEQ ID NO. 29 provided in PCT/US 2020/44635.

In other embodiments, expression vectors are used to facilitate expression of tolerogenic factors. In some embodiments, the expression vector comprises a polynucleotide sequence encoding DUX4, the polynucleotide sequence encoding DUX4 being a codon change sequence comprising one or more base substitutions to reduce the total number of CpG sites while preserving the DUX4 protein sequence. In some cases, the codon change sequence of DUX4 comprises SEQ ID NO:1 of PCT/US 2020/44635. In some cases, the codon change sequence for DUX4 is SEQ ID NO:1 of PCT/US 2020/44635. In other embodiments, the expression vector comprises a polynucleotide sequence encoding DUX4 comprising SEQ ID NO. 1 of PCT/US 2020/44635. In some embodiments, the expression vector comprises a polynucleotide sequence encoding a DUX4 polypeptide sequence having at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28 of PCT/US2020/44635 and SEQ ID NO. 29. In some embodiments, the expression vector comprises a polynucleotide sequence encoding a DUX4 polypeptide sequence selected from the group consisting of: SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28 of PCT/US2020/44635 and SEQ ID NO. 29.

The increase in DUX4 expression can be determined using known techniques such as Western blot, ELISA assay, FACS assay, immunoassay, etc.

N. additional tolerogenic factors

In certain embodiments, one or more tolerogenic factors may be inserted or reinserted into a genome-edited cell to create an immune-free universal donor cell, such as a universal donor stem cell, a universal donor T cell, or a universal donor cell. In certain embodiments, the engineered cells and/or hypoimmunogenic cells disclosed herein have been further modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, but are not limited to, one or more of the following: CD47, DUX4, CD24, CD27, CD35, 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, fasL, CCL21, CCL22, mfge8, CD16, CD52, H2-M3, CD16 Fc receptor, IL15-RF and Serpinb9. In some embodiments, the tolerogenic factors are selected from the group consisting of: CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, fasL, serpinb9, CCL21, CCL22 and Mfge8. In some embodiments, the tolerogenic factors are selected from the group consisting of: DUX4, HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor and IL-35. In some embodiments, the tolerogenic factors are selected from the group consisting of: HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor and IL-35. In some embodiments, the tolerogenic factors are selected from the group consisting of: CD47, DUX4, CD24, CD27, CD35, 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, fasL, CCL21, CCL22, mfge8, CD16, CD52, H2-M3, CD16 Fc receptor, IL15-RF and Serpinb9.

Useful genomic, polynucleotide and polypeptide information about human CD27 (which is also known as CD27L receptor, tumor necrosis factor receptor superfamily member 7, TNFSF7, T cell activating antigens S152, tp55 and T14) is provided, for example, in GeneCard identifiers GC12P008144, HGNC No.11922, NCBI Gene ID 939, uniprot No. P26842, and NCBI RefSeq No. nm_001242.4 and np_001233.1.

Useful 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 RefSeq 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.

Useful genomic, polynucleotide and polypeptide information about human CD55 (also known as complement decay acceleration factor) is provided, for example, in GeneCard identifiers GC01P207321, hgncno.2665, NCBI Gene ID 1604, uniprot No. P08174, as well as NCBI RefSeqNo.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.

Useful 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.

Useful 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.

Useful genomic, polynucleotide and polypeptide information about human HLA-C is provided, for example, in GeneCard identifiers GC06M031272, HGNC No.4933, NCBI Gene ID 3107, uniprot No. P10321, NCBI RefSeq No. NP-002108.4 and NM-002117.5.

Useful 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, and NCBI RefSeq No. NP-005507.3 and NM-005516.5.

Useful 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.

Useful 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.

Useful genomic, polynucleotide and polypeptide information about human IDO1 is provided, for example, in GeneCard identifiers GC08P039891, HGNC No.6059, NCBI Gene ID 3620, uniprot No. P14202, and NCBI RefSeq nos. np_002155.1 and nm_002164.5.

Useful genomic, polynucleotide and polypeptide information about human IL-10 is provided, for example, in GeneCard identifiers GC01M206767, HGNC No.5962, NCBI Gene ID 3586, uniprot No. P22301, and NCBI RefSeq No. NP-000563.1 and NM-000572.2.

Useful 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.

Useful genomic, polynucleotide and polypeptide information about human CCL21 is provided, for example, in GeneCard identifiers GC09M034709, HGNC No.10620, NCBI Gene ID6366, uniprot No. o00585, and NCBI RefSeq nos. np_002980.1 and nm_002989.3.

Useful 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, and NCBI RefSeq No. np_002981.2, nm_002990.4, xp_016879020.1, and xm_017023531.1.

Useful genomic, polynucleotide and polypeptide information about human Mfge is provided, for example, in GeneCard identifier GC15M088898, HGNC No.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.

Useful 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.

Methods for modulating gene and factor (protein) expression include genome editing techniques, 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 (e.g., stem cell, induced pluripotent stem cell, differentiated cell, hematopoietic stem cell, primary T cell, or CAR-T cell) has a genetic modification that inactivates B2M and CIITA genes, and expresses a plurality of exogenous polypeptides selected from the group consisting of: CD47 and DUX4, CD47 and CD24, CD47 and CD27, CD47 and CD46, CD47 and CD55, CD47 and CD59, CD47 and CD200, CD47 and HLA-C, CD and HLA-E, CD47 and HLA-E heavy chain, CD47 and HLA-G, CD and PD-L1, CD47 and IDO1, CD47 and CTLA4-Ig, CD47 and C1-inhibitor, CD47 and IL-10, CD47 and IL-35, CD47 and IL-39, CD47 and FasL, CD47 and CCL21, CD47 and CCL22, CD47 and Mfge8, CD47 and Serpinb9, and any combination thereof. In some cases, such cells also have a genetic modification that inactivates the CD142 gene.

In some cases, a gene editing system (such as a CRISPR/Cas system) is used to facilitate insertion of tolerogenic factors (such as tolerogenic factors) into a safe harbor or target locus (such as an AAVS1 locus) to actively suppress immune rejection. In some cases, tolerogenic factors are inserted into a safe harbor or target locus using expression vectors. In some embodiments, the safe harbor or target locus is an 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 expression of a target gene (e.g., CD47, DUX4, or another tolerogenic factor gene) is increased by expressing a fusion protein or protein complex comprising: (1) A site-specific binding domain specific for an exogenous target gene (e.g., CD47, DUX4, or another tolerogenic factor gene) 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; WO 02/016536 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 repeat sequences" and partially direct repeat sequences 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 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 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 gene transcription initiation site (TSS).

It is within the level of the skilled artisan to design or identify a gRNA sequence that is or comprises the sequence of the targeted gene, including the sequence of the exons 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 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, SRC PvALF, atHD2A and ERF-2. See, e.g., 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.69:3-12; 2000) Trends biochem.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 et al, (1999) Plant mol:87-99; goff et al, (1991) Plant mol:419-429; and (1999) Prol.45:41-49, nat.35.4.35.

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): 3898-394 l). 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、PRMT 2/6、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 sgRNA nucleic acid components associated with functional domains of polypeptide components are also known to those of skill in the art and are described in detail herein.

In some embodiments, the present disclosure provides a cell (e.g., primary T cells and low-immunogenicity stem cells and derivatives thereof) or population thereof comprising a genome, wherein the cell genome has been modified to express CD47. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express CD47. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be used to facilitate insertion of CD47 into a cell line. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs 200784-231885 of Table 29 of WO2016183041, which is incorporated herein by reference.

In some embodiments, the present disclosure provides a cell (e.g., primary T cells and low-immunogenicity stem cells and derivatives thereof) or population thereof comprising a genome, wherein the cell genome has been modified to express HLA-C. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express HLA-C. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be used to facilitate insertion of HLA-C into a cell line. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs 3278-5183 of Table 10 of WO2016183041, which is incorporated herein by reference.

In some embodiments, the present disclosure provides a cell (e.g., primary T cells and low-immunogenicity stem cells and derivatives thereof) or population thereof comprising a genome, wherein the cell genome has been modified to express HLA-E. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express HLA-E. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be used to facilitate insertion of HLA-E into a cell line. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs 189859-193183 of Table 19 of WO2016183041, which is incorporated herein by reference.

In some embodiments, the present disclosure provides a cell (e.g., primary T cells and low-immunogenicity stem cells and derivatives thereof) or population thereof comprising a genome, wherein the cell genome has been modified to express HLA-F. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express HLA-F. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be used to facilitate insertion of HLA-F into a cell line. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NO:688808-399754 of Table 45 of WO2016183041, which is incorporated herein by reference.

In some embodiments, the present disclosure provides a cell (e.g., primary T cells and low-immunogenicity stem cells and derivatives thereof) or population thereof comprising a genome, wherein the cell genome has been modified to express HLA-G. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express HLA-G. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be used to facilitate insertion of HLA-G into a stem cell line. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs 188372-189858 of Table 18 of WO2016183041, which is incorporated herein by reference.

In some embodiments, the present disclosure provides a cell (e.g., primary T cells and low-immunogenicity stem cells and derivatives thereof) or population thereof comprising a genome, wherein the cell genome has been modified to express PD-L1. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express PD-L1. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be used to facilitate insertion of PD-L1 into a stem cell line. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of SEQ ID NOs 193184-200783 of Table 21 of WO2016183041, which is incorporated herein by reference.

In some embodiments, the disclosure provides a cell (e.g., primary T cells and low-immunogenicity stem cells and derivatives thereof) or population thereof comprising a genome, wherein the cell genome has been modified to express CTLA4-Ig. In some embodiments, the disclosure provides a method for altering the genome of a cell to express CTLA4-Ig. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids can be used to facilitate insertion of CTLA4-Ig into a stem cell line. In certain embodiments, the at least one ribonucleic acid or the at least one pair of ribonucleic acids is selected from any of those disclosed in WO2016183041 (including the sequence listing).

In some embodiments, the present disclosure provides a cell (e.g., primary T cells and low-immunogenicity stem cells and derivatives thereof) or population thereof comprising a genome, wherein the cell genome has been modified to express a CI-inhibitor. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express a CI-inhibitor. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids may be used to facilitate insertion of the CI-inhibitor into the stem cell line. In certain embodiments, the at least one ribonucleic acid or the at least one pair of ribonucleic acids is selected from any of those disclosed in WO2016183041 (including the sequence listing).

In some embodiments, the disclosure provides a cell (e.g., primary T cells and low-immunogenicity stem cells and derivatives thereof) or population thereof comprising a genome, wherein the cell genome has been modified to express IL-35. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express IL-35. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids can be used to facilitate insertion of IL-35 into a stem cell line. In certain embodiments, the at least one ribonucleic acid or the at least one pair of ribonucleic acids is selected from any of those disclosed in WO2016183041 (including the sequence listing).

In some embodiments, the tolerizing factor is expressed in the cell using an expression vector. For example, an expression vector for expressing CD47 in a cell comprises a polynucleotide sequence encoding CD 47. The expression vector may be an inducible expression vector. The expression vector may be a viral vector, such as but not limited to a lentiviral vector. In some embodiments, the tolerogenic factors are introduced into the cells using a fusion-mediated delivery or a transposase system selected from the group consisting of: a conditional or inducible transposase, a conditional or inducible PiggyBac transposon, a conditional or inducible sleeping beauty (SB 11) transposon, a conditional or inducible Mos1 transposon, and a conditional or inducible Tol2 transposon.

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 a tolerogenic factor into a genomic locus of a low-immunogenic cell. In some cases, polynucleotides encoding tolerogenic factors are inserted into a safe harbor or target locus, such as but not limited to AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD 142), MICA, MICB, LRP1 (CD 91), HMGB1, ABO, RHD, FUT1, or KDM5D loci. In some embodiments, the polynucleotide encoding the tolerogenic factors is inserted into a B2M locus, CIITA locus, TRAC locus or TRB locus. In some embodiments, a polynucleotide encoding a tolerogenic factor is inserted into any one of the loci depicted in tables 2-5 provided herein. In certain embodiments, the polynucleotide encoding the tolerogenic factors is operably linked to a promoter.

O.Y-Linked tropocadherin 11

In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) the expression of one or more Y chromosome genes by targeting and modulating (e.g., reducing or eliminating) the expression of the Y chromosome genes. In some embodiments, modulation is performed using a gene editing system (e.g., CRISPR/Cas system). In some embodiments, the cells have a reduced ability to induce an innate and/or adaptive immune response in the recipient subject.

In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) the expression of a Y-linked tropocadherin 11 antigen by targeting and modulating (e.g., reducing or eliminating) the expression of a Y-linked tropocadherin 11 gene (e.g., PCDH 11Y). In some embodiments, modulation is performed using a CRISPR/Cas system. In some embodiments, the cell has a reduced ability to induce an immune response in a recipient subject.

In some embodiments, the target polynucleotide sequences of the present disclosure are variants of the PCDH11Y gene. In some embodiments, the target polynucleotide sequence is a homolog of the PCDH11Y gene. In some embodiments, the target polynucleotide sequence is an ortholog of the PCDH11Y gene.

In some embodiments, the cells described herein comprise a genetic modification at a locus encoding a Y-linked tropocadherin 11 antigen protein. In other words, the cell comprises a genetic modification at the PCDH11Y locus. In some cases, the nucleotide sequence encoding the Y-linked tropocadherin 11 antigen protein is listed in refseq.no. n nm_001278619.1, nm_032971.2, nm_032972.2, nm_032973.2, or xm_017030082.1, or in Genbank No.AJ276803、AF277053、AF332216、AF332217、AJ564958、AJ564959、AJ564960、AJ564961、AJ564962、AJ564963、AJ564966 or AJ 56496. In some cases, the PCDH11Y locus is described in NCBI Gene ID No. 83259. In some cases, the amino acid sequence of the Y-linked tropocadherin 11 antigen is depicted as NCBI GenBank No.CAC13122.1、AAL55729.1、AAK13468.1、AAK13469.1、CAD92429.1、CAD92430.1、CAD92431.1、CAD92432.1、CAD92433.1、CAD92434.1、CAD92437.1 or CAD92440.1. Additional description of Y-linked tropocadherin 11 antigen proteins and loci can be found in Uniprot No. Q9BZA8, HGNC Ref.No.15813 and OMIM Ref.No.400022.

In some embodiments, the engineered and/or hypoimmunogenic cells outlined herein comprise a genetic modification that targets the PCDH11Y gene. In some embodiments, the genetic modification of the targeted PCDH11Y gene is generated by gene editing the PCDH11Y gene using a gene editing tool (such as, but not limited to, CRISPR/Cas, TALE-nuclease, zinc finger nuclease, other virus-based gene editing systems, or RNA interference). In some embodiments, the gene editing targets the coding sequence of the PCDH11Y gene. In some cases, the cell does not produce a functional PCDH11Y gene product. In the absence of PCDH11Y gene product, the cells were completely devoid of Y-linked tropocadherin 11 antigen.

In some embodiments, cas9 or Cas12a editing systems are used to target the sequence of the PCDH11Y gene to introduce insertions or deletions into the gene to disrupt its function, and in some cases inactivate it. In some embodiments, a single guide RNA is used. In some embodiments, a double guide RNA is used. In some embodiments, either of the gRNA target sequences of table 2A or table 2B is used. In some cases, more than one gRNA target sequence of table 2A and/or table 2B is used for gene editing. In some embodiments, the Cas9 editing system comprises a Cas9 protein or a fragment thereof, a tracrRNA, and a crRNA. In some embodiments, the Cas12a editing system comprises a Cas12a protein or a fragment thereof and a crRNA.

In some embodiments, a frameshift insertion-deletion is introduced in any coding sequence of a gene. In some embodiments, modifications are added within the UTR, intron, or exon of the gene to disrupt the function of the PCDH11Y gene. In some embodiments, CRISPR/Cas editing is used that comprises any one or more of the gRNA target sequences of table 2A and/or table 2B.

In some embodiments, modifications are introduced into the PCDH11Y gene to inactivate the gene. In some embodiments, the coding exon, such as exon 1 or exon 2 or exon 3 of the PCDH11Y gene, is targeted. In some cases, deletions are created using a Cas editing system and a guide RNA target sequence targeting the 5' sequence of the PCDH11Y gene, as well as a guide RNA target sequence targeting an exon (such as but not limited to exon 1 or 2). In some embodiments, the cells described herein comprise homozygous modification of the PCDH11Y gene, thereby inactivating the gene.

TABLE 2A exemplary PCDH11Y gRNA target sequence-exon 1

TABLE 2B exemplary PCDH11Y gRNA target sequence-exon 2

In some embodiments, the gRNA target sequence is directed against exon 1 or exon 2 of the PCDH11Y gene. In some embodiments, the gRNA target sequence is a gRNA of table 2A and/or table 2B that induces a frameshift mutation to inactivate exon 1 or exon 2.

In some embodiments, the expression of the PCDH11Y gene is partially or completely inactivated by an insertion or deletion within exon 1 or exon 2 of the PCDH11Y gene.

Assays to test whether the PCDH11Y gene has been inactivated are known and described herein. In one embodiment, the PCDH11Y gene genetic modification and the decrease in expression of the Y-linked tropocadherin 11 antigen protein by PCR can be determined by FACS analysis. In another embodiment, western blotting of cell lysates probed with antibodies to the Y-linked tropocadherin 11 antigen protein is used to detect Y-linked tropocadherin 11 antigen protein expression. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating genetic modifications.

P.Y-Linked nerve connector protein 4

In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of a Y-linked fibronectin 4 antigen by targeting and modulating (e.g., reducing or eliminating) expression of a Y-linked fibronectin 4 gene (e.g., NLGN 4Y). In some embodiments, modulation is performed using a gene editing system (e.g., CRISPR/Cas system). In some embodiments, the cells have a reduced ability to induce an innate and/or adaptive immune response in the recipient subject.

In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of the NLGN4Y gene. In some embodiments, the target polynucleotide sequence is a homolog of the NLGN4Y gene. In some embodiments, the target polynucleotide sequence is an ortholog of the NLGN4Y gene.

In some embodiments, the cells described herein comprise a genetic modification at a locus encoding a Y-linked fibronectin 4 antigen protein. In other words, the cell comprises a genetic modification at the NLGN4Y locus. In some cases, the nucleotide sequence encoding the Y-linked fibronectin 4 antigen protein is listed in RefSeq.No.N NM_001164238.1、NM_001206850.1、NM_014893.4、XM_017030034.1、XM_017030035.1、XM_017030036.1、XM_017030037.1、XM_017030038.1、XM_017030040.1 or xm_017030041.1, or in Genbank No. af376804, AB023168, BX537428, AC010726, AC010879, AC010979, AC011903, BC032567, BC113525, or BC 113551. In some cases, the NLGN4Y locus is described in NCBI Gene ID No. 22829. In certain instances, the amino acid sequence of the Y-linked fibronectin 4 antigen is depicted as NCBI GenBank No. aam46113.1, BAA76795.2, CAD97670.1, AAH32567.1, AAI13526.1, or AAI13552.1. Additional description of Y-linked fibronectin 4 antigen proteins and loci can be found in Uniprot No. Q8NFZ3, HGNC Ref.No.15529 and OMIM Ref.No.400028.

In some embodiments, the engineered and/or hypoimmunogenic cells outlined herein comprise genetic modifications that target the NLGN4Y gene. In some embodiments, genetic modifications targeting the NLGN4Y gene are generated by gene editing the NLGN4Y gene using a gene editing tool such as, but not limited to, CRISPR/Cas, TALE-nuclease, zinc finger nuclease, other virus-based gene editing systems, or RNA interference. In some embodiments, the gene editing targets the coding sequence of the NLGN4Y gene. In some cases, the cell does not produce a functional NLGN4Y gene product. In the absence of NLGN4Y gene product, the cells were completely devoid of Y-linked fibronectin 4 antigen.

In some embodiments, cas9 or Cas12a editing systems are used to target the sequence of the NLGN4Y gene to introduce insertions or deletions into the gene to disrupt its function and, in some cases, inactivate it. In some embodiments, a single guide RNA is used. In some embodiments, a double guide RNA is used. In some embodiments, any of the gRNA target sequences of table 3, table 4, and/or table 5 are used. In some cases, more than one gRNA target sequence of table 3, table 4, and/or table 5 is used for gene editing. In some embodiments, the Cas9 editing system comprises a Cas9 protein or a fragment thereof, a tracrRNA, and a crRNA. In some embodiments, the Cas12a editing system comprises a Cas12a protein or a fragment thereof and a crRNA.

In some embodiments, a frameshift insertion-deletion is introduced in any coding sequence of a gene. In some embodiments, modifications are added within the UTR, intron, or exon of the gene to disrupt the function of the NLGN4Y gene. In some embodiments, CRISPR/Cas editing is used that comprises any one or more of the gRNA target sequences of table 3, table 4, and/or table 5.

In some embodiments, modifications are introduced into the NLGN4Y gene to inactivate the gene. In some embodiments, the coding exon, such as exon 3 or exon 4 or exon 5 of the NLGN4Y gene, is targeted. In some cases, deletions are created using Cas editing systems and guide RNA target sequences targeting sequences 5' of the NLGN4Y gene as well as guide RNA target sequences targeting exons (such as but not limited to exon 3 or exon 4 or exon 5). In some embodiments, the cells described herein comprise homozygous modification of the NLGN4Y gene, thereby inactivating the gene.

TABLE 3 exemplary NLGN4Y gRNA target sequence-exon 3

TABLE 4 exemplary NLGN4Y gRNA target sequence-exon 4

SEQ ID NO	Position of	Chain	Sequence(s)	PAM
					285	301455	1	GTTTTTAAGTACCGGTGACC	AGG
286	301455	-1	GCCTTTTGCTGCCTGGTCAC	CGG
					287	301462	-1	CATAGTTGCCTTTTGCTGCC	TGG
288	301465	1	ACCGGTGACCAGGCAGCAAA	AGG
					289	301474	1	CAGGCAGCAAAAGGCAACTA	TGG
290	301475	1	AGGCAGCAAAAGGCAACTAT	GGG
					291	301482	1	AAAAGGCAACTATGGGCTCC	TGG
292	301489	-1	TCAGTGCTTGAATCTGATCC	AGG
					293	301502	1	TGGATCAGATTCAAGCACTG	AGG
294	301505	1	ATCAGATTCAAGCACTGAGG	TGG
					295	301512	1	TCAAGCACTGAGGTGGATTG	AGG
296	301522	1	AGGTGGATTGAGGAGAATGT	CGG
					297	301531	1	GAGGAGAATGTCGGAGCCTT	TGG
298	301534	1	GAGAATGTCGGAGCCTTTGG	CGG
					299	301535	1	AGAATGTCGGAGCCTTTGGC	GGG
300	301536	1	GAATGTCGGAGCCTTTGGCG	GGG
					301	301536	-1	TCTCTTGGGGTCCCCGCCAA	AGG
302	301549	-1	CAAAGATAGTCACTCTCTTG	GGG
					303	301550	-1	CCAAAGATAGTCACTCTCTT	GGG
304	301551	-1	GCCAAAGATAGTCACTCTCT	TGG
					305	301561	1	CCCAAGAGAGTGACTATCTT	TGG
306	301566	1	GAGAGTGACTATCTTTGGCT	CGG
					307	301567	1	AGAGTGACTATCTTTGGCTC	GGG
308	301568	1	GAGTGACTATCTTTGGCTCG	GGG
					309	301569	1	AGTGACTATCTTTGGCTCGG	GGG
310	301573	1	ACTATCTTTGGCTCGGGGGC	TGG
					311	301574	1	CTATCTTTGGCTCGGGGGCT	GGG
312	301575	1	TATCTTTGGCTCGGGGGCTG	GGG
					313	301587	-1	GGTCAACAGGCTGACACAGG	AGG
314	301590	-1	CAGGGTCAACAGGCTGACAC	AGG
					315	301600	-1	AGTAGTGGGACAGGGTCAAC	AGG

TABLE 5 exemplary NLGN4Y gRNA target sequence-exon 5

In some embodiments, the gRNA target sequence is directed against exon 1 or exon 2 of the NLGN4Y gene. In some embodiments, the gRNA target sequence is a gRNA of table 3, table 4, and/or table 5 that induces a frameshift mutation to inactivate exon 3, exon 4, or exon 5.

In some embodiments, the expression of the NLGN4Y gene is partially or completely inactivated by an insertion or deletion within exon 3, exon 4, or exon 5 of the NLGN4Y gene.

Assays to test whether the NLGN4Y gene has been inactivated are known and described herein. In one embodiment, the genetic modification of the NLGN4Y gene and the reduction in expression of the Y-linked fibronectin 4 antigen protein by PCR can be determined by FACS analysis. In another embodiment, Y-linked fibronectin 4 antigen protein expression is detected using Western blotting of cell lysates probed with antibodies to the Y-linked fibronectin 4 antigen protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactivating genetic modifications.

Chimeric antigen receptor

Provided herein are low immunogenicity cells comprising Chimeric Antigen Receptors (CARs). In some embodiments, the CAR binds to CD 19. In some embodiments, the CAR binds to CD 20. In some embodiments, the CAR binds to CD 22. In some embodiments, the CAR binds to CD 19. In some embodiments, the CAR binds to CD19 and CD 22. 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 comprises a single binding domain that binds to a single target antigen. In some embodiments, the CAR comprises a single binding domain that binds to more than one target antigen (e.g., 2, 3, or more target antigens). In some embodiments, the CAR comprises two binding domains such that each binding domain binds to a different target antigen. In some embodiments, the CAR comprises two binding domains such that each binding domain binds to the same target antigen. A detailed description of exemplary CARs including CD 19-specific, CD 20-specific, CD19/CD 20-bispecific CARs, CD 22-specific, and CD19/CD 22-bispecific CARs can be found in WO2012/079000, WO 2016/149588, and WO2020/014482, the disclosures of which including the sequence listing and figures are incorporated herein by reference in their entirety. In some embodiments, the CAR comprises two binding domains such that each binding domain binds to the same target antigen.

In some embodiments, the CD 19-specific CAR comprises an anti-CD 19 single chain antibody fragment (scFv), a transmembrane domain (such as a transmembrane domain derived from human CD8 a), a 4-1BB (CD 137) costimulatory signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CD 20-specific CAR comprises an anti-CD 20 scFv, a transmembrane domain (such as a transmembrane domain derived from human CD8 a), a 4-1BB (CD 137) costimulatory signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CD19/CD20 bispecific CAR comprises an anti-CD 19 scFv, an anti-CD 20 scFv, a transmembrane domain (such as a transmembrane domain derived from human CD8 a), a 4-1BB (CD 137) costimulatory signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CD 22-specific CAR comprises an anti-CD 22scFv, a transmembrane domain (such as a transmembrane domain derived from human CD8 a), a 4-1BB (CD 137) costimulatory signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CD19/CD22 bispecific CAR comprises an anti-CD 19 scFv, an anti-CD 22scFv, a transmembrane domain (such as a transmembrane domain derived from human CD8 a), a 4-1BB (CD 137) costimulatory signaling domain, and a CD3 zeta signaling domain.

In some embodiments, the CAR comprises a commercial CAR construct carried by a T cell. Non-limiting examples of commercial CAR-T cell based therapies include briyl alendronate (brexucabtagene autoleucel) from CARTESIAN THERAPEUTICSAlkylrensaiAi Jiwei Racing (idecabtagene vicleucel)Li Jimai Lun's (lisocabtagene maraleucel)TexarensaiDESCARTES-08 and DESCARTES-11, CTL119 from Novartis, P-BMCA-101 from Poseida Therapeutics, PBCAR B and PBCAR269A from Precision Biosciences, FT819 from Fate Therapeutics and CYAD-211 from Clyad Oncology.

In some embodiments, the low immunogenicity cells described herein comprise a polynucleotide encoding a Chimeric Antigen Receptor (CAR) comprising an antigen binding domain. In some embodiments, the low immunogenicity cells described herein comprise a Chimeric Antigen Receptor (CAR) comprising an antigen binding domain. In some embodiments, the polynucleotide is or comprises a Chimeric Antigen Receptor (CAR) comprising an antigen binding domain. 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.

1. Antigen Binding Domain (ABD) targeting antigens characteristic of tumor cells or cancer cells

In some embodiments, the Antigen Binding Domain (ABD) targets an antigen characteristic of a tumor cell. In other words, 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 a tumor-associated antigen is selected from the group consisting of cell surface receptors, ion channel linked receptors, enzyme linked receptors, G protein coupled receptors, receptor tyrosine kinases, tyrosine kinase related receptors, receptor-like tyrosine phosphatases, receptor serine/threonine kinases, receptor guanylate cyclases, histidine kinase related receptors, epidermal Growth Factor Receptors (EGFR) (including ErbB1/EGFR, erbB2/HER2, erbB3/HER3 and ErbB4/HER 4), fibroblast Growth Factor Receptors (FGFR) (including FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF18 and FGF 21), vascular Endothelial Growth Factor Receptors (VEGFR) (including VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF), RET receptors and Eph receptor families (including EphA 1) 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、Bestrophins、TMEM16A、GABA receptors, glycine receptor, ABC transporter, NAV1.1, NAV1.2, NAV1.3, NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9, sphingosine-1-phosphate receptor (S1P 1R), NMDA channel, transmembrane protein, multi-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, tfg, trexp 3+, tr 3, tr 17, T5672, T5 IX 5, TAG 5, and so on-binding protein, PSMA, folate binding protein, ganglioside (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、ANTXR1、 folate receptor alpha (FRa), ERBB2 (Her 2/neu), ephA2, IL-13Ra2, epidermal Growth Factor Receptor (EGFR), 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, gpl00, bcr-abl, tyrosinase, fucosyl GM1, sLe 3, TGS5, maA-Acetyl-2 Folic acid receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF, CD97, CD179a, ALK, polysialic acid 、PLACl、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-la、MAGE-A1、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, 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, BST2, 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 an antigenic fragment or antigenic portion thereof.

ABD targeting T cell characteristic antigen

In some embodiments, the antigen binding domain targets an antigen characteristic of T cells. In some embodiments, the ABD binds an antigen associated with a T cell. In some cases, such an antigen is expressed by or located on the surface of a T cell. In some embodiments, the T cell-characteristic antigen or T cell-associated antigen is selected from a cell surface receptor, a membrane transporter (e.g., an active or passive transporter, e.g., an ion channel protein, a pore-forming protein, etc.), a transmembrane receptor, a membrane enzyme, and/or a T cell-characteristic cell adhesion protein. In some embodiments, the T cell-characteristic antigen may be 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 、AKT1、AKT2、AKT3、ATF2、BCL10、CALM1、CD3D(CD3δ)、CD3E(CD3ε)、CD3G(CD3γ)、CD4、CD8、CD28、CD45、CD80(B7-1)、CD86(B7-2)、CD247(CD3ζ)、CTLA-4(CD152)、ELK1、ERK1(MAPK3)、ERK2、FOS、FYN、GRAP2(GADS)、GRB2、HLA-DRA、HLA-DRB1、HLA-DRB3、HLA-DRB4、HLA-DRB5、HRAS、IKBKA(CHUK)、IKBKB、IKBKE、IKBKG(NEMO)、IL2、ITPR1、ITK、JUN、KRAS2、LAT、LCK、MAP2K1(MEK1)、MAP2K2(MEK2)、MAP2K3(MKK3)、MAP2K4(MKK4)、MAP2K6(MKK6)、MAP2K7(MKK7)、MAP3K1(MEKK1)、MAP3K3、MAP3K4、MAP3K5、MAP3K8、MAP3K14(NIK)、MAPK8(JNK1)、MAPK9(JNK2)、MAPK10(JNK3)、MAPK11(p38β)、MAPK12(p38γ)、MAPK13(p38δ)、MAPK14(p38α)、NCK、NFAT1、NFAT2、NFKB1、NFKB2、NFKBIA、NRAS、PAK1、PAK2、PAK3、PAK4、PIK3C2B、PIK3C3(VPS34)、PIK3CA、PIK3CB、PIK3CD、PIK3R1、PKCA、PKCB、PKCM、PKCQ、PLCY1、PRF1( perforin), PTEN, RAC1, RAF1, RELA, SDF1, SHP2, SLP76, SOS, SRC, TBK1, TCRA, TEC, TRAF6, VAV1, VAV2, or ZAP70.

ABD targeting of antigens characteristic of autoimmune diseases/disorders and/or inflammatory diseases/disorders

In some embodiments, the antigen binding domain targets an antigen that is characteristic of an autoimmune disease/disorder and/or inflammatory disease/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 disease/disorder and/or an inflammatory disease/disorder. In some embodiments, the autoimmune or inflammatory disorder is selected from chronic Graft Versus Host Disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, goodpasture's syndrome, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, collectinopathies, pemphigus vulgaris, grave's disease, autoimmune hemolytic anemia, hemophilia a, primary sjogren's syndrome, thrombotic thrombocytopenic purpura, neuromyelitis optica, erwinia 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 erythropoiesis disorder, although illustrative non-limiting examples of alloimmune disorders include allo-sensitization (see e.g., blazar, 2015, et al (j.15); 931-41) or xenogeneic sensitization from hematopoietic or solid organ transplants, blood transfusion, pregnancy and fetuses, neonatal alloimmune thrombocytopenia, neonatal hemolytic diseases, sensitization to foreign antigens, such as replacement of hereditary or acquired deficiency disorders treated with enzyme or protein replacement therapies, blood products and gene therapies may occur. 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, for example, US 2003/0077149; WO 2017/058753; WO 2017/058850; US 2021/023445; WO 2019/201995A1; EP 3 781,590 A1, the contents of which are incorporated herein by reference.

ABD targeting of antigens characteristic of senescent cells

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.

ABD targeting of antigens characteristic of infectious diseases

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.

ABD binds cell surface antigens of cells

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 CAR antigen binding domain binds a cell surface antigen characteristic of a T cell, such as a cell surface antigen on a T cell. In some embodiments, the T cell-characteristic antigen may be a cell surface receptor, a membrane transporter (e.g., an active or passive transporter, e.g., an ion channel protein, a pore-forming protein, etc.), a transmembrane receptor, a membrane enzyme, and/or a T cell-characteristic cell adhesion protein. In some embodiments, the T cell-characteristic antigen may be 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 T cell receptor. In some embodiments, the T cell receptor may be AKT1、AKT2、AKT3、ATF2、BCL10、CALM1、CD3D(CD3δ)、CD3E(CD3ε)、CD3G(CD3γ)、CD4、CD8、CD28、CD45、CD80(B7-1)、CD86(B7-2)、CD247(CD3ζ)、CTLA-4(CD152)、ELK1、ERK1(MAPK3)、ERK2、FOS、FYN、GRAP2(GADS)、GRB2、HLA-DRA、HLA-DRB1、HLA-DRB3、HLA-DRB4、HLA-DRB5、HRAS、IKBKA(CHUK)、IKBKB、IKBKE、IKBKG(NEMO)、IL2、ITPR1、ITK、JUN、KRAS2、LAT、LCK、MAP2K1(MEK1)、MAP2K2(MEK2)、MAP2K3(MKK3)、MAP2K4(MKK4)、MAP2K6(MKK6)、MAP2K7(MKK7)、MAP3K1(MEKK1)、MAP3K3、MAP3K4、MAP3K5、MAP3K8、MAP3K14(NIK)、MAPK8(JNK1)、MAPK9(JNK2)、MAPK10(JNK3)、MAPK11(p38β)、MAPK12(p38γ)、MAPK13(p38δ)、MAPK14(p38α)、NCK、NFAT1、NFAT2、NFKB1、NFKB2、NFKBIA、NRAS、PAK1、PAK2、PAK3、PAK4、PIK3C2B、PIK3C3(VPS34)、PIK3CA、PIK3CB、PIK3CD、PIK3R1、PKCA、PKCB、PKCM、PKCQ、PLCY1、PRF1( perforin), PTEN, RAC1, RAF1, RELA, SDF1, SHP2, SLP76, SOS, SRC, TBK1, TCRA, TEC, TRAF6, VAV1, VAV2, or ZAP70.

7. Transmembrane domain

In some embodiments, the CAR transmembrane domain comprises at least one transmembrane region of: the α, β or ζ chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, 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. Antigen binding domain binding

8. A signaling domain or 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 /TNFSF4;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/SLAMF5;CD229/SLAMF3;CRACC/SLAMF7;NTB-A/SLAMF6;SLAM/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;DPPIV/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, 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 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.

9. Domain inducing cytokine gene expression following successful CAR signaling

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 immunology Reports, 27, 2017, 1 month, 37 (1).

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.

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, 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.

10. ABD comprising an antibody or antigen binding portion thereof

In some embodiments, the CAR antigen binding domain 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 CAR antigen binding domain comprises a CD19 antibody, a CD22 antibody, a T cell alpha chain antibody, a T cell beta chain antibody, a T cell gamma chain antibody, a T cell delta chain antibody, a CCR7 antibody, a CD3 antibody, a CD4 antibody, a CD5 antibody, a CD7 antibody, a CD8 antibody, a CD11B antibody, a CD11c antibody, a CD16 antibody, a CD20 antibody, a CD21 antibody, a CD25 antibody, a CD28 antibody, a CD34 antibody, a CD35 antibody, a CD40 antibody, a CD45RA antibody, a CD45RO antibody, a CD52 antibody, a CD56 antibody, a CD62L antibody, a CD68 antibody, a CD80 antibody, a CD95 antibody, a CD117 antibody, a CD127 antibody, a CD133 antibody, a CD137 (4-1 BB) antibody, a CD163 antibody, a F4/80 antibody, an IL-RA antibody, a Sca-1 antibody, a CTLA-4 antibody, a GITR antibody, a GARP antibody, a LAP antibody, a granzyme B antibody, a LFA-1 antibody, an MR1 antibody, a scFv antibody, or a Fab or a fragment of a par antibody.

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 addition to the CARs described herein, a variety of chimeric antigen receptors and nucleotide sequences encoding the same are known in the art and will be suitable for fusion delivery and reprogramming of target cells in vivo and in vitro as described herein. See, e.g., WO2013040557, WO2012079000, WO2016030414, smith T et al, nature nanotechnology.2017.DOI:10.1038/NNANO.2017.57, the disclosures of which are incorporated herein by reference.

Additional description of CAR

In certain embodiments, the cell can comprise an exogenous polynucleotide encoding a CAR. CARs (also known as chimeric immune receptors, chimeric T cell receptors, or artificial T cell receptors) are receptor proteins that have been engineered to give host cells (e.g., T cells) a novel ability to target a particular protein. The receptors are chimeric in that they bind antigen binding and T cell activation functions in a single receptor. The polycistronic vectors of the present disclosure can be used to express one or more CARs in a host cell (e.g., T cell) for cell-based therapies against various target antigens. CARs expressed by one or more expression cassettes may be the same or different. In these embodiments, the CAR may comprise an extracellular binding domain (also referred to as a "binding agent"), a transmembrane domain, and an intracellular signaling domain that specifically binds to a target antigen. In certain embodiments, the CAR may further comprise one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, and/or one or more intracellular co-stimulatory domains. The domains may be directly adjacent to each other or may have one or more amino acid linking domains. The nucleotide sequence encoding the CAR may be derived from a mammalian sequence, such as a mouse sequence, a primate sequence, a human sequence, or a combination thereof. Where the nucleotide sequence encoding the CAR is non-human, the sequence of the CAR may be humanized. The nucleotide sequence encoding the CAR may also be codon optimized for expression in mammalian cells, e.g., human cells. In any of these embodiments, the nucleotide sequence encoding the CAR can have at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to a nucleotide sequence disclosed herein. Sequence variations may be due to codon optimization, humanization, restriction enzyme-based cloning scars, and/or additional amino acid residues linking functional domains, etc.

In certain embodiments, the CAR may comprise a signal peptide at the N-terminus. Non-limiting examples of signal peptides include CD8 a signal peptide, igK signal peptide, and granulocyte-macrophage colony-stimulating factor receptor subunit a (GMCSFR-a, also known as colony-stimulating factor 2 receptor subunit a (CSF 2 RA)) signal peptide, and variants thereof, the amino acid sequences of which are provided in table 6 below.

TABLE 6 exemplary sequence of Signal peptides

In certain embodiments, the extracellular binding domain of the CAR may comprise one or more antibodies specific for a target antigen or multiple target antigens. The antibody may be an antibody fragment, such as an scFv, or a single domain antibody fragment, such as a VHH. In certain embodiments, scFv may comprise the heavy chain variable region (V _H) and the light chain variable region (V _L).V_H and V _L) of an antibody linked by a linker, which may be linked in any order, i.e., V _H -linker-V _L or V _L -linker-V _H non-limiting examples of linkers include Whitlow linkers, (G ₄S)_n (n may be a positive integer, e.g., 1, 2,3, 4, 5, 6, etc.) linkers and variants thereof; CS1/SLAMF7, CD38, CD138, GPRC5D, TACI and BCMA (associated with myeloma), GD2, HER2, EGFR, EGFRvIII, B H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FR alpha, IL-13R alpha, mesothelin, MUC1, MUC16 and ROR1 (associated with solid tumors.) in any of these embodiments, the extracellular binding domain of the CAR may be codon optimized for expression in the host cell, or have a variant sequence to increase the function of the extracellular binding domain.

In certain embodiments, the CAR may comprise a hinge domain, also referred to as a spacer region. The terms "hinge" and "spacer" may be used interchangeably throughout this disclosure. Non-limiting examples of hinge domains include the CD 8a hinge domain, CD28 hinge domain, igG4 hinge-CH 2-CH3 domain, and variants thereof, the amino acid sequences of which are provided in table 7 below.

TABLE 7 exemplary sequences of hinge domains

In certain embodiments, the transmembrane domain of the CAR may comprise the following transmembrane region: the α, β or ζ chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional variants thereof, including human forms of each of these sequences. In other embodiments, the transmembrane domain may comprise 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 functional variants thereof, including human forms of each of these sequences. Table 8 provides amino acid sequences of some exemplary transmembrane domains.

TABLE 8 exemplary sequences of transmembrane domains

In some embodiments of the present invention, in some embodiments, the intracellular signaling domain and/or intracellular co-stimulatory domain of the CAR may comprise one or more 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, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD/TNFRSF 7, 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/TNFbeta OX40/TNFRSF4, OX40 ligand /TNFSF4、RELT/TNFRSF19L、TACI/TNFRSF13B、TL1A/TNFSF15、TNFα、TNF RII/TNFRSF1B、2B4/CD244/SLAMF4、BLAME/SLAMF8、CD2、CD2F-10/SLAMF9、CD48/SLAMF2、CD58/LFA-3、CD84/SLAMF5、CD229/SLAMF3、CRACC/SLAMF7、NTB-A/SLAMF6、SLAM/CD150、CD2、CD7、CD53、CD82/Kai-1、CD90/Thy1、CD96、CD160、CD200、CD300a/LMIR1、HLA I, 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、DPPIV/CD26、EphB6、TIM-1/KIM-1/HAVCR、TIM-4、TSLP、TSLP R、 lymphocyte function-associated antigen-1 (LFA-1), NKG2C, CD3 zeta, immunoreceptor 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, ligand that specifically binds to CD83, and functional variants thereof, including human forms of each of these sequences. In some embodiments, the intracellular signaling domain and/or intracellular co-stimulatory domain comprises one or more signaling domains selected from the group consisting of a CD3 zeta domain, ITAM, CD28 domain, 4-1BB domain or a functional variant thereof. Table 9 provides amino acid sequences of some exemplary intracellular co-stimulatory and/or signaling domains. In certain embodiments, as in the case of Texarensaine described below, the CD3 zeta signaling domain of SEQ ID NO:18 may have a mutation at amino acid position 14, such as a glutamine (Q) to lysine (K) mutation (see SEQ ID NO: 115).

TABLE 9 exemplary sequences of intracellular costimulatory and/or signaling domains

In certain embodiments in which the polycistronic vector encodes two or more CARs, the two or more CARs may comprise the same functional domain, or one or more different functional domains, as described. For example, two or more CARs can comprise different signal peptides, extracellular binding domains, hinge domains, transmembrane domains, costimulatory domains, and/or intracellular signaling domains to minimize the risk of recombination due to sequence similarity. Or alternatively, two or more CARs may comprise the same domain. Where identical domains and/or backbones are used, codon differences are optionally introduced at the nucleotide sequence level to minimize the risk of recombination.

CD19 CAR

In some embodiments, the CAR is a CD19 CAR ("CD 19-CAR"), and in these embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding the CD19 CAR. In some embodiments, a CD19 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD19, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and/or an intracellular signaling domain in tandem.

In some embodiments, the signal peptide of the CD19 CAR comprises a CD 8a signal peptide. In some embodiments, the CD 8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID No. 6, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 6. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO. 7, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID NO. 7. In some embodiments, the signal peptide comprises GMCSFR- α or CSF2RA signal peptide. In some embodiments, GMCSFR- α or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID No. 8 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 8.

In some embodiments, the extracellular binding domain of the CD19 CAR is specific for CD19 (e.g., human CD 19). The extracellular binding domain of a CD19 CAR may be codon optimized for expression in a host cell, or have variant sequences to increase the function of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, e.g., an scFv.

In some embodiments, the extracellular binding domain of the CD19 CAR comprises an scFv derived from an FMC63 monoclonal antibody (FMC 63) comprising a heavy chain variable region (V _H) and a light chain variable region (V _L) of FMC63 linked by a linker. FMC63 and derived scFv have been described in Nicholson et al, mol. Immun.34 (16-17): 1157-1165 (1997) and PCT application publication No. WO2018/213337, the entire contents of each of which are incorporated herein by reference. In some embodiments, the amino acid sequences of the complete FMC 63-derived scFv (also referred to as FMC63 scFv) and the different portions thereof are provided in table 10 below. In some embodiments, the CD 19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID No. 19, 20, or 25, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with an amino acid sequence set forth in SEQ ID No. 19, 20, or 25. In some embodiments, the CD 19-specific scFv may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOS.21-23 and 26-28. In some embodiments, a CD 19-specific scFv may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS.21-23. In some embodiments, a CD 19-specific scFv may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS.26-28. In any of these embodiments, the CD 19-specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence that has at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with any sequence identified. In some embodiments, the extracellular binding domain of a CD19 CAR comprises or consists of one or more CDRs as described herein.

In some embodiments, the linker connecting the V _H and V _L portions of the scFv is a Whitlow linker having the amino acid sequence set forth in SEQ ID NO. 24. In some embodiments, the Whitlow linker may be replaced with a different linker, for example a 3xG ₄ S linker having the amino acid sequence set forth in SEQ ID NO. 30, which results in a different FMC 63-derived scFv having the amino acid sequence set forth in SEQ ID NO. 29. In some of these embodiments, the CD 19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO. 29, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID NO. 29.

TABLE 10 exemplary sequences of anti-CD 19 scFv and components

In some embodiments, the extracellular binding domain of CD19CAR is derived from CD 19-specific antibodies, including, for example, SJ25C1 (Bejcek et al, cancer Res.55:2346-2351 (1995)), HD37 (Pezutto et al, J.Immunol.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)), B4HB12B (Kansas & Tedder, J.Immunol.147:4094-4102 (1991)), yazawa et al, proc.Natl. Acad. Sci.USA 102:15178-15183 (2005)), J.Pharmacol.exp. Ther.213-222 (2010), B4 (Callard:418-427 (1987)), B4 (1997), B4 (1999) and B.Immunol.335.335-148 (1989)). In any of these embodiments, the extracellular binding domain of the CD19CAR may comprise or consist of V _H、V_L and/or one or more CDRs of any antibody.

In some embodiments, the hinge domain of the CD19 CAR comprises a CD 8a hinge domain, e.g., a human CD 8a hinge domain. In some embodiments, the CD 8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 9, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 9. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 10, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 10. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with an amino acid sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch 2-Ch3 domain, e.g., a human IgG4 hinge-Ch 2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch 2-Ch3 domain comprises or consists of the amino acid sequence set forth in SEQ ID NO. 13, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to the amino acid sequence set forth in SEQ ID NO. 13.

In some embodiments, the transmembrane domain of the CD19 CAR comprises a CD 8a transmembrane domain, e.g., a human CD 8a transmembrane domain. In some embodiments, the CD 8a transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 14, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 14. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 15, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 15.

In some embodiments, the intracellular co-stimulatory domain of the CD19 CAR comprises a 4-1BB co-stimulatory domain. 4-1BB (also known as CD 137) transmits potent costimulatory signals to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. In some embodiments, the 4-1BB costimulatory domain is human. In some embodiments, the 4-1BB co-stimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO. 16, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID NO. 16. In some embodiments, the intracellular co-stimulatory domain comprises a CD28 co-stimulatory domain. CD28 is another costimulatory molecule on T cells. In some embodiments, the CD28 co-stimulatory domain is human. In some embodiments, the CD28 co-stimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 17, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 17. In some embodiments, the intracellular co-stimulatory domain of the CD19 CAR comprises a 4-1BB co-stimulatory domain and a CD28 co-stimulatory domain as described.

In some embodiments, the intracellular signaling domain of the CD19 CAR comprises a CD3zeta (ζ) signaling domain. Cd3ζ binds to T Cell Receptor (TCR) generating a signal and contains an immunoreceptor tyrosine-based activation motif (ITAM). The CD3zeta signaling domain refers to an amino acid residue from the zeta chain cytoplasmic domain that is sufficient to functionally transmit the initial signal necessary for T cell activation. In some embodiments, the CD3zeta signaling domain is human. In some embodiments, the CD3zeta signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 18, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 18.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR, the CD19 CAR comprising, for example, a CD19 CAR comprising: a CD19 specific scFv having the sequence set forth in SEQ ID No. 19 or SEQ ID No. 29, a CD8 a hinge domain of SEQ ID No.9, a CD8 a transmembrane domain of SEQ ID No. 14, a 4-1BB costimulatory domain of SEQ ID No. 16, a CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the disclosed sequences). In any of these embodiments, the CD19 CAR can additionally comprise a signal peptide as described (e.g., a CD8 a signal peptide).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR, the CD19 CAR comprising, for example, a CD19 CAR comprising: a CD19 specific scFv having the sequence set forth in SEQ ID No. 19 or SEQ ID No. 29, an IgG4 hinge domain of SEQ ID No. 11 or SEQ ID No. 12, a CD28 transmembrane domain of SEQ ID No. 15, a 4-1BB costimulatory domain of SEQ ID No. 16, a CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the disclosed sequences). In any of these embodiments, the CD19 CAR can additionally comprise a signal peptide as described (e.g., a CD8 a signal peptide).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR, the CD19 CAR comprising, for example, a CD19 CAR comprising: a CD19 specific scFv having the sequence set forth in SEQ ID No. 19 or SEQ ID No. 29, a CD28 hinge domain of SEQ ID No. 10, a CD28 transmembrane domain of SEQ ID No. 15, a CD28 costimulatory domain of SEQ ID No. 17, a CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identity to the disclosed sequences). In any of these embodiments, the CD19 CAR can additionally comprise a signal peptide as described (e.g., a CD8 a signal peptide).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19 CAR set forth in SEQ ID NO:116 or having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the nucleotide sequence set forth in SEQ ID NO:116 (see Table 11). The encoded CD19 CAR has the corresponding amino acid sequence set forth in SEQ ID No. 117, or has at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 117, with the following components: CD8 a signal peptide, FMC63 scFv (V _L -Whitlow linker-V _H), CD8 a hinge domain, CD8 a transmembrane domain, 4-1BB costimulatory domain, and CD3 zeta signaling domain.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a commercially available embodiment of the CD19 CAR. Non-limiting examples of commercial embodiments of CD19 CARs expressed and/or encoded by T cells include temozolomide, li Jimai's colese, alzem, and briyl olmesartan.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a texarenate or a portion thereof. The temsiren comprises a CD19CAR having the following composition: CD8 a signal peptide, FMC63 scFv (V _L-3xG₄ S linker-V _H), CD8 a hinge domain, CD8 a transmembrane domain, 4-1BB co-stimulatory domain and CD3 zeta signaling domain. The nucleotide and amino acid sequences of CD19CAR in texarensai are provided in table 11 and the comments of the sequences are provided in table 12.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding Li Jimai th, or a portion thereof. Li Jimai the pharmaceutical composition comprises a CD19CAR having the following composition: GMCSFR-alpha or CSF2RA signal peptide, FMC63 scFv (V _L -Whitlow linker-V _H), igG4 hinge domain, CD28 transmembrane domain, 4-1BB costimulatory domain, and CD3 zeta signaling domain. The nucleotide and amino acid sequences of CD19CAR in Li Jimai's rence are provided in table 11 and the comments of the sequences are provided in table 13.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding alemtuquone or a portion thereof. The alopecie comprises a CD19CAR having the following composition: GMCSFR-alpha or CSF2RA signal peptide, FMC63 scFv (V _L -Whitlow linker-V _H), CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain and CD3 zeta signaling domain. The nucleotide and amino acid sequences of CD19CAR in alemtujopsis are provided in table 11 and the comments of the sequences are provided in table 14.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding briyl alendronate or a portion thereof. The briyl alendronate comprises a CD19 CAR having the following composition: GMCSFR-alpha signal peptide, FMC63 scFv, CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3 zeta signaling domain.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19CAR set forth in SEQ ID No. 31, 33, or 35 or having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to a nucleotide sequence set forth in SEQ ID No. 31, 33, or 35. The encoded CD19CAR has a corresponding amino acid sequence set forth in SEQ ID No. 32, 34, or 36, respectively, or has at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to an amino acid sequence set forth in SEQ ID No. 32, 34, or 36, respectively.

Table 11. Exemplary sequences of cd19 CAR

TABLE 12 annotation of Tixarensai CD19 CAR sequence

TABLE 13 annotation of Li Jimai Lemci CD19 CAR sequence

TABLE 14 annotation of the Alcalamine CD19 CAR sequence

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD19CAR set forth in SEQ ID No. 31, 33, or 35 or having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to a nucleotide sequence set forth in SEQ ID No. 31, 33, or 35. The encoded CD19CAR has a corresponding amino acid sequence set forth in SEQ ID No. 32, 34, or 36, respectively, having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to the amino acid sequence set forth in SEQ ID No. 32, 34, or 36, respectively.

CD20 CAR

In some embodiments, the CAR is a CD20 CAR ("CD 20-CAR"), and in these embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding the CD20 CAR. CD20 is an antigen that is found on the surface of B cells early in the pre-B phase and at progressively increasing levels until the B cells mature, as well as on cells of most B cell tumors. CD20 positive cells are sometimes also found in cases of hodgkin's disease, myeloma and thymoma. In some embodiments, a CD20 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD20, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and/or an intracellular signaling domain in tandem.

In some embodiments, the signal peptide of the CD20 CAR comprises a CD 8a signal peptide. In some embodiments, the CD 8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID No. 6, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 6. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO. 7, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID NO. 7. In some embodiments, the signal peptide comprises GMCSFR- α or CSF2RA signal peptide. In some embodiments, GMCSFR- α or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID No. 8 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 8.

In some embodiments, the extracellular binding domain of the CD20 CAR is specific for CD20 (e.g., human CD 20). The extracellular binding domain of a CD20 CAR may be codon optimized for expression in a host cell, or have variant sequences to increase the function of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, e.g., an scFv.

In some embodiments, the extracellular binding domain of the CD20CAR is derived from a CD20 specific antibody, including, for example, leu16, IF5, 1.5.3, rituximab, otouzumab, timox, ofatuzumab, tositumumab, ornitumumab, veltuzumab, rituximab, and ore Li Zhushan antibodies. In some embodiments, the CD20CAR is derived from a CD 20-specific CAR, including, for example, MB-106, UCART20, or C-CAR066, as detailed in table 15A. In any of these embodiments, the extracellular binding domain of the CD20CAR may comprise or consist of V _H、V_L and/or one or more CDRs of any of the antibodies or CARs detailed in table 15A.

Table 15A. Exemplary CD 20-specific CARs

In some embodiments, the extracellular binding domain of the CD20 CAR comprises an scFv derived from a Leu16 monoclonal antibody comprising a heavy chain variable region (V _H) and a light chain variable region (V _L) of Leu16 linked by a linker. See Wu et al, protein engineering.14 (12): 1025-1033 (2001). In some embodiments, the linker is a 3xG ₄ S linker. In other embodiments, the linker is a Whitlow linker as described herein. In some embodiments, the amino acid sequences of the different portions of the complete Leu 16-derived scFv (also referred to as the Leu16 scFv) and their different portions are provided in table 15B below. In some embodiments, the CD 20-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID No. 37, 38, or 42, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) with an amino acid sequence set forth in SEQ ID No. 37, 38, or 42. In some embodiments, the CD 20-specific scFv may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 39-41, 43 and 44. In some embodiments, a CD 20-specific scFv may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS: 39-41. In some embodiments, a CD 20-specific scFv may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS: 43-44. In any of these embodiments, the CD 20-specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence that has at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with any sequence identified. In some embodiments, the extracellular binding domain of a CD20 CAR comprises or consists of one or more CDRs as described herein.

Table 15B exemplary sequences of anti-CD 20 scFv and Components

In some embodiments, the hinge domain of the CD20 CAR comprises a CD 8a hinge domain, e.g., a human CD 8a hinge domain. In some embodiments, the CD 8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 9, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 9. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 10, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 10. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with an amino acid sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch 2-Ch3 domain, e.g., a human IgG4 hinge-Ch 2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch 2-Ch3 domain comprises or consists of the amino acid sequence set forth in SEQ ID NO. 13, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to the amino acid sequence set forth in SEQ ID NO. 13.

In some embodiments, the transmembrane domain of the CD20 CAR comprises a CD 8a transmembrane domain, e.g., a human CD 8a transmembrane domain. In some embodiments, the CD 8a transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 14, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 14. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 15, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 15.

In some embodiments, the intracellular co-stimulatory domain of the CD20 CAR comprises a 4-1BB co-stimulatory domain, e.g., a human 4-1BB co-stimulatory domain. In some embodiments, the 4-1BB co-stimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO.16, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID NO. 16. In some embodiments, the intracellular co-stimulatory domain comprises a CD28 co-stimulatory domain, e.g., a human CD28 co-stimulatory domain. In some embodiments, the CD28 co-stimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 17, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 17.

In some embodiments, the intracellular signaling domain of the CD20 CAR comprises a CD3zeta (ζ) signaling domain, e.g., a human CD3 ζ signaling domain. In some embodiments, the CD3zeta signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 18, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 18.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, the CD20 CAR comprising, for example, a CD20 CAR comprising: a CD20 specific scFv having the sequence set forth in SEQ ID No. 37, a CD8 a hinge domain of SEQ ID No. 9, a CD8 a transmembrane domain of SEQ ID No. 14, a 4-1BB costimulatory domain of SEQ ID No. 16, a CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identity to the disclosed sequences).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, the CD20 CAR comprising, for example, a CD20 CAR comprising: a CD20 specific scFv having the sequence set forth in SEQ ID No. 37, a CD28 hinge domain of SEQ ID No. 10, a CD8 a transmembrane domain of SEQ ID No. 14, a 4-1BB costimulatory domain of SEQ ID No. 16, a CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the disclosed sequences).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, the CD20 CAR comprising, for example, a CD20 CAR comprising: a CD20 specific scFv having the sequence set forth in SEQ ID No. 37, an IgG4 hinge domain of SEQ ID No. 11 or SEQ ID No. 12, a CD8 a transmembrane domain of SEQ ID No. 14, a 4-1BB costimulatory domain of SEQ ID No. 16, a CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identity to the disclosed sequences).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, the CD20 CAR comprising, for example, a CD20 CAR comprising: a CD20 specific scFv having the sequence set forth in SEQ ID No. 37, a CD8 a hinge domain of SEQ ID No. 9, a CD28 transmembrane domain of SEQ ID No. 15, a 4-1BB costimulatory domain of SEQ ID No. 16, a CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the disclosed sequences).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, the CD20 CAR comprising, for example, a CD20 CAR comprising: a CD20 specific scFv having the sequence set forth in SEQ ID No. 37, a CD28 hinge domain of SEQ ID No. 10, a CD28 transmembrane domain of SEQ ID No. 15, a 4-1BB costimulatory domain of SEQ ID No. 16, a CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the disclosed sequences).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD20 CAR, the CD20 CAR comprising, for example, a CD20 CAR comprising: a CD20 specific scFv having the sequence set forth in SEQ ID No. 37, an IgG4 hinge domain of SEQ ID No. 11 or SEQ ID No. 1, a CD28 transmembrane domain of SEQ ID No. 15, a 4-1BB costimulatory domain of SEQ ID No. 16, a CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identity to the disclosed sequences).

CD22 CAR

In some embodiments, the CAR is a CD22 CAR ("CD 22-CAR"), and in these embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding the 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' slymphoma) and is absent on the cell surface or stem cells at the early stages of B cell development. In some embodiments, the CD22 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD22, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and/or an intracellular signaling domain in tandem.

In some embodiments, the signal peptide of the CD22 CAR comprises a CD 8a signal peptide. In some embodiments, the CD 8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID No. 6, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 6. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO. 7, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID NO. 7. In some embodiments, the signal peptide comprises GMCSFR- α or CSF2RA signal peptide. In some embodiments, GMCSFR- α or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID No. 8 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 8.

In some embodiments, the extracellular binding domain of the CD22 CAR is specific for CD22 (e.g., human CD 22). The extracellular binding domain of a CD22 CAR may be codon optimized for expression in a host cell, or have variant sequences to increase the function of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, e.g., an scFv.

In some embodiments, the extracellular binding domain of the CD22 CAR is derived from a CD22 specific antibody, including, for example, SM03, oantituzumab, epalizumab, mositumomab, and pinatuzumab. In any of these embodiments, the extracellular binding domain of the CD22 CAR can comprise or consist of V _H、V_L and/or one or more CDRs of any antibody.

In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv derived from an m971 monoclonal antibody (m 971) comprising a heavy chain variable region (V _H) and a light chain variable region (V _L) of m971 linked by a linker. In some embodiments, the linker is a 3xG ₄ S linker. In other embodiments, whitlow linkers may be used instead. In some embodiments, the amino acid sequences of the entire m 971-derived scFv (also referred to as the m971 scFv) and the different portions thereof are provided in table 16 below. In some embodiments, the CD 22-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID No. 45, 46, or 50, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with an amino acid sequence set forth in SEQ ID No. 45, 46, or 50. In some embodiments, the CD 22-specific scFv may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOS.47-49 and 51-53. In some embodiments, a CD 22-specific scFv may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS.47-49. In some embodiments, a CD 22-specific scFv may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS: 51-53. In any of these embodiments, the CD 22-specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence that has at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with any sequence identified. In some embodiments, the extracellular binding domain of a CD22 CAR comprises or consists of one or more CDRs as described herein.

In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv 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 derived from m971-L7 comprises V _H and V _L of m971-L7 linked by a 3xG ₄ S linker. In other embodiments, whitlow linkers may be used instead. In some embodiments, the amino acid sequences of the complete m971-L7 derived scFv (also referred to as m971-L7 scFv) and the different portions thereof are provided in Table 16 below. In some embodiments, the CD 22-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID No. 54, 55, or 59, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with an amino acid sequence set forth in SEQ ID No. 54, 55, or 59. In some embodiments, the CD 22-specific scFv may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 56-58 and 60-62. In some embodiments, a CD 22-specific scFv may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 56-58. In some embodiments, a CD 22-specific scFv may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 60-62. In any of these embodiments, the CD 22-specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence that has at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with any sequence identified. In some embodiments, the extracellular binding domain of a CD22 CAR comprises or consists of one or more CDRs as described herein.

TABLE 16 exemplary sequences of anti-CD 22 scFv and components

In some embodiments, the extracellular binding domain of the CD22 CAR comprises immunotoxin HA22 or BL22. Immunotoxins BL22 and HA22 are therapeutic agents comprising CD 22-specific scFv 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 immunotoxin) is a mutated, higher affinity form of BL22 (Ho et al, j. Biol. Chem.,280 (1): 607-17 (2005)). Suitable sequences of 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 incorporated by reference in their entirety.

In some embodiments, the hinge domain of the CD22 CAR comprises a CD 8a hinge domain, e.g., a human CD 8a hinge domain. In some embodiments, the CD 8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 9, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 9. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 10, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 10. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with an amino acid sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch 2-Ch3 domain, e.g., a human IgG4 hinge-Ch 2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch 2-Ch3 domain comprises or consists of the amino acid sequence set forth in SEQ ID NO. 13, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to the amino acid sequence set forth in SEQ ID NO. 13.

In some embodiments, the transmembrane domain of the CD22 CAR comprises a CD 8a transmembrane domain, e.g., a human CD 8a transmembrane domain. In some embodiments, the CD 8a transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 14, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 14. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 15, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 15.

In some embodiments, the intracellular co-stimulatory domain of the CD22 CAR comprises a 4-1BB co-stimulatory domain, e.g., a human 4-1BB co-stimulatory domain. In some embodiments, the 4-1BB co-stimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO.16, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID NO. 16. In some embodiments, the intracellular co-stimulatory domain comprises a CD28 co-stimulatory domain, e.g., a human CD28 co-stimulatory domain. In some embodiments, the CD28 co-stimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 17, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 17.

In some embodiments, the intracellular signaling domain of the CD22 CAR comprises a CD3zeta (zeta) signaling domain, e.g., a human CD3zeta signaling domain. In some embodiments, the CD3zeta signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 18, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 18.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, the CD22 CAR comprising, for example, a CD22 CAR comprising: a CD 22-specific scFv having the sequence set forth in SEQ ID No. 45 or SEQ ID No. 54, the CD 8a hinge domain of SEQ ID No. 9, the CD 8a transmembrane domain of SEQ ID No. 14, the 4-1BB costimulatory domain of SEQ ID No. 16, the CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the disclosed sequences).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, the CD22 CAR comprising, for example, a CD22 CAR comprising: a CD 22-specific scFv having the sequence set forth in SEQ ID No. 45 or SEQ ID No. 54, the CD28 hinge domain of SEQ ID No. 10, the CD8 a transmembrane domain of SEQ ID No. 14, the 4-1BB costimulatory domain of SEQ ID No. 16, the CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identity to the disclosed sequences).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, the CD22 CAR comprising, for example, a CD22 CAR comprising: a CD 22-specific scFv having the sequence set forth in SEQ ID No. 45 or SEQ ID No. 54, an IgG4 hinge domain of SEQ ID No. 11 or SEQ ID No. 12, a CD8 a transmembrane domain of SEQ ID No. 14, a 4-1BB costimulatory domain of SEQ ID No. 16, a CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the disclosed sequences).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, the CD22 CAR comprising, for example, a CD22 CAR comprising: a CD 22-specific scFv having the sequence set forth in SEQ ID No. 45 or SEQ ID No. 54, the CD 8a hinge domain of SEQ ID No. 9, the CD28 transmembrane domain of SEQ ID No. 15, the 4-1BB costimulatory domain of SEQ ID No. 16, the CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identity to the disclosed sequences).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, the CD22 CAR comprising, for example, a CD22 CAR comprising: a CD 22-specific scFv having the sequence set forth in SEQ ID No. 45 or SEQ ID No. 54, the CD28 hinge domain of SEQ ID No. 10, the CD28 transmembrane domain of SEQ ID No. 15, the 4-1BB costimulatory domain of SEQ ID No. 16, the CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identity to the disclosed sequences).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a CD22 CAR, the CD22 CAR comprising, for example, a CD22 CAR comprising: a CD 22-specific scFv having the sequence set forth in SEQ ID No. 45 or SEQ ID No. 54, an IgG4 hinge domain of SEQ ID No. 11 or SEQ ID No. 12, a CD28 transmembrane domain of SEQ ID No. 15, a 4-1BB costimulatory domain of SEQ ID No. 16, a CD3 zeta signaling domain of SEQ ID No. 18 and/or variants thereof (i.e. sequences having at least 80% identity, e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the disclosed sequences).

BCMACAR

In some embodiments, the CAR is BCMACAR ("BCMA-CAR"), and in these embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding 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 may comprise a signal peptide, an extracellular binding domain that specifically binds BCMA, a hinge domain, a transmembrane domain, an intracellular co-stimulatory domain, and/or an intracellular signaling domain in tandem.

In some embodiments, the signal peptide of BCMACAR comprises a CD 8a signal peptide. In some embodiments, the CD 8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID No. 6, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 6. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO. 7, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID NO. 7. In some embodiments, the signal peptide comprises GMCSFR- α or CSF2RA signal peptide. In some embodiments, GMCSFR- α or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID No. 8 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 8.

In some embodiments, the extracellular binding domain of BCMACAR is specific for BCMA (e.g., human BCMA). BCMACAR extracellular binding domain can be codon optimized for expression in a host cell, or have variant sequences to increase the extracellular binding domain function.

In some embodiments, the extracellular binding domain comprises an immunogenic active portion of an immunoglobulin molecule, e.g., an scFv. In some embodiments, the extracellular binding domain of BCMACAR is derived from BCMA-specific antibodies, including, for example, bei Lan tamab, erlenmeratab, terlipstatin, LCAR-B38M, and sidase. In any of these embodiments, the extracellular binding domain of BCMACAR may comprise or consist of V _H、V_L and/or one or more CDRs of any antibody.

In some embodiments, the extracellular binding domain of BCMACAR comprises a scFv derived from C11D5.3, which is a murine monoclonal antibody as described in Carpenter et al, clin.cancer Res.19 (8): 2048-2060 (2013). See also PCT application publication No. WO 2010/104949. The c11d5.3 derived scFv may comprise a heavy chain variable region (V _H) and a light chain variable region (V _L) of c11d5.3 linked by a Whitlow linker, the amino acid sequences of which are provided in table 17. In some embodiments, the BCMA specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 63, 64, or 68, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 63, 64, or 68. In some embodiments, the BCMA specific extracellular binding domain may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 65-67 and 69-71. In some embodiments, the BCMA specific extracellular binding domain may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 65-67. In some embodiments, the BCMA specific extracellular binding domain may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 69-71. In any of these embodiments, the BCMA specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to any sequence identified. In some embodiments, the extracellular binding domain of BCMACAR comprises or consists of one or more CDRs as described herein.

In some embodiments, the extracellular binding domain of BCMACAR comprises an scFv derived from another murine monoclonal antibody C12A3.2, such as described by Carpenter et al, clin.cancer Res.19 (8): 2048-2060 (2013) and PCT application publication No. WO2010/104949, the amino acid sequences of which are also provided in Table 17 below. In some embodiments, the BCMA specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 72, 73, or 77, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 72, 73, or 77. In some embodiments, the BCMA specific extracellular binding domain may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 74-76 and 78-80. In some embodiments, the BCMA specific extracellular binding domain may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 74-76. In some embodiments, the BCMA specific extracellular binding domain may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS: 78-80. In any of these embodiments, the BCMA specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to any sequence identified. In some embodiments, the extracellular binding domain of BCMACAR comprises or consists of one or more CDRs as described herein.

In some embodiments, the extracellular binding domain of BCMACAR comprises a murine monoclonal antibody with high specificity for human BCMA, designated BB2121 in Friedman et al, hum. Gene Ther.29 (5): 585-601 (2018). See also PCT application publication No. WO2012163805.

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. Oncol.11 (1): 141 (2018), also known as LCAR-B38M. See also PCT application publication No. WO2018/028647.

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), also referred to as FHVH. See also PCT application publication No. WO2019/006072. The amino acid sequences of FHVH and its CDRs are provided in table 17 below. In some embodiments, the BCMA specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 81 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 81. In some embodiments, the BCMA specific extracellular binding domain may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 82-84. In any of these embodiments, the BCMA specific extracellular binding domain can comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to any sequence identified. In some embodiments, the extracellular binding domain of BCMACAR comprises or consists of one or more CDRs as described herein.

In some embodiments, the extracellular binding domain of BCMACAR comprises an scFv derived from CT103A (or CAR 0085), as described in U.S. patent No. 11,026,975B2, the amino acid sequences of which are provided in table 17 below. In some embodiments, the BCMA specific extracellular binding domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 118, 119, or 123, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 118, 119, or 123. In some embodiments, the BCMA specific extracellular binding domain may comprise one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 120-122 and 124-126. In some embodiments, the BCMA specific extracellular binding domain may comprise a light chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 120-122. In some embodiments, the BCMA specific extracellular binding domain may comprise a heavy chain having one or more CDRs having the amino acid sequences set forth in SEQ ID NOS 124-126. In any of these embodiments, the BCMA specific scFv can comprise one or more CDRs comprising one or more amino acid substitutions or comprising a sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to any sequence identified. In some embodiments, the extracellular binding domain of BCMACAR comprises or consists of one or more CDRs as described herein.

Additionally, BCMA-directed CARs and binding agents have been described in U.S. application publication nos. 2020/0246681 A1 and 2020/0339699A1, the entire contents of each of which are incorporated herein by reference.

TABLE 17 exemplary sequences of anti-BCMA binders and components

In some embodiments, the hinge domain of BCMACAR comprises a CD 8a hinge domain, e.g., a human CD 8a hinge domain. In some embodiments, the CD 8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 9, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 9. In some embodiments, the hinge domain comprises a CD28 hinge domain, e.g., a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 10, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 10. In some embodiments, the hinge domain comprises an IgG4 hinge domain, e.g., a human IgG4 hinge domain. In some embodiments, the IgG4 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) with an amino acid sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12. In some embodiments, the hinge domain comprises an IgG4 hinge-Ch 2-Ch3 domain, e.g., a human IgG4 hinge-Ch 2-Ch3 domain. In some embodiments, the IgG4 hinge-Ch 2-Ch3 domain comprises or consists of the amino acid sequence set forth in SEQ ID NO. 13, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to the amino acid sequence set forth in SEQ ID NO. 13.

In some embodiments, the transmembrane domain of BCMACAR comprises a CD 8a transmembrane domain, e.g., a human CD 8a transmembrane domain. In some embodiments, the CD 8a transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 14, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 14. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain, e.g., a human CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 15, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 15.

In some embodiments, the intracellular co-stimulatory domain of BCMACAR comprises a 4-1BB co-stimulatory domain, e.g., a human 4-1BB co-stimulatory domain. In some embodiments, the 4-1BB co-stimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID NO. 16, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID NO. 16. In some embodiments, the intracellular co-stimulatory domain comprises a CD28 co-stimulatory domain, e.g., a human CD28 co-stimulatory domain. In some embodiments, the CD28 co-stimulatory domain comprises or consists of the amino acid sequence set forth in SEQ ID No. 17, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 17.

In some embodiments, the intracellular signaling domain of BCMACAR comprises a CD3zeta (ζ) signaling domain, e.g., a human CD3 ζ signaling domain. In some embodiments, the CD3zeta signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID No. 18, or comprises or consists of an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to an amino acid sequence set forth in SEQ ID No. 18.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding BCMACAR, including BCMACAR comprising any of, for example, a BCMA-specific extracellular binding domain as described, a CD8 a hinge domain of SEQ ID NO:9, a CD8 a transmembrane domain of SEQ ID NO:14, a 4-1BB costimulatory domain of SEQ ID NO:16, a CD3 zeta signaling domain of SEQ ID NO:18, and/or variants thereof (i.e., sequences having at least 80% identity, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identity to the disclosed sequences). In any of these embodiments, BCMACAR may additionally comprise a signal peptide as described (e.g., a CD8 a signal peptide).

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding BCMACAR, including BCMACAR comprising any of, for example, a BCMA-specific extracellular binding domain as described, a CD 8a hinge domain of SEQ ID NO:9, a CD 8a transmembrane domain of SEQ ID NO:14, a CD28 co-stimulatory domain of SEQ ID NO:17, a CD3 zeta signaling domain of SEQ ID NO:18, and/or variants thereof (i.e., sequences having at least 80% identity, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99 identity to the disclosed sequences). In any of these embodiments BCMACAR may additionally comprise a signal peptide as described.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding BCMACAR set forth in SEQ ID NO:127 or having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the nucleotide sequence set forth in SEQ ID NO:127 (see Table 18). The coded BCMACAR has the corresponding amino acid sequence set forth in SEQ ID No. 128, or has at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 128, with the following components: CD 8a signal peptide, CT103A scFv (V _L -Whitlow linker-V _H), CD 8a hinge domain, CD 8a transmembrane domain, 4-1BB costimulatory domain, and CD3 zeta signaling domain.

In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding a commercially available BCMACAR embodiment, including, for example, ai Jiwei renieratene (also known as bb 2121). In some embodiments, the polycistronic vector comprises an expression cassette comprising a nucleotide sequence encoding Ai Jiwei th, or a portion thereof. Ai Jiwei the pharmaceutical composition comprises BCMACAR having the following composition: BB2121 binding agent, CD8 alpha hinge domain, CD8 alpha transmembrane domain, 4-1BB costimulatory domain, and CD3 zeta signaling domain.

TABLE 18 exemplary sequences of BCMAAR

Characterization of hypoimmunogenic cells

In some embodiments, the population of low-immunogenicity stem cells retains pluripotency as compared to a control stem cell (e.g., a wild-type stem cell or an immunogenic stem cell). In some embodiments, the population of low-immunogenic stem cells retains differentiation potential compared to a control stem cell (e.g., a wild-type stem cell or an immunogenic stem cell).

In some embodiments, the population of low-immunogenic cells (such as low-immunogenic differentiated cells and CAR-T cells) administered elicits reduced or lower levels of immune activation in the subject or patient. In some cases, the level of immune activation elicited by the low-immunogenicity 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 immune activation produced by administration of the immunogenic cells. In some embodiments, the administered population of low-immunogenicity cells fails to elicit immune activation in the subject or patient.

In some embodiments, the population of low-immunogenic cells (such as low-immunogenic differentiated cells and CAR-T cells) administered elicit a reduced or lower level of T cell response in the subject or patient. In some cases, the low-immunogenicity cell elicits a T cell response 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 the T cell response level produced by administration of the immunogenic cell. In some embodiments, the administered population of low-immunogenicity cells fails to elicit a T cell response to the cells in the subject or patient.

In some embodiments, the population of low-immunogenic cells (such as low-immunogenic differentiated cells and CAR-T cells) administered elicit a reduced or lower level of NK cell response in the subject or patient. In some cases, the low immunogenic cells elicit NK cell response 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 NK cell response levels produced by administration of the immunogenic cells. In some embodiments, the administered population of low-immunogenicity cells fails to elicit an NK cell response to the cells in the subject or patient.

In some embodiments, the population of low-immunogenic cells (such as low-immunogenic differentiated cells and CAR-T cells) administered initiates reduced or lower levels of macrophage phagocytosis in the subject or patient. In some cases, the level of NK cell response elicited by the hypoimmunogenic 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 as compared to the level of phagocytosis by macrophages produced by administration of the immunogenic cells. In some embodiments, the administered population of low-immunogenicity cells fails to elicit macrophage phagocytosis of cells in the subject or patient.

In some embodiments, the population of low-immunogenic cells (such as low-immunogenic differentiated cells and CAR-T cells) administered induces reduced or lower levels of systemic TH1 activation in the subject or patient. In some cases, the low-immunogenicity cells elicit a level of systemic TH1 activation 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 the level of systemic TH1 activation produced by administration of the immunogenic cells. In some embodiments, the administered population of low-immunogenicity cells fails to elicit systemic TH1 activation in the subject or patient.

In some embodiments, the administered population of low-immunogenic cells (such as low-immunogenic differentiated cells and CAR-T cells) induces a reduced or lower level of NK cell killing in the subject or patient. In some cases, the NK cell killing level elicited by the low-immunogenicity 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 NK cell killing level produced by administration of the immunogenic cells. In some embodiments, the low immunogenic cell population administered fails to trigger NK cell killing in the subject or patient.

In some embodiments, the administered population of low-immunogenic cells, such as low-immunogenic differentiated cells and CAR-T cells, induces a reduced or lower level of immune activation of Peripheral Blood Mononuclear Cells (PBMCs) in the subject or patient. In some cases, the level of immune activation of the PBMCs elicited by the low-immunogenicity 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 immune activation of the PBMCs produced by administration of the immunogenic cells. In some embodiments, the administered population of hypoimmunogenic cells fails to elicit immune activation of PBMCs in the subject or patient.

In some embodiments, the population of low-immunogenic cells (such as low-immunogenic differentiated cells and CAR-T cells) administered elicit reduced or lower levels of donor-specific IgG antibodies in the subject or patient. In some cases, the low immunogenic 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 the donor-specific IgG antibody level produced by administration of the immunogenic cell. In some embodiments, the low immunogenic cell population administered is incapable of eliciting donor-specific IgG antibodies in the subject or patient.

In some embodiments, the population of low-immunogenic cells (such as low-immunogenic differentiated cells and CAR-T cells) administered elicit reduced or lower levels of donor-specific IgM antibodies in the subject or patient. In some cases, the low immunogenic cell elicits donor-specific IgM antibody 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 donor-specific IgM antibody levels produced by administration of the immunogenic cell. In some embodiments, the administered population of low-immunogenicity cells is incapable of eliciting donor-specific IgM antibodies in the subject or patient.

In some embodiments, the administered population of low-immunogenic cells (such as low-immunogenic differentiated cells and CAR-T cells) elicit reduced or lower levels of IgM and IgG antibody production in the subject or patient. In some cases, the low immunogenic 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 the immunogenic cells. In some embodiments, the administered population of low-immunogenicity cells is incapable of eliciting IgM and IgG antibody production in the subject or patient.

In some embodiments, the population of low-immunogenic cells (such as low-immunogenic differentiated cells and CAR-T cells) administered induces a reduced or lower level of cytotoxic T cell killing in the subject or patient. In some cases, the low-immunogenicity cell-induced cytotoxic T cell killing level 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 cytotoxic T cell killing level produced by administration of the immunogenic cell. In some embodiments, the administered population of low-immunogenicity cells fails to elicit cytotoxic T cell killing in the subject or patient.

In some embodiments, the population of low-immunogenic cells administered, such as low-immunogenic differentiated cells and CAR-T cells, induces reduced or lower levels of Complement Dependent Cytotoxicity (CDC) in the subject or patient. In some cases, the low immunogenic cells elicit CDC 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 CDC levels produced by administration of the immunogenic cells. In some embodiments, the administered population of low-immunogenicity cells fails to elicit CDC in the subject or patient.

S. therapeutic cells from primary T cells

Provided herein are low immunogenicity cells, including but not limited to primary T cells that evade immune recognition. In some embodiments, the engineered and/or hypoimmunogenic cells are produced (e.g., generated, cultured, or derived) from T cells (such as primary T cells). In some cases, primary T cells are obtained (e.g., harvested, extracted, withdrawn, or taken) from a subject or individual. In some embodiments, 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 engineered and/or hypoimmunogenic cells do not activate an innate and/or adaptive immune response in the patient (e.g., the recipient after administration). Methods of treating a disorder by administering a population of hypoimmunogenic cells to a subject (e.g., a recipient) or patient in need thereof are provided. In some embodiments, the engineered and/or low immunogenicity cells described herein comprise T cells engineered (e.g., modified) to express chimeric antigen receptors, including but not limited to chimeric antigen receptors described herein. In some cases, the T cells are a population or subpopulation of primary T cells from one or more individuals. 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 disclosure relates to a low immunogenicity primary T cell that overexpresses CD47 and CAR and has reduced expression of one or more Y chromosome genes and reduced expression or lack of expression of one or more MHC class I and/or MHC class II human leukocyte antigen molecules and has reduced expression or lack of expression of TCR complex molecules. The cells outlined herein overexpress CD47 and CAR and evade immune recognition. In some embodiments, the primary T cells exhibit reduced expression of one or more Y chromosome genes and reduced levels or activity of MHC class I antigen molecules, MHC class II antigen molecules, and/or TCR complex molecules. In certain embodiments, the primary T cells overexpress CD47 and CAR, and have a genetic modification in the PCDH11Y gene. In certain embodiments, the primary T cells overexpress CD47 and CAR, and have genetic modifications in the NLGN4Y gene. In certain embodiments, the primary T cells overexpress CD47 and CAR, and have a genetic modification in the PCDH11Y gene and a genetic modification in the NLGN4Y gene. In certain embodiments, the primary T cells overexpress CD47 and CAR, and have genomic modifications in the B2M gene. In some embodiments, the T cells overexpress CD47 and CAR, and have genomic modifications in the CIITA gene. In some embodiments, the primary T cells overexpress CD47 and CAR, and have genomic modifications in the TRAC gene. In some embodiments, the primary T cells overexpress CD47 and CAR, and have genomic modifications in the TRB gene. In some embodiments, the T cell overexpresses CD47 and CAR, and has genomic modifications in one or more of the following genes: PCDH11Y, NLGN4Y, B2M, CIITA, TRAC and TRB gene.

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 certain embodiments, the T cell expresses 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, the T cell is a modified (e.g., engineered) T cell. In some cases, the modified T cell comprises a modification that causes the cell to 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 modified T cell comprises a modification that causes the cell 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. Useful modifications to primary T cells are described in detail in US2016/0348073 and WO2020/018620, the disclosures of which are incorporated herein in their entirety.

In some embodiments, the engineered and/or low immunogenicity cells described herein comprise T cells engineered (e.g., modified) to express chimeric antigen receptors, including but not limited to chimeric antigen receptors described herein. In some cases, the T cells are a population or subpopulation of primary T cells from one or more individuals. In some embodiments, T cells described herein, such as engineered or modified T cells, include reduced expression of endogenous T cell receptors. In some embodiments, T cells described herein, such as engineered or modified T cells, include reduced expression of cytotoxic T lymphocyte-associated protein 4 (CTLA-4). In other embodiments, T cells described herein, such as engineered or modified T cells, include reduced expression of programmed cell death (PD-1). In certain embodiments, T cells described herein, such as engineered or modified T cells, include reduced expression of CTLA-4 and PD-1. Methods of reducing or eliminating CTLA-4, PD-1, and expression of both CTLA-4 and PD-1 can include any methods recognized by those skilled in the art, such as, but not limited to, genetic modification techniques using rare-cutting endonucleases and RNA silencing or RNA interference techniques. Non-limiting examples of rare-cutting endonucleases include any Cas protein, TALEN, zinc finger nuclease, meganuclease, and homing endonuclease. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the CTLA-4 and/or PD-1 locus.

In some embodiments, a T cell described herein, such as an engineered or modified T cell, comprises enhanced PD-L1 expression.

In some embodiments, the low immunogenicity T 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 or target 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 a B2M, CIITA, TRAC, TRB, PD-1 or CTLA-4 gene.

In some embodiments, the low immunogenicity T cell comprises a polynucleotide encoding a CAR that is expressed in the cell using an expression vector. In some embodiments, the CAR is introduced into the cell using a viral expression vector that mediates integration of the CAR sequence into the genome of the cell. For example, an expression vector for expressing a CAR in a cell comprises a polynucleotide sequence encoding the CAR. The expression vector may be an inducible expression vector. The expression vector may be a viral vector, such as but not limited to a lentiviral vector.

The low immunogenicity 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 leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic cancer, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

T. therapeutic cells differentiated from low immunogenic pluripotent stem cells

Provided herein are low-immunogenicity cells, including cells derived from pluripotent stem cells that evade immune recognition. In some embodiments, the cells do not activate an innate and/or adaptive immune response in the patient or subject (e.g., the recipient after administration). Methods of treating a disorder comprising repeatedly administering to a subject in need thereof a population of hypoimmunogenic cells are provided.

In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of one or more Y chromosome genes and reduced expression of MHC class I human leukocyte antigen molecules. In other embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of one or more Y chromosome genes and reduced expression of MHC class II human leukocyte antigen molecules. In certain embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of one or more Y chromosome genes and reduced expression of TCR complexes. In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of one or more Y chromosome genes and reduced expression of MHC class I and class II human leukocyte antigen molecules. In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of one or more Y chromosome genes and reduced expression of MHC class I and class II human leukocyte antigen molecules and TCR complexes.

In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of one or more Y chromosome genes and reduced expression of MHC class I and/or class II human leukocyte antigen molecules, and to exhibit increased CD47 expression. In some cases, the cells overexpress CD47 by having one or more CD47 transgenes. In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of one or more Y chromosome genes and reduced expression of MHC class I and class II human leukocyte antigen molecules, and to exhibit increased CD47 expression. In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of one or more Y chromosome genes and reduced expression of MHC class I and class II human leukocyte antigen molecules and TCR complexes, and to exhibit increased CD47 expression.

In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of one or more Y chromosome genes and reduced expression of MHC class I and/or class II human leukocyte antigen molecules, to exhibit increased CD47 expression, and to exogenously express chimeric antigen receptors. In some cases, the cell overexpresses a CD47 polypeptide by having one or more CD47 transgenes. In some cases, the cell overexpresses the CAR polypeptide by having one or more CAR transgenes. In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of one or more Y chromosome genes and reduced expression of MHC class I and class II human leukocyte antigen molecules, to exhibit increased CD47 expression, and to exogenously express chimeric antigen receptors. In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of one or more Y chromosome genes and reduced expression of MHC class I and II human leukocyte antigen molecules and TCR complexes, to exhibit increased CD47 expression, and to exogenously express chimeric antigen receptors.

Such pluripotent stem cells are low-immunogenicity stem cells. Such differentiated cells are hypoimmunogenic cells.

Any of the pluripotent stem cells described herein can differentiate into any cell of an organism or tissue. In some embodiments, the cells exhibit reduced expression of one or more Y chromosome genes, reduced expression of MHC class I and/or class II human leukocyte antigen molecules, and reduced expression of TCR complexes. In some cases, expression of one or more Y chromosome genes is reduced compared to an unmodified or wild-type cell of the same cell type. In some cases, the expression of MHC class I and/or class II human leukocyte antigen molecules is reduced compared to an unmodified or wild-type cell of the same cell type. In some cases, the expression of the TCR complex is reduced compared to an unmodified or wild-type cell of the same cell type. In some embodiments, the cells exhibit increased CD47 expression. In some cases, the expression of CD47 in a cell encompassed by the present disclosure is increased compared to an unmodified or wild-type cell of the same cell type. In some embodiments, the cell exhibits exogenous CAR expression. Described herein are methods for reducing the levels of MHC class I and/or class II human leukocyte antigen molecules and TCR complexes and increasing expression of CD47 and CARs.

In some embodiments, the cells used in the methods described herein evade immune recognition and response when administered to a patient (e.g., a recipient subject). The cells may 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 pluripotent stem cells and any cells differentiated from such pluripotent stem cells evade immune recognition include, but are not limited to, IFN-gamma Elispot assays, microglial killing assays, cell implantation animal models, cytokine release assays, ELISA, real-time quantitative microelectronic biosensor systems using bioluminescence imaging or chromium release assays or for cell analysisRTCA system, agilent) to perform killing assays, mixed lymphocyte reactions, immunofluorescence assays, and the like.

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.

1. T lymphocytes differentiated from low-immunogenicity pluripotent cells

T lymphocytes (T cells, including primary T cells) provided herein are derived from HIP cells (e.g., low immunogenicity ipscs) described herein. Methods for generating T cells (including CAR-T cells) from pluripotent stem cells (e.g., ipscs) are described, for example, in Iriguchi et al, nature Communications, 430 (2021); themeli et al, CELL STEM CELL,16 (4): 357-366 (2015); themeli et al, nature Biotechnology, 31:928-933 (2013).

T lymphocyte-derived hypoimmunogenic cells include, but are not limited to, primary T cells that evade immune recognition. In some embodiments, the low immunogenicity cells are produced (e.g., generated, cultured, or derived) by T cells (such as primary T cells). In some cases, primary T cells are obtained (e.g., harvested, extracted, withdrawn, or taken) from a subject or individual. In some embodiments, 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 hypoimmunogenic cells do not activate an immune response in the patient (e.g., the recipient after administration). Methods of treating a disorder by administering a population of hypoimmunogenic cells to a subject (e.g., a recipient) or patient in need thereof are provided. In some embodiments, the low immunogenicity cells described herein comprise T cells engineered (e.g., modified) to express chimeric antigen receptors, including but not limited to the chimeric antigen receptors described herein. In some cases, the T cells are a population or subpopulation of primary T cells from one or more individuals. 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 HIP-derived T cells comprise a Chimeric Antigen Receptor (CAR). Any suitable CAR may be included in hyHIP-derived T cells, including the CARs described herein. In some embodiments, the low immunogenicity-induced pluripotent stem cell-derived T 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 or target locus. In some embodiments, the polynucleotide is inserted into a B2M, CIITA, TRAC, TRB, PD-1 or CTLA-4 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).

The HIP-derived T cells provided herein can be used to treat suitable autoimmune diseases/disorders and/or inflammatory diseases/disorders, including but not limited to diseases of the nervous, gastrointestinal and endocrine systems, as well as skin and other connective tissue, eyes, blood and blood vessels. Examples of autoimmune diseases include, but are not limited to, hashimoto's thyroiditis, systemic lupus erythematosus, sjogren's syndrome, graves ' disease, scleroderma, rheumatoid arthritis, multiple sclerosis, MS associated with EBV infection, myasthenia gravis, and diabetes.

2. NK cells derived from low-immunogenicity pluripotent cells

Natural Killer (NK) cells provided herein are derived from HIP cells described herein (e.g., low immunogenicity iPSCs).

NK cells (also defined as "large granular lymphocytes") represent a cell lineage differentiated from common lymphoid progenitor cells (also producing B lymphocytes and T lymphocytes). Unlike T cells, NK cells do not naturally contain CD3 on the plasma membrane. Importantly, NK cells do not express TCRs and often lack other antigen-specific cell surface receptors (as well as TCRs and CD3, which do not express immunoglobulin B cell receptors, but instead often express CD16 and CD 56). The cytotoxic activity of NK cells does not require sensitization, but can be enhanced by activation using a variety of cytokines, including IL-2. NK cells are generally thought to lack the appropriate or complete signaling pathways necessary for antigen receptor-mediated signaling and are therefore not thought to be capable of antigen receptor-dependent signaling, activation, and expansion. NK cells are cytotoxic and balance activating and inhibitory receptor signaling to regulate their cytotoxic activity. For example, NK cells expressing CD16 may bind to the Fc domain of an antibody that binds to an infected cell, resulting in NK cell activation. In contrast, activity against cells expressing high levels of MHC class I proteins/molecules is reduced. Upon contact with target cells, NK cells release proteins such as perforins and enzymes such as proteases (granzymes). Perforin can form pores in the cell membrane of target cells, thereby inducing apoptosis or cell lysis.

There are many techniques available for generating NK cells from pluripotent stem cells (e.g., ipscs), including CAR-NK cells; see, e.g., zhu et al, methods Mol biol.2019;2048:107-119; knorr et al STEM CELLS TRANSL Med.2013 2 (4): 274-83.Doi:10.5966/sctm.2012-0084; zeng et al, stem Cell reports.2017dec12;9 (6) 1796-1812; ni et al, methods Mol biol.2013;1029:33-41; bernareggi et al, exp Hematol.2019:13-23; shankar et al STEM CELL RES ter 2020;11 234, which is incorporated by reference in its entirety, in particular methods and reagents for differentiation. Differentiation may be determined, as known in the art, typically by assessing the presence of NK cell-related and/or specific markers, including but not limited to CD56、KIRs、CD16、NKp44、NKp46、NKG2D、TRAIL、CD122、CD27、CD244、NK1.1、NKG2A/C、NCR1、Ly49、CD49b、CD11b、KLRG1、CD43、CD62L and/or CD226.

3. Cardiac cell

Provided herein are cardiac cell types differentiated from low immunogenicity-induced pluripotent (HIP) cells for subsequent transplantation or implantation into a subject (e.g., 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 ca2+ concentration 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 induced pluripotent stem cells by in vitro differentiation comprises: (a) Culturing a population of low immunogenicity induced pluripotent stem cells in a medium comprising a GSK inhibitor; (b) Culturing a population of low immunogenicity induced pluripotent stem cells in a medium comprising a WNT antagonist to produce a population of precordial cells; and (c) culturing the pre-cardiac cell population in a medium comprising insulin to produce an immunocompromised 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 benzoate diacrylate, trimethylolpropane ethoxylate (1 EO/QH) methyl, tricyclo [5.2.1.02,6] decane dimethanol diacrylate, neopentyl glycol ethoxylate 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 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.

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, 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.

4. Neural cell

Provided herein are different neural cell types differentiated from low immunogenicity induced pluripotent stem (HIP) cells 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 nerve cell types include, but are not limited to, brain endothelial cells, neurons (e.g., dopaminergic neurons), glial cells, and the like.

In some embodiments, differentiation of induced pluripotent stem cells is performed by exposing or contacting the cells to specific factors known to produce a specific cell lineage in order to target their differentiation to a specific, desired lineage and/or cell type of interest. In some embodiments, terminally differentiated cells exhibit a particular phenotypic characteristic or characteristic. 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

5. Brain endothelial cells

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 conditions, 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). 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.

In some embodiments, brain Endothelial Cells (ECs), precursors and progenitors thereof differentiate 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 EC or neural cell production. 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, von Willebrand factor (von Willebrand 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, advanced glycation 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-CC 2 binding cassette transporter ABCG2, multi-drug resistance-related protein 1ABCC1, small anion-receptor-related protein ABCC1, small anion-related protein Guan Duote and multi-drug resistance-related protein ABCC 4.

In some embodiments, the brain EC is characterized by having one or more features selected from the group consisting of: high expression of tight junctions, high resistance, low fenestration, small perivascular gaps, 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).

6. Dopaminergic neurons

In some embodiments, the low immunogenicity induced pluripotent stem (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, DA neurons from low immunogenicity induced pluripotent stem (HIP) cells are administered to a patient, e.g., a human patient, to treat a neurodegenerative disease or condition. In some cases, the neurodegenerative disease or condition 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 differentiate 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, neurostimularin (neurturin), knockout serum substitutes, N-acetylcysteine, c-kit ligands, modified forms thereof, mimics thereof, analogs 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: the ionotropic glutamate 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、 hypothalamic secretin, NADPH, NGF, ach, VAChT, PAX, EMX2p75, CORIN, TUJ1, NURR1 and/or any combination thereof.

7. Glial cells

In some embodiments, the described 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 produced 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 disclosure may be found in WO2020/018615, the disclosure of which is incorporated herein by reference in its entirety.

8. Endothelial cells

Provided herein are low-immunogenicity pluripotent cells differentiated into various endothelial cell types for subsequent transplantation or implantation into a subject (e.g., 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.

In some embodiments, endothelial cells differentiated from the subject's low immunogenicity pluripotent cells are administered to a patient in need thereof, e.g., a human patient. Endothelial cells may be administered to a patient suffering from a disease or condition 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 injury, hypertension, angina, and myocardial infarction due to coronary artery disease, renal vascular hypertension, renal failure due to renal arterial stenosis, lower limb claudication, 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 graft.

For example, endothelial cells can be used in cell therapies for repairing ischemic tissue, forming blood vessels and heart valves, engineering vascular prostheses, repairing damaged blood vessels, and inducing 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 certain embodiments, provided herein is a method of repairing or replacing a tissue in need of vascular cells or vascularization. The methods involve administering to a human patient in need of such treatment a composition containing isolated endothelial cells to promote angiogenesis in such tissue. 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 grafts 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, HIP derivatives are transplanted using techniques known in the art, depending on the cell type and the end use of the cells. In some embodiments, the cellular HIPs provided herein that differentiate from a subject can be transplanted intravenously or by injection at a specific location in a patient. 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 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 (Endoplin), CD146, endocan (ESM-l), endoglyx-l, endostatin (Endomucin), 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, VE-l, MRB (magic round robin-bout), nucleolin, PAL-E (TEM-endothelium, VCK-35), VCM-35), vascular endothelial cell adhesion markers (VEGFR-1), VEGFR-l, VEGFR-1, VEGFR-l, GATA2 (VEGFR-l), GBP-l (guanylate binding protein-l), GRO-alpha, HEX, ICAM-2 (intercellular adhesion molecule 2), LM02, VE-l, MRB (magic round robin-bout), nuclear nucleolin, PAL-E (TEM-35, VEM-35, VCM-35, vascular endothelial cell adhesion markers (VEM-35), vascular markers (VER-6-35, vascular endothelial cell adhesion markers, vascular markers) (VEP-6, vascular markers), vascular endothelial cell markers (VEP-6, vascular endothelial cell markers, vascular endothelial cell tumor-endothelial (VEP-endothelial cell tumor-6 (VEP 6).

In some embodiments, endothelial cells are genetically modified to express exogenous genes encoding proteins of interest (such as, but not limited to, enzymes, hormones, receptors, ligands, or drugs) useful for treating a disorder/condition 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), 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 implanted graft. Non-limiting illustrative examples include secreting or expressing thrombolytic agents to prevent intraluminal clot formation, smooth muscle proliferation inhibitors to prevent luminal narrowing due to smooth muscle hypertrophy, and expressing and/or secreting endothelial cell mitogens or autocrine factors to stimulate endothelial cell proliferation and improve the extent or duration of the endothelial cell lining of the graft lumen.

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 at the target anatomical site.

In other embodiments, the endothelial cells are 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 transplanted endothelial cells after completion of tumor therapy.

In some embodiments, the endothelial cells described herein 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, angina pectoris, myocardial infarction resulting from coronary artery disease, renal vascular hypertension, renal failure resulting from renal arterial stenosis, other vascular conditions or diseases.

In some embodiments, the low immunogenicity pluripotent cells 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 a population of low-immunogenicity endothelial cells from a population of low-immunogenicity induced pluripotent stem (HIP) cells by in vitro differentiation comprises: (a) Culturing a population of HIP cells in a first medium comprising a GSK inhibitor; (b) Culturing the population of HIP 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 hypoimmunogenic endothelial 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 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.

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.

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

Additional description of endothelial cells 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.

9. Thyroid cells

In some embodiments, the low-immunogenicity pluripotent cells 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 for the production of thyroid cells from human pluripotent stem cells and methods and reagents 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.

10. Liver cell

In some embodiments, the low immunogenicity mobile pluripotent stem (HIP) cells differentiate into hepatocytes to address loss of hepatocyte function or cirrhosis. There are a number of techniques that can be used to differentiate HIP 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).

11. Islet cells

In some embodiments, islet cells (also referred to as pancreatic beta cells) are derived from the low-immunogenicity induced pluripotent stem (HIP) cells described herein. In some cases, the low-immunogenicity, pluripotent cells differentiated into various islet cell types are transplanted or implanted into a subject (e.g., 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 islet cell types include, but are not limited to, islet progenitor cells, immature islet cells, mature islet cells, and the like. In some embodiments, pancreatic cells described herein are administered to a subject to treat diabetes.

In some embodiments, the islet cells are derived from a low-immunogenicity pluripotent cell described herein. Useful methods for differentiating pluripotent stem cells into islet cells are described, for example, in US9,683,215; US9,157,062; and US8,927,280.

In some embodiments, the islet cells produced by the methods as disclosed herein secrete insulin. In some embodiments, the islet cells exhibit at least two characteristics of endogenous islet cells, such as, but not limited to, secretion of insulin in response to glucose and expression of a beta cell marker.

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.l, nkx6.2, pax4, pax6, ptfla, isll, sox9, soxl, and FoxA2.

In some embodiments, the isolated islet cells produce insulin in response to an increase in glucose. In various embodiments, the isolated islet cells secrete insulin in response to an increase in 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 low-immunogenicity pluripotent cells 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 on successful differentiation of beta cells from human iPSCs, in particular 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 generating a population of low-immunogenicity islet cells from a population of low-immunogenicity induced pluripotent stem (HIP) cells by in vitro differentiation comprises: (a) Culturing a population of HIP cells in a first 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 to produce an immature islet cell population; and (b) culturing the immature islet cell population in a second medium, different from the first medium, to produce a hyperimmune islet 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 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.

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

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

12. Retinal Pigment Epithelial (RPE) cells

Provided herein are Retinal Pigment Epithelial (RPE) cells derived from the described low immunogenicity induced pluripotent stem (HIP) cells. For example, human RPE cells can be produced by differentiating human HIP cells. In some embodiments, the low-immunogenicity pluripotent cells differentiated into various RPE cell types are transplanted or implanted into a subject (e.g., 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.

The term "RPE" cell refers to a retinas epithelial cell having a gene expression profile similar or substantially similar to that of a native RPE cell. Such RPE cells derived from pluripotent stem cells may have a polygonal, planar lamellar morphology of natural RPE cells when grown to confluence on planar substrates.

The RPE cells may be implanted in a patient suffering from macular degeneration or in a patient 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.

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.

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 a population of low-immunogenicity Retinal Pigment Epithelium (RPE) cells from a population of low-immunogenicity pluripotent cells by in vitro differentiation comprises: (a) Culturing a population of low-immunogenicity pluripotent cells in a first medium comprising any 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 a population of low-immunogenicity 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 for use in the present disclosure may be found in WO2020/018615, the disclosure of which is incorporated herein by reference in its entirety.

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.

T lymphocytes

T lymphocytes (T cells) provided herein are derived from the described low immunogenicity induced pluripotent stem (HIP) cells. Methods for generating T cells (including CAR-T cells) from pluripotent stem cells (e.g., ipscs) are described, for example, in Iriguchi et al, nature Communications, 430 (2021); themeli et al, CELL STEM CELL,16 (4): 357-366 (2015); themeli et al, nature Biotechnology, 31:928-933 (2013).

In some embodiments, the low immunogenicity induced pluripotent stem cell-derived T cell comprises a Chimeric Antigen Receptor (CAR). Any suitable CAR may be included in the low immunogenicity induced pluripotent stem cell-derived T cells, including the CARs described herein. In some embodiments, the low immunogenicity-induced pluripotent stem cell-derived T 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 or target locus. In some embodiments, the polynucleotide is inserted into a B2M, CIITA, TRAC, TRB, PD-1 or CTLA-4 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).

HIP-derived 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 leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic cancer, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

U.genetic modification method

In some embodiments, a vector herein is a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule, including into a cell or cell genome. The transferred nucleic acid is typically linked to, e.g., inserted into, a vector nucleic acid molecule. The vector may include sequences that direct autonomous replication in the cell, or may include sequences sufficient to allow integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication defective retroviruses and lentiviruses. Non-viral vectors may require a delivery vehicle to facilitate entry of the nucleic acid molecule into the cell.

Viral vectors may comprise nucleic acid molecules, including virally derived nucleic acid elements, which generally facilitate transfer or integration of the nucleic acid molecule into the cell genome or into viral particles that mediate nucleic acid transfer. The viral particles will typically include various viral components, and sometimes host cell components in addition to the nucleic acids. The viral vector may comprise, for example, a virus or viral particle (e.g., as naked DNA) that is capable of transferring nucleic acid into a cell or into a transferred nucleic acid. Viral vectors and transfer plasmids may comprise structural and/or functional genetic elements derived primarily from viruses. Retroviral vectors may comprise viral vectors or plasmids containing structural and functional genetic elements derived primarily from retroviruses or parts thereof.

In some vectors described herein, at least a portion of one or more protein coding regions necessary to facilitate replication or replication may not be present as compared to the corresponding wild-type virus. This makes viral vector replication defective. In some embodiments, the vector is capable of transducing a target non-dividing host cell and/or integrating its genome into the host genome.

In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of the following: the 5' promoter (e.g., to control expression of the entire packaged RNA), the 5' ltr (e.g., which includes R (polyadenylation tail signal) and/or U5 including primer activation signal), the primer binding site, the psi packaging signal, the RRE element for nuclear export, the promoter directly upstream of the transgene to control transgene expression, the transgene (or other exogenous agent element), the polypurine region, and the 3' ltr (e.g., which includes mutated U3, R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more of cPPT, WPRE, and/or an insulator element.

Retroviruses typically replicate by reverse transcribing their genomic RNA into linear double-stranded DNA copies and subsequently covalently integrating their genomic DNA into the host genome. The structure of the wild-type retroviral genome typically comprises a 5 'Long Terminal Repeat (LTR) and a 3' LTR with a packaging signal between or within them that enables the genome to be packaged, a primer binding site, an integration site that enables integration into the host cell genome, and gag, pol, and env genes encoding packaging components that facilitate viral particle assembly. More complex retroviruses have additional features such as rev and RRE sequences in HIV that enable efficient export of the RNA transcript of the integrated provirus from the nucleus of the infected target cell to the cytoplasm. In provirus, the viral gene is flanked at both ends by regions called Long Terminal Repeats (LTRs). LTRs are involved in proviral integration and transcription. The LTR also acts as an enhancer-promoter sequence and can control the expression of viral genes. Encapsidation of retroviral RNA occurs through the psi sequence located at the 5' end of the viral genome.

The LTRs themselves are typically similar (e.g., identical) sequences, which can be divided into three elements, designated U3, R, and U5. U3 is derived from a sequence unique to the 3' end of RNA. R is derived from sequences repeated at both ends of the RNA, and U5 is derived from sequences unique to the 5' end of the RNA. The size of these three elements can vary considerably among different retroviruses.

For viral genomes, the transcription initiation site is typically at the boundary between U3 and R in one LTR, while the poly (A) addition (termination) site is at the boundary between R and U5 in the other LTR. U3 contains most of the transcriptional control elements of provirus, including promoters and multiple enhancer sequences that respond to cellular and, in some cases, viral transcriptional activator proteins. Some retroviruses contain any one or more of the following genes encoding proteins involved in regulating gene expression: tot, rev, tax and rex.

Regarding the structural genes gag, pol and env themselves, gag encodes the internal structural proteins of the virus. Gag proteins are proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes a Reverse Transcriptase (RT) which contains a DNA polymerase that mediates genome replication, a related RNase H and an Integrase (IN). The env gene encodes the Surface (SU) glycoprotein and Transmembrane (TM) protein of virions, which form complexes that interact specifically with cellular receptor proteins. This interaction promotes infection, for example, by fusing the proviral membrane with the cell membrane.

In the replication defective retroviral vector genome gag, pol and env may be absent or nonfunctional. The R regions at both ends of the RNA are typically repetitive sequences. U5 and U3 represent unique sequences at the 5 'and 3' ends of the RNA genome, respectively. Retroviruses may also contain additional genes encoding proteins other than gag, pol, and env. Examples of additional genes include one or more of vif, vpr, vpx, vpu, tat, rev and nef (in HIV). EIAV has, inter alia, an additional gene S2.

Illustrative retroviruses suitable for use in particular embodiments include, but are not limited to: moloney murine leukemia virus (M-MuLV), moloney murine sarcoma virus (MoMSV), harv murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline Leukemia Virus (FLV), foamy virus (spumavirus), friedel murine (Friend murine) leukemia virus, murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentiviruses.

In some embodiments, the retrovirus is a gamma retrovirus. In some embodiments, the retrovirus is epsilon retrovirus. In some embodiments, the retrovirus is an alpha retrovirus. In some embodiments, the retrovirus is a beta retrovirus. In some embodiments, the retrovirus is a delta retrovirus. In some embodiments, the retrovirus is a foamy retrovirus. In some embodiments, the retrovirus is an endogenous retrovirus. In some embodiments, the retrovirus is a lentivirus.

In some embodiments, the retroviral or lentiviral vector further comprises one or more insulator elements, such as the insulator elements described herein. In various embodiments, the vector comprises a promoter operably linked to a polynucleotide encoding an exogenous agent. The vector may have one or more LTRs, any of which comprises one or more modifications, such as one or more nucleotide substitutions, additions or deletions. The vector may also comprise one or more helper elements (e.g., cPPT/FLAP) that increase transduction efficiency, viral packaging (e.g., psi (Y) packaging signal, RRE), and/or other elements that increase expression of the exogenous gene (e.g., poly (a) sequences), and may optionally comprise WPRE or HPRE. In some embodiments, the lentiviral nucleic acid comprises, e.g., from 5 'to 3', one or more (e.g., all) of: promoters (e.g., CMV), R sequences (e.g., comprising TAR), U5 sequences (e.g., for integration), PBS sequences (e.g., for reverse transcription), DIS sequences (e.g., for genome dimerization), psi packaging signals, partial gag sequences, RRE sequences (e.g., for nuclear export), cPPT sequences (e.g., for nuclear import), promoters driving expression of exogenous agents, genes encoding exogenous agents, WPRE sequences (e.g., for efficient transgene expression), PPT sequences (e.g., for reverse transcription), R sequences (e.g., for polyadenylation and termination), and U5 signals (e.g., for integration).

Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); the viscina-meidi virus (VMV) virus; goat arthritis-encephalitis virus (CAEV); equine Infectious Anemia Virus (EIAV); feline Immunodeficiency Virus (FIV); bovine Immunodeficiency Virus (BIV); and Simian Immunodeficiency Virus (SIV). In some embodiments, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used. Lentiviral vectors may comprise viral vectors or plasmids containing structural and functional genetic elements or portions thereof, including LTRs derived primarily from lentiviruses.

In embodiments, a lentiviral vector (e.g., a lentiviral expression vector) may comprise a lentiviral transfer plasmid (e.g., as naked DNA) or an infectious lentiviral particle. With respect to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is understood that the sequences of these elements may be present in the form of RNA in lentiviral particles and may be present in the form of DNA in DNA plasmids.

In embodiments, a lentiviral vector is a vector having sufficient retroviral genetic information to allow packaging of an RNA genome into a viral particle capable of infecting a target cell in the presence of a packaging component. Infection of the target cell may include reverse transcription and integration into the target cell genome. RLVs typically carry non-viral coding sequences that will be delivered by the vector to the target cell. In embodiments, the RLV is unable to replicate independently within the target cell to produce infectious retroviral particles. In general, RLV lacks functional gag-pol and/or env genes and/or other genes involved in replication. The vector may be configured as a split intron vector, for example, as described in PCT patent application WO 99/15683, which is incorporated herein by reference in its entirety.

In some embodiments, the lentiviral vector comprises a minimal viral genome, e.g., the viral vector has been manipulated to remove non-essential elements and retain essential elements in order to provide the functionality required to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell, e.g., as described in WO 98/17815, which is incorporated herein by reference in its entirety.

The minimal lentiviral genome may comprise, for example, (5 ') R-U5-one or more first nucleotide sequences-U3-R (3'). However, plasmid vectors for use in generating lentiviral genomes in a source cell may also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in the source cell. These regulatory sequences may comprise native sequences associated with the transcribed retroviral sequence, e.g., the 5' u3 region, or they may comprise a heterologous promoter, such as another viral promoter, e.g., the CMV promoter. Some lentiviral genomes contain additional sequences that facilitate efficient viral production. For example, in the case of HIV, rev and RRE sequences may be included.

In some embodiments, the rare-cutting endonuclease is introduced into a cell containing the target polynucleotide sequence in the form of a nucleic acid encoding the rare-cutting endonuclease. 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.

The present disclosure contemplates altering a target polynucleotide sequence using the gene editing system of the present disclosure (e.g., CRISPR/Cas) in any manner available to those of skill in the art. 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.

The CRISPR/Cas systems of the present disclosure can be used to alter any target polynucleotide sequence in a cell. One of skill in the art will readily appreciate that the desired target polynucleotide sequence to be altered in any particular cell may correspond to any genomic sequence whose expression correlates with a disorder or otherwise facilitates entry of a pathogen into the cell. For example, the desired target polynucleotide sequence that is altered in a cell may be a polynucleotide sequence corresponding to a genomic sequence containing a disease-associated single polynucleotide polymorphism. In such examples, the CRISPR/Cas system of the present disclosure can be used to correct a disease-associated SNP in a cell by replacing the disease-associated SNP with a wild-type allele. As another example, a polynucleotide sequence of a target gene responsible for entry or proliferation of a pathogen into a cell may be a suitable target for deletion or insertion to disrupt the function of the target gene, thereby preventing entry or proliferation of the pathogen into the cell.

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, a CRISPR/Cas system of the present disclosure includes a Cas protein and at least one to two ribonucleic acids capable of directing and hybridizing the Cas protein to a target motif of a target polynucleotide sequence. As used herein, "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues (i.e., a polymer of amino acids) joined by peptide bonds, and include modified amino acids (e.g., phosphorylated, glycosylated, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing.

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 comprise moieties (e.g., pegylation, glycosylation, lipidation, acetylation, capping, etc.).

In some embodiments, the Cas protein comprises a core Cas protein, an isoform thereof, or any Cas-like protein having similar function or activity of any Cas protein or isoform thereof. 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 type V Cas protein. In some embodiments, the Cas protein comprises a Cas protein of the e.coli (e.coli) subtype (also known 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). Examples of Cas proteins include, but are not limited to: cas3, cas8a, cas5, cas8b, cas8c, cas10d, cse1, cse2, csy1, csy2, csy3 and/or GSU0054. In some embodiments, the Cas protein comprises Cas3, cas8a, cas5, cas8b, cas8c, cas10d, cse1, cse2, csy1, csy2, csy3, and/or GSU0054. Examples of Cas proteins include, but are not limited to: cas9, csn2, and/or Cas4. In some embodiments, the Cas protein comprises Cas9, csn2, and/or Cas4. In some embodiments, examples of Cas proteins include, but are not limited to: cas10, csm2, cmr5, cas10, csx11, and/or Csx10. In some embodiments, the Cas protein comprises Cas10, csm2, cmr5, cas10, csx11, and/or Csx10. In some embodiments, examples of Cas proteins include, but are not limited to: csf1. In some embodiments, the Cas protein comprises Csf1. In some embodiments, examples of Cas proteins include, but are not limited to: cas12a, cas12b, cas12C, C2C4, C2C8, C2C5, C2C10, and C2C9; and CasX (Cas 12 e) and CasY (Cas 12 d). See also, for example, koonin et al, curr Opin microbiol 2017;37:67-78, "Diversity, classification and evolution of CRISPR-CAS SYSTEMS". In some embodiments, the Cas protein comprises Cas12a, cas12b, cas12c, cas12d, cas12e, cas12d, and/or Cas12e. In some embodiments, the Cas protein comprises, in some embodiments, cas protein comprises Cas13, cas13a, C2, cas13b, cas13C, and/or Cas13d.

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, 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 some embodiments, the Cas protein is complexed with one to two 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.

The methods of the present disclosure 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 of the present disclosure can be selected to hybridize to a variety of different target motifs, depending on the particular CRISPR/Cas system and sequence of the 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 an offset 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 sequences that can be used for CRISPR/Cas-based gene targeting described herein are provided in table 19. Such sequences can be found in WO2016183041, 5, 9 filed 2016, the disclosure of which includes tables, appendices and sequence listings, which are incorporated herein by reference in their entirety.

TABLE 19 exemplary gRNA sequences that can be used to target genes

Other exemplary gRNA sequences that can be used for CRISPR/Cas-based targeting of the genes described herein are provided in PCT/US22/30394 filed 5/20 at 2022, the disclosures of which including tables, appendices, and sequence listings are incorporated herein by reference in their entirety.

In some embodiments, cells of the present technology 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 one nuclease catalytic domain to cleave a nucleic acid target sequence. The catalytic domain is preferably a nuclease domain, and more preferably a domain having endonuclease activity, such as I-TevI, colE7, nucA and Fok-I. In various embodiments, the TALE domain can be fused to meganucleases, such as I-CreI and I-Onul or functional variants thereof. In a more preferred embodiment, 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 sequence 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. Preferably, the RVD associated with identifying the different nucleotides 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, preferably in a sequence-specific manner, due to the 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 & Shi, science271:1081-1085 (1996)).

In some embodiments, the cells of the present disclosure are prepared using homing endonucleases. 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 40 bp. The homing endonuclease according to the present technology may for example correspond to a LAGLIDADG endonuclease, an HNH endonuclease or a GIY-YIG endonuclease. A preferred homing endonuclease according to the present disclosure may be an I-CreI variant.

In some embodiments, cells of the present technology 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 make cells of the present technology. 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, CIITA can be knockdown in pluripotent stem cells by introducing CIITA SIRNA into the cells or transducing a virus expressing CIITA SHRNA into the cells. In some embodiments, RNA interference is employed to reduce or inhibit expression of at least one selected from the group consisting of CIITA, B2M, NLRC, TCR- α and TCR- β.

In some embodiments, the cells provided herein are genetically modified to reduce expression of one or more immune factors (including a target polypeptide) to create immune-free or hypoimmunogenic cells. In certain embodiments, the cells disclosed herein (e.g., stem cells, induced pluripotent stem cells, differentiated cells, hematopoietic stem cells, primary T cells, and CAR-T cells) comprise one or more genetic modifications to reduce expression of one or more target polynucleotides. Non-limiting examples of such target polynucleotides and polypeptides include CIITA, B2M, NLRC, CTLA-4, PD-1, HLA-A, HLA-BM, HLA-C, RFX-ANK, NFY-A, RFX, RFX-AP, NFY-B, NFY-C, IRF1, and TAP1.

In some embodiments, the CRISPR/Cas system is used for genetic modification. 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 at the time of administration.

I. Gene editing system

In some embodiments, methods for genetically modifying cells 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, as well as nickase systems, base editing systems, primer editing systems, and gene writing systems known in the art.

i.ZFN

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.

ii.TALEN

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

Meganucleases are enzymes in the endonuclease family, characterized in that they are capable of recognizing and cleaving 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.

Transposase (IV)

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.

V. CRISPR/Cas System

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 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 "primordial spacer" 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 the crRNA directs the Cas9 complex to cleave the complementary target DNA sequence, provided that they are adjacent to a short sequence called a "protospacer 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 20 below.

TABLE 20 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.

Incision enzyme

The nuclease domain of Cas (particularly Cas 9), the nuclease can be independently mutated to produce an enzyme called DNA "nickase". Nickases are able to introduce single-strand cleavage with the same specificity as conventional CRISPR/Cas nuclease systems (including, for example, CRISPR/Cas 9). Nicking enzymes can be used to generate double strand breaks that can be used in gene editing systems (Mali et al, nat Biotech,31 (9): 833-838 (2013); mali et al Nature Methods,10:957-963 (2013); mali et al Science,339 (6121): 823-826 (2013)). In some cases, when two Cas nickases are used, a long overhang rather than a blunt end will be created on each cut end, which allows additional control over precise gene integration and insertion (Mali et al, nat Biotech,31 (9): 833-838 (2013); mali et al Nature Methods,10:957-963 (2013); mali et al, science,339 (6121): 823-826 (2013)). Since both nicking Cas enzymes must efficiently cleave their target DNA, paired nicking enzymes can have lower off-target effects than systems based on double-stranded cleaving Cas (Ran et al, cell,155 (2): 479-480 (2013); mali et al, nat Biotech,31 (9): 833-838 (2013); mali et al Nature Methods,10:957-963 (2013); mali et al, science,339 (6121): 823-826 (2013)).

Recombinant expression method of tolerogenic factors and/or chimeric antigen receptors

For all of these techniques, well-known recombinant techniques are used to generate recombinant nucleic acids as outlined herein. In certain embodiments, a recombinant nucleic acid encoding 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, a hybrid promoter that combines elements of more than one promoter, or a synthetic promoter. The expression construct may be present on an episome (such as a plasmid) in the cell, or the expression construct may be inserted into a chromosome, such as a locus. In some embodiments, the expression vector includes a selectable marker gene to allow selection of transformed host cells. Some 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 some embodiments, the expression vector is designed for selection of the host cell to be transformed, the particular variant polypeptide desired to be expressed, the copy number of the vector, the ability to control that copy number, and/or the expression of any other protein encoded by the vector (such as an antibiotic marker).

Examples of suitable mammalian promoters include, for example, promoters from the following genes: elongation factor 1 alpha (EF 1 alpha) promoter, CAG 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 Nature273:113-120 (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 (4) insertion of a proteolytic cleavage site (self-cleaving peptide) between genes or insertion of an Internal Ribosome Entry Site (IRES) between genes.

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., AAV transduction, lentiviral transduction) or otherwise delivered on a viral vector (e.g., fusogenic mediated delivery). In some embodiments, the polynucleotide is introduced into the cell via fusogenic mediated delivery or a transposase system selected from the group consisting of: a conditional or inducible transposase, a conditional or inducible PiggyBac transposon, a conditional or inducible sleeping beauty (SB 11) transposon, a conditional or inducible Mos1 transposon, and a conditional or inducible Tol2 transposon.

In some embodiments, the cells provided herein are genetically modified to include one or more exogenous polynucleotides inserted into one or more genomic loci of a low immunogenicity cell. In some embodiments, the exogenous polynucleotide encodes a protein of interest, such as a chimeric antigen receptor. The exogenous polynucleotide 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 exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction (e.g., using a vector). In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying an exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis VSV-G envelope and carries an exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentiviral-based viral vector.

Unlike certain methods of introducing polynucleotides described herein into cells, which typically involve activating cells, such as activated T cells (e.g., CD8 ⁺ T cells), polynucleotides can be introduced into non-activated T cells using suitable techniques. Suitable techniques include, but are not limited to, activating T cells (CD 8 ⁺ T cells) with one or more antibodies that bind CD3, CD8, and/or CD28, or fragments or portions thereof that may or may not bind to beads (e.g., scFv and VHH). Surprisingly, the fusogenic mediated introduction of polynucleotides into T cells is performed in non-activated T cells (e.g., CD8 ⁺ T cells) that have not been previously contacted with one or more activating antibodies or fragments or portions thereof (e.g., CD3, CD8, and/or CD 28). In some embodiments, the fusion-precursor mediated introduction of the polynucleotide into the T cell is performed in vivo (e.g., after the T cell has been administered to a subject). In other embodiments, the fusion-precursor mediated introduction of the polynucleotide into the T cell is performed in vitro (e.g., prior to administration of the T cell to a subject).

Provided herein are non-activated T cells comprising reduced expression of one or more Y chromosome genes HLA-A, HLa-B, HLA-C, CIITA, TCR- α, and/or TCR- β relative to a wild type T cell, wherein the non-activated T cells further comprise a first gene encoding CD 47. In some embodiments, the non-activated T cell comprises reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4, HLA-A, HLa-B, HLA-C, CIITA, TCR-a, and/or TCR- β relative to a wild-type T cell, wherein the non-activated T cell further comprises a first exogenous polynucleotide encoding CD 47.

In some embodiments, the non-activated T cells are not treated with an anti-CD 3 antibody, an anti-CD 28 antibody, a T cell activating cytokine, or a soluble T cell costimulatory molecule. In some embodiments, the non-activated T cells do not express an activation marker. In some embodiments, the non-activated T cells express CD3 and CD28, and wherein CD3 and/or CD28 are inactive.

In some embodiments, the anti-CD 3 antibody is OKT3. In some embodiments, the anti-CD 28 antibody is CD28.2. In some embodiments, the T cell activating cytokine is selected from the group of T cell activating cytokines consisting of IL-2, IL-7, IL-15, and IL-21. In some embodiments, the soluble T cell costimulatory molecule is selected from the group of soluble T cell costimulatory molecules consisting of an anti-CD 28 antibody, an anti-CD 80 antibody, an anti-CD 86 antibody, an anti-CD 137L antibody, and an anti-ICOS-L antibody.

In some embodiments, the non-activated T cells are primary T cells. In other embodiments, the non-activated T cells differentiate from engineered and/or hypoimmunogenic cells of the present disclosure. In some embodiments, the T cell is a CD8 ⁺ T cell.

In some embodiments, the first exogenous polynucleotide encodes CD47.

In some embodiments, the non-activated T cell further comprises a second exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR). In some embodiments, the CAR is selected from the group consisting of a CD 19-specific CAR, a CD 20-specific CAR, a BCMA-specific CAR, and a CD 22-specific CAR.

In some embodiments, the first and/or second exogenous polynucleotide is carried by a viral vector (including lentiviral vectors). In some embodiments, the first and/or second exogenous polynucleotide is carried by a lentiviral vector comprising a CD8 binding agent. In some embodiments, the first and/or second exogenous polynucleotide is introduced into the cell using a fusion-mediated delivery or a transposase system selected from the group consisting of: a conditional or inducible transposase, a conditional or inducible PiggyBac transposon, a conditional or inducible sleeping beauty (SB 11) transposon, a conditional or inducible Mos1 transposon, and a conditional or inducible Tol2 transposon.

In some embodiments, the first and/or second exogenous polynucleotide is inserted into a particular locus of at least one allele of a T cell. In some embodiments, the specific locus is selected from the group consisting of: safe harbor or target loci, B2M loci, CIITA loci, TRAC loci, and TRB loci. In some embodiments, the second exogenous polynucleotide encoding the CAR is inserted into a specific locus selected from the group consisting of: safe harbor or target loci, B2M loci, CIITA loci, TRAC loci, and TRB loci. In some embodiments, the first exogenous polynucleotide encoding CD47 is inserted into a specific locus selected from the group consisting of: safe harbor or target loci, B2M loci, CIITA loci, TRAC loci, and TRB loci. In some embodiments, the second exogenous polynucleotide encoding the CAR and the first exogenous polynucleotide encoding the CD47 are inserted into different loci. In some embodiments, the second exogenous polynucleotide encoding the CAR and the first exogenous polynucleotide encoding CD47 are inserted into the same locus. In some embodiments, the second exogenous polynucleotide encoding the CAR and the first exogenous polynucleotide encoding CD47 are inserted into the B2M locus. In some embodiments, the second exogenous polynucleotide encoding the CAR and the first exogenous polynucleotide encoding CD47 are inserted into the CIITA locus. In some embodiments, the second exogenous polynucleotide encoding the CAR and the first exogenous polynucleotide encoding CD47 are inserted into the TRAC locus. In some embodiments, the second exogenous polynucleotide encoding the CAR and the first exogenous polynucleotide encoding CD47 are inserted into the TRB locus. In some embodiments, the second exogenous polynucleotide encoding the CAR and the first exogenous polynucleotide encoding CD47 are inserted into a safe harbor or target locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of: CCR5 locus, CXCR4 locus, PPP1R12C locus, albumin locus, SHS231 locus, CLYBL locus, rosa locus, F3 (CD 142) locus, MICA locus, MICB locus, LRP1 (CD 91) locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus and KDM5D locus.

In some embodiments, the non-activated T cells do not express HLA-A, HLA-B, and/or HLA-C antigens. In some embodiments, the non-activated T cells do not express B2M. In some embodiments, the non-activated T cells do not express HLA-DP, HLA-DQ, and/or HLA-DR antigens. In some embodiments, the non-activated T cells do not express CIITA. In some embodiments, the non-activated T cells do not express TCR- α. In some embodiments, the non-activated T cells do not express TCR- β. In some embodiments, the non-activated T cells do not express TCR- α and TCR- β.

In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and/or a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding CAR inserted into the TRAC locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and/or a second exogenous polynucleotide encoding a CAR inserted into a TRB locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into a TRB locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and/or a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and/or a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding CAR inserted into the CIITA locus.

In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and/or a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding CAR inserted into the TRAC locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and/or a second exogenous polynucleotide encoding a CAR inserted into a TRB locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into a TRB locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and/or a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and/or a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. In some embodiments, the non-activated T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding CAR inserted into the CIITA locus.

Provided herein are engineered T cells comprising reduced expression of HLA-A, HLa-B, HLA-C, CIITA, TCR-a, and/or TCR- β relative to wild type T cells, wherein the engineered T cells further comprise a first exogenous polynucleotide encoding CD47 carried by a lentiviral vector comprising a CD8 binding agent. In some embodiments, the engineered T cell comprises reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4, HLA-A, HLa-B, HLA-C, CIITA, TCR-a, and/or TCR- β relative to a wild-type T cell, wherein the engineered T cell further comprises a first exogenous polynucleotide encoding CD47 carried by a lentiviral vector comprising a CD8 binding agent.

In some embodiments, the engineered T cell is a primary T cell. In other embodiments, the engineered T cells differentiate from the hypoimmunogenic cells of the present disclosure. In some embodiments, the T cell is a CD8 ⁺ T cell. In some embodiments, the T cell is a CD4 ⁺ T cell.

In some embodiments, the engineered T cells do not express an activation marker. In some embodiments, the engineered T-cells express CD3 and CD28, and wherein CD3 and/or CD28 are inactive.

In some embodiments, the engineered T cells are not treated with an anti-CD 3 antibody, an anti-CD 28 antibody, a T cell activating cytokine, or a soluble T cell costimulatory molecule. In some embodiments, the anti-CD 3 antibody is OKT3, wherein the anti-CD 28 antibody is CD28.2, wherein the T cell activating cytokine is selected from the group of T cell activating cytokines consisting of IL-2, IL-7, IL-15 and IL-21, and wherein the soluble T cell co-stimulatory molecule is selected from the group of soluble T cell co-stimulatory molecules consisting of anti-CD 28 antibody, anti-CD 80 antibody, anti-CD 86 antibody, anti-CD 137L antibody and anti-ICOS-L antibody. In some embodiments, the engineered T cells have not been treated with one or more T cell activating cytokines selected from the group consisting of IL-2, IL-7, IL-15, and IL-21. In some cases, the cytokine is IL-2. In some embodiments, one or more cytokines is IL-2, and another is selected from the group consisting of IL-7, IL-15 and IL-21.

In some embodiments, the non-activated T cell further comprises a second exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR). In some embodiments, the CAR is selected from the group consisting of a CD 19-specific CAR and a CD 22-specific CAR.

In some embodiments, the engineered T cell further comprises a second exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR). In some embodiments, the first and/or second exogenous polynucleotide is inserted into a particular locus of at least one allele of a T cell. In some embodiments, the specific locus is selected from the group consisting of: safe harbor or target loci, B2M loci, CIITA loci, TRAC loci, and TRB loci. In some embodiments, the first exogenous polynucleotide encoding CD47 is inserted into a specific locus selected from the group consisting of: safe harbor or target loci, B2M loci, CIITA loci, TRAC loci, and TRB loci. In some embodiments, the second exogenous polynucleotide encoding the CAR is inserted into a specific locus selected from the group consisting of: safe harbor or target loci, B2M loci, CIITA loci, TRAC loci, and TRB loci. In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding CAR are inserted into different loci. In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding CAR are inserted into the same locus. In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding CAR are inserted into a B2M locus, CIITA locus, TRAC locus, TRB locus, or safe harbor or target locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of: CCR5 locus, CXCR4 locus, PPP1R12C locus, albumin locus, SHS231 locus, CLYBL locus, rosa locus, F3 (CD 142) locus, MICA locus, MICB locus, LRP1 (CD 91) locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus and KDM5D locus.

In some embodiments, the CAR is selected from the group consisting of a CD 19-specific CAR and a CD 22-specific CAR. In some embodiments, the CAR is a CD19 specific CAR. In some embodiments, the CAR is a CD 22-specific CAR. In some embodiments, the CAR comprises an antigen binding domain that binds to any one selected from the group consisting of CD19, CD20, CD22, CD38, CD123, CD138, and BCMA.

In some embodiments, the engineered T cell does not express HLA-A, HLa-B, and/or HLa-C antigens, wherein the engineered T cell does not express B2M, wherein the engineered T cell does not express HLa-DP, HLa-DQ, and/or HLa-DR antigens, wherein the engineered T cell does not express CIITA, and/or wherein the engineered T cell does not express TCR-a and TCR- β.

In some embodiments, the first and/or second exogenous polynucleotide is inserted into at least one allele of the T cell using viral transduction. In some embodiments, the first and/or second exogenous polynucleotide is inserted into at least one allele of the T cell using a lentiviral-based viral vector.

In some embodiments, the engineered T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRAC^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and/or a second exogenous polynucleotide encoding a CAR inserted into a TRAC locus, a TRB locus, a B2M locus, or a CIITA locus. In some embodiments, the engineered T cell is a PCDH11Y ^{indel of insertion / indel of insertion}、NLGN4Y^{indel of insertion / indel of insertion}、B2M^{indel of insertion / indel of insertion}、CIITA^{indel of insertion / indel of insertion}、TRB^{indel of insertion / indel of insertion} cell comprising a first exogenous polynucleotide encoding CD47 and/or a second exogenous polynucleotide encoding a CAR inserted into a TRAC locus, a TRB locus, a B2M locus, or a CIITA locus.

In some embodiments, the non-activated T cells and/or engineered T cells of the disclosure are in a subject. In other embodiments, the non-activated T cells and/or engineered T cells of the disclosure are in vitro.

In some embodiments, the non-activated T cells and/or engineered T cells of the disclosure express a CD8 binding agent. In some embodiments, the CD8 binding agent is an anti-CD 8 antibody. In some embodiments, the anti-CD 8 antibody is selected from the group consisting of: mouse anti-CD 8 antibodies, rabbit anti-CD 8 antibodies, human anti-CD 8 antibodies, humanized anti-CD 8 antibodies, camelid (e.g., llama, alpaca, camel) anti-CD 8 antibodies, and fragments thereof. In some embodiments, the fragment thereof is an scFv or VHH. In some embodiments, the CD8 binding agent binds to the CD 8a chain and/or the CD8 β chain.

In some embodiments, the CD8 binding agent is fused to a transmembrane domain that is incorporated into the viral envelope. In some embodiments, the lentiviral vector is pseudotyped with a viral fusion protein. In some embodiments, the viral fusion protein comprises one or more modifications to reduce binding to its native receptor.

In some embodiments, the viral fusion protein is fused to a CD8 binding agent. In some embodiments, the viral fusion protein comprises a nipah virus F glycoprotein and a nipah virus G glycoprotein fused to a CD8 binding agent. In some embodiments, the lentiviral vector does not comprise a T cell activating molecule or a T cell costimulatory molecule. In some embodiments, the non-activated T cells and/or engineered T cells of the disclosure are in a subject. In other embodiments, the non-activated T cells and/or engineered T cells of the disclosure are in vitro.

In some embodiments, the viral fusion protein is fused to a CD8 binding agent. In some embodiments, the viral fusion protein comprises a nipah virus F glycoprotein and a nipah virus G glycoprotein fused to a CD8 binding agent. In some embodiments, the lentiviral vector does not comprise a T cell activating molecule or a T cell costimulatory molecule. In some embodiments, the lentiviral vector encodes a first exogenous polynucleotide and/or a second exogenous polynucleotide.

In some embodiments, upon transfer into the first subject, the non-activated T cells or engineered T cells exhibit one or more responses selected from the group consisting of: (a) a T cell response, (b) an NK cell response, and (c) a macrophage response, which is reduced as compared to a wild-type cell after transfer to the second subject. In some embodiments, the first subject and the second subject are different subjects. In some embodiments, the macrophage response is phagocytosis.

In some embodiments, the non-activated T cells or engineered T cells exhibit one or more selected from the group consisting of: (a) reduced TH1 activation in a subject, (b) reduced NK cell killing in a subject, and (c) reduced killing of intact PBMCs in a subject.

In some embodiments, the non-activated T cells or engineered T cells, when transferred into the subject, elicit one or more items selected from the group consisting of: (a) a decrease in donor-specific antibodies in a subject, (b) a decrease in IgM or IgG antibodies in a subject, and (c) a decrease in Complement Dependent Cytotoxicity (CDC) in a subject.

In some embodiments, the non-activated T cells or engineered T cells are transduced with a lentiviral vector comprising a CD8 binding agent in a subject. In some embodiments, the lentiviral vector carries a gene encoding CAR and/or CD 47.

In some embodiments, the gene encoding CAR and/or CD47 is introduced into the cell using a gene therapy vector or a transposase system selected from the group consisting of a transposase, a PiggyBac transposon, a sleeping beauty (SB 11) transposon, a Mos1 transposon, and a Tol2 transposon. In some embodiments, the gene therapy vector is a retrovirus or fusion.

Provided herein are pharmaceutical compositions comprising non-activated T cells and/or engineered T cell populations of the present disclosure and a pharmaceutically acceptable additive, carrier, diluent or excipient.

The methods provided herein comprise administering to a subject a composition comprising non-activated T cells and/or engineered T cell populations of the present disclosure, or one or more pharmaceutical compositions of the present disclosure.

In some embodiments, the T cell activation treatment is not administered to the subject before, after, and/or concurrently with the administration of the composition. In some embodiments, the T cell activation therapy comprises lymphocyte depletion.

Provided herein are methods of treating a subject suffering from cancer comprising administering to the subject a composition comprising non-activated T cells and/or an engineered T cell population of the present disclosure, or one or more pharmaceutical compositions of the present disclosure, wherein no T cell activation treatment is administered to the subject prior to, after, and/or concurrently with administration of the composition. In some embodiments, the T cell activation therapy comprises lymphocyte depletion.

Provided herein are methods for expanding T cells capable of recognizing and killing tumor cells of a subject in need thereof, comprising administering to the subject a composition comprising non-activated T cells and/or engineered T cell populations of the present disclosure, or one or more pharmaceutical compositions of the present disclosure, wherein no T cell activation treatment is administered to the subject prior to, after, and/or concurrently with administration of the composition. In some embodiments, the T cell activation therapy comprises lymphocyte depletion.

Provided herein are methods of treating a subject suffering from an autoimmune disease/disorder and/or inflammatory disease/disorder comprising administering to the subject a composition comprising non-activated T cells and/or an engineered T cell population of the present disclosure, or one or more pharmaceutical compositions of the present disclosure, wherein no T cell activation treatment is administered to the subject prior to, after, and/or concurrently with administration of the composition. In some embodiments, the T cell activation therapy comprises lymphocyte depletion.

Provided herein are dosage regimens for treating a condition, disease, or disorder in a subject comprising administering a composition comprising non-activated T cells and/or an engineered T cell population of the present disclosure, or one or more pharmaceutical compositions of the present disclosure, and a pharmaceutically acceptable additive, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered in about 1-3 therapeutically effective doses.

Provided herein are dosage regimens for treating a condition, disease, or disorder in a subject comprising administering a composition comprising non-activated T cells and/or an engineered T cell population of the present disclosure, or one or more pharmaceutical compositions of the present disclosure, and a pharmaceutically acceptable additive, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered in about 1-3 clinically effective doses.

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, other immunoassays, reverse transcriptase polymerase chain reaction (RT-PCR), and the like.

W. production of induced pluripotent Stem cells

The present technology provides methods for producing low-immunogenicity pluripotent cells. In some embodiments, the method comprises generating pluripotent stem cells. 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.

Generally, ipscs are made 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.

Determination of low immunogenicity phenotype and multipotent retention

Once the engineered and/or low immunogenic 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.

An in vivo assay may be performed to assess the immunogenicity of the cells outlined herein. In some embodiments, the survival and immunogenicity of the hypoimmunogenic cells are determined using an allogeneic humanized immunodeficiency mouse model. In some cases, low immunogenicity pluripotent stem cells were transplanted into allogeneic humanized NSG-SGM3 mice and assayed for cell rejection, cell viability, and teratoma formation. In some cases, the implanted low-immunogenicity pluripotent stem cells 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 some embodiments, 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.

As will be appreciated by those of skill in the art, successful reduction of MHC I function (HLA I when the cell is derived from a human cell) in a pluripotent cell can be measured using techniques known in the art and as described below; for example, FACS techniques using labeled antibodies that bind to HLA complexes; for example, commercially available HLA-A, HLA-B and HLA-C antibodies that bind to the alpha chain of a human major histocompatibility HLA class I antigen molecule are used.

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 to one or more HLA cell surface components as discussed above.

Successful reduction of MHC II function (HLA II when the cells are derived from human cells) in pluripotent cells or derivatives thereof 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 addition, cells can be tested to confirm that HLA II complexes are not expressed on the cell surface. Again, such assays are performed as 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 I and II (or MHC I and II) molecules, the engineered and/or hypoimmunogenic cells of the present technology have reduced susceptibility to macrophage phagocytosis and NK cell killing. The resulting hypoimmunogenic cells "escape" immune macrophages and the innate pathway due to the reduction or lack of TCR complexes and the expression of one or more CD47 transgenes.

Exogenous polynucleotide

In some embodiments, the engineered and/or low-immunogenicity cells provided herein are genetically modified to include one or more exogenous polynucleotides inserted into one or more genomic loci of the low-immunogenicity cells. In some embodiments, the exogenous polynucleotide encodes a protein of interest, such as a chimeric antigen receptor. The exogenous polynucleotide 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).

The exogenous polynucleotide may be inserted into any suitable genomic locus of the low-immunogenicity cell. In some embodiments, the exogenous polynucleotide is inserted into a safe harbor or target locus as described herein. Suitable safe harbor and target loci include, but are not limited to, CCR5 genes, CXCR4 genes, PPP1R12C (also known as AAVS 1) genes, albumin genes, SHS231 loci, CLYBL genes, rosa genes (e.g., rosa 26), 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, PDGFRa genes, OLIG2 genes, GFAP genes, and KDM5D genes (also known as HY). In some embodiments, the exogenous polynucleotide is inserted into an intron, exon, or coding sequence region of a safe harbor or target 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. In some embodiments, the polynucleotide is inserted into the B2M, CIITA, TRAC, TRB, PD-1 or CTLA-4 locus. Exemplary genomic loci for insertion of exogenous polynucleotides are described in table 21.

Table 21: exemplary genomic loci for insertion of exogenous polynucleotides

Table 22: non-limiting examples of Cas9 guide RNAs

For Cas9 guides, the spacer sequences for all Cas9 guides are provided in table 23, where 20nt guide sequences are described as corresponding to unique guide sequences, and can be any of those described herein, including, for example, those listed in table 22.

Table 23: cas9 guide RNAs

In some embodiments, the low-immunogenicity cells comprising the exogenous polynucleotide are derived from low-immunogenicity induced pluripotent cells (HIPs), e.g., as described herein. Such hypoimmunogenic cells include, for example, cardiac cells, neural cells, brain endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid cells, islet cells (beta cells), retinal pigment epithelial cells, NK cells, and T cells. In some embodiments, the low immunogenicity cell comprising the exogenous polynucleotide is a pancreatic beta cell, a T cell (e.g., a primary T cell), or a glial progenitor cell.

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 or target locus or safe harbor or target locus as disclosed herein, or into a genomic locus that causes silencing or reduced expression of an endogenous gene. In some embodiments, the polynucleotide is inserted into the B2M, CIITA, TRAC, TRB, PD1 or CTLA4 locus.

In some embodiments, the low-immunogenicity cells comprising the exogenous polynucleotide are primary T cells or T cells derived from low-immunogenicity pluripotent cells (e.g., low-immunogenicity ipscs). In exemplary embodiments, the exogenous polynucleotide is a chimeric antigen receptor (e.g., any CAR described herein). In some embodiments, the exogenous polynucleotide is operably linked to a promoter for expressing the exogenous polynucleotide in a low immunogenicity cell.

Pharmaceutically acceptable carrier

In some embodiments, the pharmaceutical compositions provided herein further comprise a pharmaceutically acceptable 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); salts such as sodium chloride; 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 comprises one or more electrolyte base solutions selected from the group consisting of: lactic acidRinger ' ssolution, plasmalyte-A ^TM, iscove's modified Dulbecco's medium, normosol-R ^TM、Veen-D^TM,And Hank balanced salt solution (phenol red free). These base solutions closely approximate the composition of mammalian extracellular physiological fluids.

In some embodiments, the pharmaceutical composition comprises one or more cryoprotectants selected from the group consisting of: arabinogalactan, glycerol, polyvinylpyrrolidone (PVP), dextrose, dextran, trehalose, sucrose, raffinose, hydroxyethyl starch (HES), propylene glycol, human Serum Albumin (HSA) and dimethyl sulfoxide (DMSO). In some embodiments, the pharmaceutically acceptable buffer is neutral buffered saline or phosphate buffered saline. In some embodiments, the pharmaceutical compositions provided herein compriseOne or more of CSB, plasma-Lyte-A ^TM, HSA, DMSO and trehalose.

Is an optimized solution of an intracellular sample containing an osmotic agent (osmotic agent)/osmotic agent (oncotic agent), a radical scavenger, and an energy source to minimize apoptosis, minimize ischemia/reperfusion injury, and maximize post-thaw recovery of the maximum number of living functional cells.Serum and protein free and non-immunogenic. /(I)CGMP-compliant manufacture from USP grade or higher feedstocks. /(I)Is a series of solutions pre-formulated with 0%, 2%, 5% or 10% DMSO.CSB isDMSO-free version of (a). In some embodiments, the pharmaceutical composition comprises a concentration of about 0-100%, 5-95%, 10-90%, 15-85%, 20-80%, 30-80%, 40-80%, 50-80%, 60-80%, 70-80%, 25-75%, 30-70%, 35-65%, 40-60%, or 45-55% weightBase solution of CSB. In some embodiments, the pharmaceutical composition comprises/>, at a concentration of about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% weight/weightBase solution of CSB.

Plasmalyte-A ^TM is a non-polymeric plasma compatibilizer and contains essential salts and nutrients similar to those found in culture media, but does not contain additional ingredients found in tissue culture media that are not approved for human infusion or are not available in U.S. P. grades (e.g., phenol red). Plasmalyte-A ^TM contains about 140mEq/L sodium (Na), about 5mEq/L potassium (K), about 3mEq/L magnesium (Mg), about 98mEq/L chlorine (Cl), about 27mEq/L acetate and about 23mEq/L gluconate. (PlasmaLyte-A ^TM is commercially available from Baxter, hyland Division, glendale Calif. product No. 2B 2543). In some embodiments, the pharmaceutical composition comprises a PlasmaLyte-a ^TM base solution at a concentration of about 0-100％、5-95％、10-90％、15-85％、15-80％、15-75％、15-70％、15-65％、15-60％、15-55％、15-50％、15-45％、15-40％、15-35％、15-30％、15-25％、20-80％、20-75％、20-70％、20-65％、20-60％、20-55％、20-50％、20-45％、20-40％、20-35％、20-30％、25-75％、30-70％、35-65％、40-60％ or 45-55% w/w. In some embodiments, the pharmaceutical composition comprises a PlasmaLyte-a ^TM base solution at a concentration of about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% weight/weight.

In some embodiments, the pharmaceutical composition comprises Human Serum Albumin (HSA) at a concentration of about 0-10％、0.3-9.3％、0.3-8.3％、0.3-7.3％、0.3-6.3％、0.3-5.3％、0.3-4.3％、0.3-3.3％、0.3-2.3％、0.3-1.3％、0.6-8.3％、0.9-7.3％、1.2-6.3％、1.5-5.3％、1.8-4.3％ or 2.1-3.3% w/v. In some embodiments, the pharmaceutical composition comprises HSA at a concentration of about 0％、0.3％、0.6％、0.9％、1.2％、1.5％、1.8％、2.1％、2.4％、2.7％、3.0％、3.3％、3.6％、3.9％、4.3％、4.6％、4.9％、5.3％、5.6％、5.9％、6.3％、6.6％、6.9％、7.3％、7.6％、7.9％、8.3％、8.6％、8.9％、9.3％、9.6％、9.9％ or 10% w/v.

In some embodiments, the pharmaceutical composition comprises Dimethylsulfoxide (DMSO) at a concentration of about 0-10%, 0.5-9.5%, 1-9%, 1.5-8.5%, 2-8%, 3-8%, 4-8%, 5-8%, 6-8%, 7-8%, 2.5-7.5%, 3-7%, 3.5-6.5%, 4-6%, or 4.5-5.5% volume/volume. In some embodiments, the pharmaceutical composition comprises HSA at a concentration of about 0％、0.25％、0.5％、0.75％、1.0％,1.25％、1.5％、1.75％、2.0％、2.25％、2.5％、2.75％、3.0％、3.25％、3.5％、3.75％、4.0％、4.25％、4.5％、4.75％、5.0％、5.25％、5.5％、5.75％、6.0％、6.25％、6.5％、6.75％、7.0％、7.25％、7.5％、7.75％、8.0％、8.25％、8.5％、8.75％、9.0％、9.25％、9.5％、9.75％ or 10.0% v/v.

In some embodiments, the pharmaceutical composition comprises trehalose at a concentration of about 0-500mM, 50-450mM, 100-400mM, 150-350mM, or 200-300 mM. In some embodiments, the pharmaceutical composition comprises trehalose at a concentration of about 0mM、10mM、20mM、30mM、40mM、50mM、60mM、70mM、80mM、90mM、100mM、125mM、150mM、175mM、200mM、225mM、250mM、275mM、300mM、325mM、350mM、375mM、400mM、425mM、450mM、475mM or 500 mM.

Exemplary pharmaceutical composition components are shown in table 24.

Table 24. Exemplary pharmaceutical composition components.

* Additional HSA besides PlasmaLyte.

In some embodiments, the pharmaceutical composition comprises the engineered and/or hypoimmunogenic cells 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).

AA, formulation 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 T cells, T cells differentiated from low-immunogenicity induced pluripotent stem cells, and other cells differentiated from low-immunogenicity induced pluripotent stem cells described herein. In some embodiments, the pharmaceutical composition comprises at least about 1x 10²、5x 10²、1x 10³、5x 10³、1x 10⁴、5x 10⁴、1x 10⁵、5x 10⁵、1x 10⁶、5x 10⁶、1x 10⁷、5x 10⁷、1x 10⁸、5x 10⁸、1x 10⁹、5x 10⁹、1x 10¹⁰ or 5x10 ¹⁰ cells. In some embodiments, the pharmaceutical composition comprises up to about 1x 10²、5x 10²、1x 10³、5x 10³、1x 10⁴、5x 10⁴、1x 10⁵、5x 10⁵、1x 10⁶、5x 10⁶、1x 10⁷、5x 10⁷、1x 10⁸、5x 10⁸、1x 10⁹、5x 10⁹、1x 10¹⁰ or 5x10 ¹⁰ cells. In some embodiments, the pharmaceutical composition comprises up to about 6.0x 10 ⁸ cells. In some embodiments, the pharmaceutical composition comprises up to about 8.0x 10 ⁸ cells. In some embodiments, the pharmaceutical composition comprises at least about 1x 10²-5x 10²、5x 10²-1x 10³、1x 10³-5x 10³、5x 10³-1x 10⁴、1x 10⁴-5x 10⁴、5x 10⁴-1x 10⁵、1x 10⁵-5x 10⁵、5x 10⁵-1x 10⁶、1x 10⁶-5x 10⁶、5x 10⁶-1x 10⁷、1x 10⁷-5x 10⁷、5x 10⁷-1x 10⁸、1x 10⁸-5x 10⁸、5x 10⁸-1x 10⁹、1x 10⁹-5x 10⁹、5x 10⁹-1x 10¹⁰ or 1x10 ¹⁰-5x 10¹⁰ cells. In an exemplary embodiment, the pharmaceutical composition comprises about 1.0x10 ⁶ to about 2.5x10 ⁸ cells. In certain embodiments, 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 low immunogenicity induced pluripotent stem 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 certain 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 therapeutically or clinically effective 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 therapeutically or clinically effective dose. In some cases, a therapeutically or clinically effective dose comprises from about 1.0x10 ⁵ to about 2.5x10 ⁸ primary T cells described herein in a volume of from about 10ml to 50 ml. In some cases, the therapeutically or clinically effective dose comprises 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 instances, a therapeutically or clinically effective dose includes from about 1.0x10 ⁵ to about 2.5x10 ⁸ T cells differentiated from a low immunogenicity induced pluripotent stem cell as described herein in a volume of from about 10ml to 50 ml. In some embodiments, the therapeutically or clinically effective dose is 1.0x 10⁵、1.1x 10⁵、1.2x 10⁵、1.3x 10⁵、1.4x10⁵、1.5x 10⁵、1.6x 10⁵、1.7x 10⁵、1.8x 10⁵、1.9x 10⁵、2.0x 10⁵、2.1x 10⁵、2.2x 10⁵、2.3x 10⁵、2.4x 10⁵、2.5x 10⁵、1.0x 10⁶、1.1x 10⁶、1.2x 10⁶、1.3x 10⁶、1.4x 10⁶、1.5x 10⁶、1.6x 10⁶、1.7x 10⁶、1.8x 10⁶、1.9x 10⁶、2.0x 10⁶、2.1x 10⁶、2.2x 10⁶、2.3x 10⁶、2.4x 10⁶、2.5x 10⁶、1.0x 10⁷、1.1x 10⁷、1.2x 10⁷、1.3x 10⁷、1.4x 10⁷、1.5x 10⁷、1.6x 10⁷、1.7x 10⁷、1.8x 10⁷、1.9x 10⁷、2.0x 10⁷、2.1x 10⁷、2.2x 10⁷、2.3x 10⁷、2.4x 10⁷、2.5x 10⁷、1.0x 10⁸、1.1x 10⁸、1.2x 10⁸、1.3x 10⁸、1.4x 10⁸、1.5x 10⁸、1.6x 10⁸、1.7x 10⁸、1.8x 10⁸、1.9x 10⁸、2.0x 10⁸、2.1x 10⁸、2.2x 10⁸、2.3x 10⁸、2.4x 10⁸ or 2.5x10 ⁸ T cells differentiated from the low-immunogenicity induced pluripotent stem cells described herein in a volume of about 10ml to 50 ml. In other cases, the therapeutically or clinically effective 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 low immunogenicity induced pluripotent stem cells. In still other cases, the therapeutically effective dose or clinically effective 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 low-immunogenicity induced pluripotent stem cells.

In some embodiments, the pharmaceutical composition is administered as a monotherapy effective dose or a clinically effective dose of about 1.0x10 ⁵ to about 1.0x10 ⁷ cells (such as primary T cells and T cells differentiated from low immunogenicity induced pluripotent stem cells) per kg body weight for 50kg or less subjects. In some embodiments, the pharmaceutical composition is administered as a single therapeutically or clinically effective dose of about 0.5x 10 to about 1.0x 10, about 1.0x 10 to about 1.0x 10, about 5.0x 10 to about 1x 10, about 1.0x 10 to about 1x 10, about 5.0x 10 to about 1.0x 10, about 1.0x 10 to about 5.0x 10, about 2.0x 10 to about 5.0x 10, about 3.0x 10 to about 5.0x 10, about 4.0x 10 to about 5.0x 10, about 5.0x 10 to about 5.0x 10, about 6.0x 10 to about 7.0x 10 to about 5.0x 10, about 9 kg/kg of body weight of the cell or the subject. In some embodiments, for a subject of 50kg or less, the therapeutically effective dose or clinically effective dose is 0.5x 10⁵、0.6x 10⁵、0.7x 10⁵、0.8x 10⁵、0.9x 10⁵、1.0x 10⁵、1.1x 10⁵、1.2x 10⁵、1.3x 10⁵、1.4x 10⁵、1.5x 10⁵、1.6x 10⁵、1.7x 10⁵、1.8x 10⁵、1.9x 10⁵、2.0x 10⁵、2.1x 10⁵、2.2x 10⁵、2.3x 10⁵、2.4x 10⁵、2.5x 10⁵、2.6x 10⁵、2.7x 10⁵、2.8x 10⁵、2.9x 10⁵、3.0x 10⁵、3.1x 10⁵、3.2x 10⁵、3.3x 10⁵、3.4x 10⁵、3.5x 10⁵、3.6x 10⁵、3.7x 10⁵、3.8x 10⁵、3.9x 10⁵、4.0x 10⁵、4.1x 10⁵、4.2x 10⁵、4.3x 10⁵、4.4x 10⁵、4.5x 10⁵、4.6x 10⁵、4.7x 10⁵、4.8x 10⁵、4.9x 10⁵、5.0x 10⁵、0.5x 10⁶、0.6x 10⁶、0.7x 10⁶、0.8x 10⁶、0.9x 10⁶、1.0x 10⁶、1.1x 10⁶、1.2x 10⁶、1.3x 10⁶、1.4x 10⁶、1.5x 10⁶、1.6x 10⁶、1.7x 10⁶、1.8x 10⁶、1.9x 10⁶、2.0x 10⁶、2.1x 10⁶、2.2x 10⁶、2.3x 10⁶、2.4x 10⁶、2.5x 10⁶、2.6x 10⁶、2.7x 10⁶、2.8x 10⁶、2.9x 10⁶、3.0x 10⁶、3.1x 10⁶、3.2x 10⁶、3.3x 10⁶、3.4x 10⁶、3.5x 10⁶、3.6x 10⁶、3.7x 10⁶、3.8x 10⁶、3.9x 10⁶、4.0x 10⁶、4.1x 10⁶、4.2x 10⁶、4.3x 10⁶、4.4x 10⁶、4.5x 10⁶、4.6x 10⁶、4.7x 10⁶、4.8x 10⁶、4.9x 10⁶、5.0x 10⁶、5.1x 10⁶、5.2x 10⁶、5.3x 10⁶、5.4x 10⁶、5.5x 10⁶、5.6x 10⁶、5.7x 10⁶、5.8x 10⁶、5.9x 10⁶、6.0x 10⁶、6.1x 10⁶、6.2x 10⁶、6.3x 10⁶、6.4x 10⁶、6.5x 10⁶、6.6x 10⁶、6.7x 10⁶、6.8x 10⁶、6.9x 10⁶、7.0x 10⁶、7.1x 10⁶、7.2x 10⁶、7.3x 10⁶、7.4x 10⁶、7.5x 10⁶、7.6x 10⁶、7.7x 10⁶、7.8x 10⁶、7.9x 10⁶、8.0x 10⁶、8.1x 10⁶、8.2x 10⁶、8.3x 10⁶、8.4x 10⁶、8.5x 10⁶、8.6x 10⁶、8.7x 10⁶、8.8x 10⁶、8.9x 10⁶、9.0x 10⁶、9.1x 10⁶、9.2x 10⁶、9.3x 10⁶、9.4x 10⁶、9.5x 10⁶、9.6x 10⁶、9.7x 10⁶、9.8x 10⁶、9.9x 10⁶、0.5x 10⁷、0.6x 10⁷、0.7x 10⁷、0.8x 10⁷、0.9x 10⁷ or 1.0x10 ⁷ cells/kg body weight. In some embodiments, for a subject of 50kg or less, the therapeutically effective dose or clinically effective dose is from about 0.2x 10 ⁶ to about 5.0x 10 ⁶ cells/kg body weight. In certain embodiments, the therapeutically effective dose or clinically effective dose ranges from less than about 0.2x 10 ⁶ to about 5.0x 10 ⁶ cells/kg body weight for a subject of 50kg or less. Or clinically effective dose in exemplary embodiments, the volume of the monotherapy effective dose or clinically effective dose is about 10ml to 50ml. In some embodiments, a therapeutically effective dose or a clinically effective dose is administered intravenously.

In exemplary embodiments, the cells are administered in a single therapeutically effective dose of about 1.0x10 ⁶ to about 5.0x10 ⁸ cells (such as primary T cells and T cells differentiated from low immunogenicity induced pluripotent stem cells) for subjects exceeding 50 kg. In some embodiments, the pharmaceutical composition is administered as a single therapeutically or clinically effective dose of about 0.5x 10 to about 1.0x 10, about 1.0x 10 to about 1.0x 10, about 5.0x 10 to about 1.0x 10, about 1.0x 10 to about 5.0x 10, about 1.0x 10 to about 1.0x 10, about 1.0x 10 to about 5.0x 10, about 2.0x 10 to about 5.0x 10, about 3.0x 10 to about 5.0x 10, about 4.0x 10 to about 5.0x 10, about 5.0x 10 to about 5.0x 10, about 6.0x 10 to about 7.0x 10 to about 5.0x 10, about 7.0x 10 to about 9 kg of body weight of the subject or less than about 50kg of the subject. In some embodiments, the therapeutically effective dose or clinically effective dose is 1.0x 10⁶、1.1x 10⁶、1.2x 10⁶、1.3x 10⁶、1.4x 10⁶、1.5x 10⁶、1.6x 10⁶、1.7x 10⁶、1.8x 10⁶、1.9x 10⁶、2.0x 10⁶、2.1x 10⁶、2.2x 10⁶、2.3x 10⁶、2.4x 10⁶、2.5x 10⁶、2.6x 10⁶、2.7x 10⁶、2.8x 10⁶、2.9x 10⁶、3.0x 10⁶、3.1x 10⁶、3.2x 10⁶、3.3x 10⁶、3.4x 10⁶、3.5x 10⁶、3.6x 10⁶、3.7x 10⁶、3.8x 10⁶、3.9x 10⁶、4.0x 10⁶、4.1x 10⁶、4.2x 10⁶、4.3x 10⁶、4.4x 10⁶、4.5x 10⁶、4.6x 10⁶、4.7x 10⁶、4.8x 10⁶、4.9x 10⁶、5.0x 10⁶、5.1x 10⁶、5.2x 10⁶、5.3x 10⁶、5.4x 10⁶、5.5x 10⁶、5.6x 10⁶、5.7x 10⁶、5.8x 10⁶、5.9x 10⁶、6.0x 10⁶、6.1x 10⁶、6.2x 10⁶、6.3x 10⁶、6.4x 10⁶、6.5x 10⁶、6.6x 10⁶、6.7x 10⁶、6.8x 10⁶、6.9x 10⁶、7.0x 10⁶、7.1x 10⁶、7.2x 10⁶、7.3x 10⁶、7.4x 10⁶、7.5x 10⁶、7.6x 10⁶、7.7x 10⁶、7.8x 10⁶、7.9x 10⁶、8.0x 10⁶、8.1x 10⁶、8.2x 10⁶、8.3x 10⁶、8.4x 10⁶、8.5x 10⁶、8.6x 10⁶、8.7x 10⁶、8.8x 10⁶、8.9x 10⁶、9.0x 10⁶、9.1x 10⁶、9.2x 10⁶、9.3x 10⁶、9.4x 10⁶、9.5x 10⁶、9.6x 10⁶、9.7x 10⁶、9.8x 10⁶、9.9x 10⁶、1.0x 10⁷、1.1x 10⁷、1.2x 10⁷、1.3x 10⁷、1.4x 10⁷、1.5x 10⁷、1.6x 10⁷、1.7x 10⁷、1.8x 10⁷、1.9x 10⁷、2.0x 10⁷、2.1x 10⁷、2.2x 10⁷、2.3x 10⁷、2.4x 10⁷、2.5x 10⁷、2.6x 10⁷、2.7x 10⁷、2.8x 10⁷、2.9x 10⁷、3.0x 10⁷、3.1x 10⁷、3.2x 10⁷、3.3x 10⁷、3.4x 10⁷、3.5x 10⁷、3.6x 10⁷、3.7x 10⁷、3.8x 10⁷、3.9x 10⁷、4.0x 10⁷、4.1x 10⁷、4.2x 10⁷、4.3x 10⁷、4.4x 10⁷、4.5x 10⁷、4.6x 10⁷、4.7x 10⁷、4.8x 10⁷、4.9x 10⁷、5.0x 10⁷、5.1x 10⁷、5.2x 10⁷、5.3x 10⁷、5.4x 10⁷、5.5x 10⁷、5.6x 10⁷、5.7x 10⁷、5.8x 10⁷、5.9x 10⁷、6.0x 10⁷、6.1x 10⁷、6.2x 10⁷、6.3x 10⁷、6.4x 10⁷、6.5x 10⁷、6.6x 10⁷、6.7x 10⁷、6.8x 10⁷、6.9x 10⁷、7.0x 10⁷、7.1x 10⁷、7.2x 10⁷、7.3x 10⁷、7.4x 10⁷、7.5x 10⁷、7.6x 10⁷、7.7x 10⁷、7.8x 10⁷、7.9x 10⁷、8.0x 10⁷、8.1x 10⁷、8.2x 10⁷、8.3x 10⁷、8.4x 10⁷、8.5x 10⁷、8.6x 10⁷、8.7x 10⁷、8.8x 10⁷、8.9x 10⁷、9.0x 10⁷、9.1x 10⁷、9.2x 10⁷、9.3x 10⁷、9.4x 10⁷、9.5x 10⁷、9.6x 10⁷、9.7x 10⁷、9.8x 10⁷、9.9x 10⁷、1.0x 10⁸、1.1x 10⁸、1.2x 10⁸、1.3x 10⁸、1.4x 10⁸、1.5x 10⁸、1.6x 10⁸、1.7x 10⁸、1.8x 10⁸、1.9x 10⁸、2.0x 10⁸、2.1x 10⁸、2.2x 10⁸、2.3x 10⁸、2.4x 10⁸、2.5x 10⁸、2.6x 10⁸、2.7x 10⁸、2.8x 10⁸、2.9x 10⁸、3.0x 10⁸、3.1x 10⁸、3.2x 10⁸、3.3x 10⁸、3.4x 10⁸、3.5x 10⁸、3.6x 10⁸、3.7x 10⁸、3.8x 10⁸、3.9x 10⁸、4.0x 10⁸、4.1x 10⁸、4.2x 10⁸、4.3x 10⁸、4.4x 10⁸、4.5x 10⁸、4.6x 10⁸、4.7x 10⁸、4.8x 10⁸、4.9x 10⁸ or 5.0x10 ⁸ cells/kg body weight for a subject of 50kg or less. In certain embodiments, the cells are administered in a single therapeutically effective dose or a clinically effective 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 therapeutically effective dose or a clinically effective dose, ranging from less than about 1.0x 10 ⁷ to about 2.5x 10 ⁸ cells for subjects exceeding 50 kg. In some embodiments, the cells are administered in a single therapeutically effective dose or a clinically effective dose, ranging from greater than about 1.0x 10 ⁷ to about 2.5x 10 ⁸ cells for subjects exceeding 50 kg. In some embodiments, the dose is administered intravenously. In exemplary embodiments, the volume of the monotherapy effective dose or the clinically effective dose is about 10ml to 50ml. In some embodiments, a therapeutically effective dose or a clinically effective dose is administered intravenously.

In exemplary embodiments, a therapeutically or clinically effective 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 therapeutically effective dose or a clinically effective dose of the pharmaceutical composition is present in a single infusion bag. In other embodiments, a single therapeutically effective dose or a clinically effective 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), wherein the multiple doses together comprise a therapeutically effective dose or a clinically effective dose regimen. 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 certain 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, a first dosage regimen is administered to a subject at a first time point, followed by a second dosage regimen 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 first dose regimen comprises low immune (HIP) T cells or primary T cells expressing the first CAR and the second dose regimen comprises low immune (HIP) T cells or primary T cells expressing the 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 low immune (HIP) T cells or primary T cells expressing the first CAR and the second dose regimen comprises low immune (HIP) 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 the course of a disease (e.g., an autoimmune disease), and at least two of the dosage regimens comprise the same type of low immune (HIP) T cells or primary T cells described herein. In other embodiments, at least two of the plurality of dosage regimens comprise different types of low immunity (HIP) T cells or primary T cells described herein.

In some embodiments, a CD 19-specific (CD 19) CAR-T cell described herein is administered to a subject at a dose of about 50x 10 ⁶ to about 110x 10 ⁶ (e.g., ,50x 10⁶、51x 10⁶、52x 10⁶、53x 10⁶、54x 10⁶、55x 10⁶、56x 10⁶、57x 10⁶、58x 10⁶、59x 10⁶、60x 10⁶、61x 10⁶、62x 10⁶、63x 10⁶、64x 10⁶、65x 10⁶、66x 10⁶、67x 10⁶、68x 10⁶、69x 10⁶、70x 10⁶、71x 10⁶、72x 10⁶、73x 10⁶、74x 10⁶、75x 10⁶、76x 10⁶、77x 10⁶、78x 10⁶、79x 10⁶、80x 10⁶、81x 10⁶、82x 10⁶、83x 10⁶、84x 10⁶、85x 10⁶、86x 10⁶、87x 10⁶、88x 10⁶、89x 10⁶、90x 10⁶、91x 10⁶、92x 10⁶、93x 10⁶、94x 10⁶、95x 10⁶、96x 10⁶、97x 10⁶、98x 10⁶、99x 10⁶、100x 10⁶、101x 10⁶、102x 10⁶、103x 10⁶、104x 10⁶、105x 10⁶、106x 10⁶、107x 10⁶、108x 10⁶、109x 10⁶ or 110x 10 ⁶) live CD 19-specific CAR-T cells. In some embodiments, the dose is a therapeutically effective amount of a live CD19 specific CAR-T cell. In other embodiments, the dose is a clinically effective amount of live CD 19-specific CAR-T cells. In some embodiments, the live CD 19-specific CAR-T cells include cd4+ T cells expressing a CD 19-specific CAR and cd8+ T cells expressing a CD 19-specific CAR in a ratio of about 1:1. In some embodiments, the CD19 specific CAR of the cell is Li Jimai @ lameeStructural equivalents thereof or functional equivalents thereof.

In some embodiments, from about 50x 10 ⁶ to about 110x 10 ⁶ (e.g., ,50x 10⁶、51x 10⁶、52x 10⁶、53x 10⁶、54x 10⁶、55x 10⁶、56x 10⁶、57x 10⁶、58x 10⁶、59x 10⁶、60x 10⁶、61x 10⁶、62x 10⁶、63x 10⁶、64x 10⁶、65x 10⁶、66x 10⁶、67x 10⁶、68x 10⁶、69x 10⁶、70x 10⁶、71x 10⁶、72x 10⁶、73x 10⁶、74x 10⁶、75x 10⁶、76x 10⁶、77x 10⁶、78x 10⁶、79x 10⁶、80x 10⁶、81x 10⁶、82x 10⁶、83x 10⁶、84x 10⁶、85x 10⁶、86x 10⁶、87x 10⁶、88x 10⁶、89x 10⁶、90x 10⁶、91x 10⁶、92x 10⁶、93x 10⁶、94x 10⁶、95x 10⁶、96x 10⁶、97x 10⁶、98x 10⁶、99x 10⁶、100x 10⁶、101x 10⁶、102x 10⁶、103x 10⁶、104x 10⁶、105x 10⁶、106x 10⁶、107x 10⁶、108x 10⁶、109x 10⁶ or 110x 10 ⁶) live CD 19-specific CAR-T cells described herein are administered to a subject. In some embodiments, the dose is a therapeutically effective amount of a live CD19 specific CAR-T cell. In other embodiments, the dose is a clinically effective amount of live CD 19-specific CAR-T cells. In some cases, 50% of the live CD 19-specific CAR-T cells are cd4+ T cells that express a CD 19-specific CAR, and 50% of the live CD 19-specific CAR-T cells are cd8+ T cells that express a CD 19-specific CAR. In some embodiments, the CD19 specific CAR of the cell is Li Jimai @ lameeStructural equivalents thereof or functional equivalents thereof.

In some embodiments, the CD 19-specific CAR-T cells described herein are administered to a subject at a dose of about 2x 10 ⁶/kg body weight. In some embodiments, the maximum dose administered is about 2x 10 ⁸ live CD 19-specific CAR-T cells. In some embodiments, the dose is a therapeutically effective amount of a live CD19 specific CAR-T cell. In other embodiments, the dose is a clinically effective amount of live CD 19-specific CAR-T cells. In some embodiments, the CD19 specific CAR of the cell is associated with aliskirenThe same CD19 specific CAR, a structural equivalent thereof or a functional equivalent thereof.

In some embodiments, the CD 19-specific CAR-T cells described herein are administered to a subject at a dose of about 2x 10 ⁶/kg body weight. In some embodiments, a maximum dose of about 2x 10 ⁸ live CD 19-specific CAR-T cells is administered to a patient weighing about 100kg and more. In some embodiments, the dose is a therapeutically effective amount of a live CD19 specific CAR-T cell. In other embodiments, the dose is a clinically effective amount of live CD 19-specific CAR-T cells. In some embodiments, the CD19 specific CAR of the cell is a CAR that is associated with briyl alendronateThe same CD19 specific CAR, a structural equivalent thereof or a functional equivalent thereof.

In some embodiments, a CD 19-specific CAR-T cell described herein is administered to a subject at a dose of up to about 2x 10 ⁸ live CD 19-specific CAR-T cells. In some embodiments, for a subject weighing about 50kg or less, about 0.2x 10 ⁶ to about 5.0x 10 ⁶ (e.g., about 0.2x 10⁶、0.4x 10⁶、0.5x 10⁶、0.6x 10⁶、0.8x 10⁶、0.9x 10⁶、1.0x 10⁶、1.2x 10⁶、1.4x 10⁶、1.5x 10⁶、1.6x 10⁶、1.8x 10⁶、1.9x 10⁶、2.0x 10⁶、2.2x 10⁶、2.4x 10⁶、2.5x 10⁶、2.6x 10⁶、2.8x 10⁶、2.9x 10⁶、3.0x 10⁶、3.2x 10⁶、3.4x 10⁶、3.5x 10⁶、3.6x 10⁶、3.8x 10⁶、3.9x 10⁶、4.0x 10⁶、4.2x 10⁶、4.4x 10⁶、4.5x 10⁶、4.6x 10⁶、4.8x 10⁶、4.9x 10⁶ or 5.0x 10 ⁶) live CD 19-specific CAR-T cells/kg body weight are administered to the subject. In some embodiments, for a subject having a body weight greater than about 50kg, about 0.1x 10 ⁸ to about 2.5x 10 ⁸ (e.g., about 0.1x 10⁶、0.2x 10⁶、0.4x 10⁶、0.5x 10⁶、0.6x 10⁶、0.8x 10⁶、0.9x 10⁶、1.0x 10⁶、1.2x 10⁶、1.4x 10⁶、1.5x 10⁶、1.6x 10⁶、1.8x 10⁶、1.9x 10⁶、2.0x 10⁶、2.2x 10⁶、2.4x 10⁶ or 2.5x 10 ⁶) live CD 19-specific CAR-T cells are administered to the subject. In some embodiments, about 0.6x10 ⁸ to about 6.0x10 ⁸ (e.g., about 0.6x 10⁸、0.8x 10⁸、0.9x 10⁸、1.0x 10⁸、1.2x 10⁸、1.4x 10⁸、1.5x 10⁸、1.6x 10⁸、1.8x 10⁸、1.9x 10⁸、2.0x 10⁸、2.2x 10⁸、2.4x 10⁸、2.5x 10⁸、2.6x 10⁸、2.8x 10⁸、2.9x 10⁸、3.0x 10⁸、3.2x 10⁸、3.4x 10⁸、3.5x 10⁸、3.6x 10⁸、3.8x 10⁸、3.9x 10⁸、4.0x 10⁸、4.2x 10⁸、4.4x 10⁸、4.5x 10⁸、4.6x 10⁸、4.8x 10⁸、4.9x 10⁸、5.0x 10⁸、5.2x 10⁸、5.4x 10⁸、5.5x 10⁸、5.6x 10⁸、5.8x 10⁸、5.9x 10⁸ or 6.0x10 ⁸) live CD 19-specific CAR-T cells are administered to a subject. In some embodiments, the dose is a therapeutically effective amount of a live CD19 specific CAR-T cell. In other embodiments, the dose is a clinically effective amount of live CD 19-specific CAR-T cells. In some embodiments, the CD19 specific CAR of the cell is with temozolomideThe same CD19 specific CAR, a structural equivalent thereof or a functional equivalent thereof.

In some embodiments, a single dose of any CD 19-specific CAR-T cell described herein comprises from about 50x 10 ⁶ to about 110x 10 ⁶ (e.g., ,50x 10⁶、51x 10⁶、52x 10⁶、53x 10⁶、54x 10⁶、55x 10⁶、56x 10⁶、57x 10⁶、58x 10⁶、59x 10⁶、60x 10⁶、61x 10⁶、62x 10⁶、63x 10⁶、64x 10⁶、65x 10⁶、66x 10⁶、67x 10⁶、68x 10⁶、69x 10⁶、70x 10⁶、71x 10⁶、72x 10⁶、73x 10⁶、74x 10⁶、75x 10⁶、76x 10⁶、77x 10⁶、78x 10⁶、79x 10⁶、80x 10⁶、81x 10⁶、82x 10⁶、83x 10⁶、84x 10⁶、85x 10⁶、86x 10⁶、87x 10⁶、88x 10⁶、89x 10⁶、90x 10⁶、91x 10⁶、92x 10⁶、93x 10⁶、94x 10⁶、95x 10⁶、96x 10⁶、97x 10⁶、98x 10⁶、99x 10⁶、100x 10⁶、101x 10⁶、102x 10⁶、103x 10⁶、104x 10⁶、105x 10⁶、106x 10⁶、107x 10⁶、108x 10⁶、109x 10⁶ or 110x 10 ⁶) live CD 19-specific CAR-T cells. In some embodiments, the dose is a therapeutically effective amount of a live CD19 specific CAR-T cell. In other embodiments, the dose is a clinically effective amount of live CD 19-specific CAR-T cells. In some embodiments, the live CD 19-specific CAR-T cells include cd4+ T cells expressing a CD 19-specific CAR and cd8+ T cells expressing a CD 19-specific CAR in a ratio of about 1:1. In some embodiments, the CD 19-specific CAR is a CAR that is in communication with Li JimaiThe same CD19 specific CAR, a structural equivalent thereof or a functional equivalent thereof.

In some embodiments, a single dose of any CD 19-specific CAR-T cell described herein comprises about 2x 10 ⁸ live CD 19-specific CAR-T cells. In some embodiments, a single infusion bag of any CD 19-specific CAR-T cell described herein comprises about 2x 10 ⁸ live CD 19-specific CAR-T cells in about 68mL of cell suspension. In some embodiments, the CD 19-specific CAR is associated with aliskirenThe same CD19 specific CAR, a structural equivalent thereof or a functional equivalent thereof. /(I)

In some embodiments, a single dose of any CD 19-specific CAR-T cell described herein comprises about 2x 10 ⁸ live CD 19-specific CAR-T cells. In some embodiments, a single infusion bag of any CD 19-specific CAR-T cell described herein comprises about 2x 10 ⁸ live CD 19-specific CAR-T cells in about 68mL of cell suspension. In some embodiments, the CD 19-specific CAR is with briyl-alendronateThe same CD19 specific CAR, a structural equivalent thereof or a functional equivalent thereof.

In some embodiments, for a subject weighing 50kg or less, a single dose of any CD 19-specific CAR-T cell described herein comprises from about 0.2x 10 ⁶ to about 5.0x10 ⁶ (e.g., about 0.2x 10⁶、0.3x 10⁶、0.4x 10⁶、0.5x 10⁶、0.6x 10⁶、0.7x 10⁶、0.8x 10⁶、0.9x 10⁶、1.0x 10⁶、1.1x 10⁶、1.2x 10⁶、1.3x 10⁶、1.4x 10⁶、1.5x 10⁶、1.6x 10⁶、1.7x 10⁶、1.8x 10⁶、1.9x 10⁶、2.0x 10⁶、2.1x 10⁶,2.2x 10⁶、2.3x 10⁶、2.4x 10⁶、2.5x 10⁶、2.6x 10⁶、2.7x 10⁶、2.8x 10⁶、2.9x 10⁶、3.0x 10⁶、3.1x 10⁶、3.2x 10⁶、3.3x 10⁶、3.4x 10⁶、3.5x 10⁶、3.6x 10⁶、3.7x 10⁶、3.8x 10⁶、3.9x 10⁶、4.0x 10⁶、4.1x 10⁶、4.2x 10⁶、4.3x 10⁶、4.4x 10⁶、4.5x 10⁶、4.6x 10⁶、4.7x 10⁶、4.8x 10⁶、4.9x 10⁶ or 5.0x10 ⁶) live CD 19-specific CAR-T cells per kg body weight. In some embodiments, for subjects having a body weight greater than 50kg, a single dose of any CD 19-specific CAR-T cell described herein comprises from about 0.1x 10 ⁸ to about 2.5x 10 ⁸ (e.g., about 0.1x 10⁶、0.2x 10⁶、0.3x 10⁶、0.4x 10⁶、0.5x 10⁶、0.6x 10⁶、0.7x 10⁶、0.8x 10⁶、0.9x 10⁶、1.0x 10⁶、1.1x 10⁶、1.2x 10⁶、1.3x 10⁶、1.4x 10⁶、1.5x 10⁶、1.6x 10⁶、1.7x 10⁶、1.8x 10⁶、1.9x 10⁶、2.0x 10⁶、2.1x 10⁶、2.2x 10⁶、2.3x 10⁶、2.4x 10⁶ or 2.5x 10 ⁶) live CD 19-specific CAR-T cells per kg body weight. In some embodiments, a single dose of any CD 19-specific CAR-T cell described herein comprises from about 0.6x10 ⁸ to about 6.0x10 ⁸ (e.g., about 0.6x 10⁸、0.7x 10⁸、0.8x 10⁸、0.9x 10⁸、1.0x 10⁸、1.1x 10⁸,1.2x 10⁸、1.3x 10⁸、1.4x 10⁸、1.5x 10⁸、1.6x 10⁸、1.7x 10⁸、1.8x 10⁸、1.9x 10⁸、2.0x 10⁸、2.1x 10⁸、2.2x 10⁸、2.3x 10⁸、2.4x 10⁸、2.5x 10⁸、2.6x 10⁸、2.7x 10⁸、2.8x 10⁸、2.9x 10⁸、3.0x 10⁸、3.1x 10⁸、3.2x 10⁸、3.3x 10⁸、3.4x 10⁸、3.5x 10⁸、3.6x 10⁸、3.7x 10⁸、3.8x 10⁸、3.9x 10⁸、4.0x 10⁸、4.1x 10⁸、4.2x 10⁸、4.3x 10⁸、4.4x 10⁸、4.5x 10⁸、4.6x 10⁸、4.7x 10⁸、4.8x 10⁸、4.9x 10⁸、5.0x 10⁸、5.1x 10⁸、5.2x 10⁸、5.3x 10⁸、5.4x 10⁸、5.5x 10⁸、5.6x 10⁸、5.7x 10⁸、5.8x 10⁸、5.9x 10⁸ or 6.0x10 ⁸) live CD 19-specific CAR-T cells. In some embodiments, a single infusion bag of any CD 19-specific CAR-T cell described herein comprises about 0.6x10 ⁸ to about 6.0x10 ⁸ (e.g., about 0.6x 10⁸、0.7x 10⁸、0.8x 10⁸、0.9x 10⁸、1.0x 10⁸、1.1x 10⁸、1.2x 10⁸、1.3x 10⁸、1.4x 10⁸、1.5x 10⁸、1.6x 10⁸、1.7x 10⁸、1.8x 10⁸、1.9x 10⁸、2.0x 10⁸、2.1x 10⁸、2.2x 10⁸、2.3x 10⁸、2.4x 10⁸、2.5x 10⁸、2.6x 10⁸、2.7x 10⁸、2.8x 10⁸、2.9x 10⁸、3.0x 10⁸、3.1x 10⁸、3.2x 10⁸、3.3x 10⁸、3.4x 10⁸、3.5x 10⁸、3.6x 10⁸、3.7x 10⁸、3.8x 10⁸、3.9x 10⁸、4.0x 10⁸、4.1x 10⁸、4.2x 10⁸、4.3x 10⁸、4.4x 10⁸、4.5x 10⁸、4.6x 10⁸、4.7x 10⁸、4.8x 10⁸、4.9x 10⁸、5.0x 10⁸、5.1x 10⁸、5.2x 10⁸、5.3x 10⁸、5.4x 10⁸、5.5x 10⁸、5.6x 10⁸、5.7x 10⁸、5.8x 10⁸、5.9x 10⁸ or 6.0x10 ⁸) live CD 19-specific CAR-T cells in about 10mL to about 50mL of cell suspension. In some embodiments, the dose is a therapeutically effective amount of a live CD19 specific CAR-T cell. In other embodiments, the dose is a clinically effective amount of live CD 19-specific CAR-T cells. In some embodiments, the CD19 specific CAR of the cell is with temozolomideThe same CD19 specific CAR, a structural equivalent thereof or a functional equivalent thereof.

In some embodiments, BCMA-specific (BCMA) CAR-T cells described herein are administered to a subject at a dose of about 250x 10 ⁶ to about 500x 10 ⁶ (e.g., ,250x 10⁶、255x 10⁶、260x10⁶、265x 10⁶、270x 10⁶、275x 10⁶、280x 10⁶、285x 10⁶、290x 10⁶、295x 10⁶、300x 10⁶、305x 10⁶、310x 10⁶、315x 10⁶、320x 10⁶、325x 10⁶、330x 10⁶、335x 10⁶、340x 10⁶、345x 10⁶、350x 10⁶、355x 10⁶、360x 10⁶、365x 10⁶、370x 10⁶、375x 10⁶、380x 10⁶、385x 10⁶、390x 10⁶、395x 10⁶、400x 10⁶、405x 10⁶、410x 10⁶、415x 10⁶、420x 10⁶、425x 10⁶、430x 10⁶、435x 10⁶、440x 10⁶、445x 10⁶、450x 10⁶、455x 10⁶、460x 10⁶、465x 10⁶、470x 10⁶、475x 10⁶、480x 10⁶、485x 10⁶、490x 10⁶、495x 10⁶ or 500x 10 ⁶) live BCMA-specific CAR-T cells. In some embodiments, the dose is a therapeutically effective amount of live BCMA-specific CAR-T cells. In other embodiments, the dose is a clinically effective amount of live BCMA-specific CAR-T cells. In some embodiments, the live BCMA-specific CAR-T cells comprise cd4+ T cells expressing a BCMA-specific CAR and cd8+ T cells expressing a BCMA-specific CAR in a ratio of about 1:1. In some embodiments, the BCMA specific CAR of the cell is Ai Jiwei @ lameeStructural equivalents thereof or functional equivalents thereof.

In some embodiments, from about 250x 10 ⁶ to about 500x 10 ⁶ (e.g., ,250x 10⁶、255x 10⁶、260x 10⁶、265x 10⁶、270x 10⁶、275x 10⁶、280x 10⁶、285x 10⁶、290x 10⁶、295x 10⁶、300x 10⁶、305x 10⁶、310x 10⁶、315x 10⁶、320x 10⁶、325x 10⁶、330x 10⁶、335x 10⁶、340x 10⁶、345x 10⁶、350x 10⁶、355x 10⁶、360x 10⁶、365x 10⁶、370x 10⁶、375x 10⁶、380x 10⁶、385x 10⁶、390x 10⁶、395x 10⁶、400x 10⁶、405x 10⁶、410x 10⁶、415x 10⁶、420x 10⁶、425x 10⁶、430x 10⁶、435x 10⁶、440x 10⁶、445x 10⁶、450x 10⁶、455x 10⁶、460x 10⁶、465x 10⁶、470x 10⁶、475x 10⁶、480x 10⁶、485x 10⁶、490x 10⁶、495x 10⁶ or 500x 10 ⁶) live BCMA specific CAR-T cells described herein are administered to a subject. In some embodiments, the dose is a therapeutically effective amount of live BCMA-specific CAR-T cells. In other embodiments, the dose is a clinically effective amount of live BCMA-specific CAR-T cells. In some cases, 50% of the live BCMA-specific CAR-T cells are cd4+ T cells that express BCMA-specific CARs, and 50% of the live BCMA-specific CAR-T cells are cd8+ T cells that express BCMA-specific CARs. In some embodiments, the BCMA specific CAR of the cell is Ai Jiwei @ lameeStructural equivalents thereof or functional equivalents thereof.

In some embodiments, BCMA-specific CAR-T cells described herein are administered to a subject at a dose of up to about 5x 10 ⁸ live BCMA-specific CAR-T cells. In some embodiments, about 2.5x 10 ⁸ to about 5.0x10 ⁸ (e.g., about 0.2x 10⁸、0.4x 10⁸、0.5x 10⁸、0.6x 10⁸、0.8x 10⁸、0.9x 10⁸、1.0x 10⁸、1.2x 10⁸、1.4x 10⁸、1.5x 10⁸、1.6x 10⁸、1.8x 10⁸、1.9x 10⁸、2.0x 10⁸、2.2x 10⁸、2.4x 10⁸、2.5x 10⁸、2.6x 10⁸、2.8x 10⁸、2.9x 10⁸、3.0x 10⁸、3.2x 10⁸、3.4x 10⁸、3.5x 10⁸、3.6x 10⁸、3.8x 10⁸、3.9x 10⁸、4.0x 10⁸、4.2x 10⁸、4.4x 10⁸、4.5x 10⁸、4.6x 10⁸、4.8x 10⁸、4.9x 10⁸ or 5.0x10 ⁸) live BCMA-specific CAR-T cells per kg body weight are administered to the subject. In some embodiments, the dose is a therapeutically effective amount of live BCMA-specific CAR-T cells. In other embodiments, the dose is a clinically effective amount of live BCMA-specific CAR-T cells. In some embodiments, the BCMA specific CAR of the cell is a conjugate with Ai Jiwei @ lameeThe same BCMA specific CAR, a structural equivalent thereof, or a functional equivalent thereof.

In some embodiments, a single dose of any BCMA-specific CAR-T cell described herein comprises about 250x 10 ⁶ to about 500x 10 ⁶ (e.g., ,250x 10⁶、255x10⁶、260x 10⁶、265x 10⁶、270x 10⁶、275x 10⁶、280x 10⁶、285x 10⁶、290x 10⁶、295x 10⁶、300x 10⁶、305x 10⁶、310x 10⁶、315x 10⁶、320x 10⁶、325x 10⁶、330x 10⁶、335x 10⁶、340x 10⁶、345x 10⁶、350x 10⁶、355x 10⁶、360x 10⁶、365x 10⁶、370x 10⁶、375x 10⁶、380x 10⁶、385x 10⁶、390x 10⁶、395x 10⁶、400x 10⁶、405x 10⁶、410x 10⁶、415x 10⁶、420x 10⁶、425x 10⁶、430x 10⁶、435x 10⁶、440x 10⁶、445x 10⁶、450x 10⁶、455x 10⁶、460x 10⁶、465x 10⁶、470x 10⁶、475x 10⁶、480x 10⁶、485x 10⁶、490x 10⁶、495x 10⁶ or 500x 10 ⁶) live BCMA-specific CAR-T cells. In some embodiments, the dose is a therapeutically effective amount of live BCMA-specific CAR-T cells. In other embodiments, the dose is a clinically effective amount of live BCMA-specific CAR-T cells. In some embodiments, the live BCMA-specific CAR-T cells comprise cd4+ T cells expressing a BCMA-specific CAR and cd8+ T cells expressing a BCMA-specific CAR in a ratio of about 1:1. In some embodiments, the BCMA specific CAR is a CAR that is in communication with Ai Jiwei amThe same BCMA specific CAR, a structural equivalent thereof, or a functional equivalent thereof.

In some embodiments, a single dose of any BCMA-specific CAR-T cell described herein comprises about 250x 10 ⁶ to about 500x 10 ⁶ (e.g., ,250x 10⁶、255x10⁶、260x 10⁶、265x 10⁶、270x 10⁶、275x 10⁶、280x 10⁶、285x 10⁶、290x 10⁶、295x 10⁶、300x 10⁶、305x 10⁶、310x 10⁶、315x 10⁶、320x 10⁶、325x 10⁶、330x 10⁶、335x 10⁶、340x 10⁶、345x 10⁶、350x 10⁶、355x 10⁶、360x 10⁶、365x 10⁶、370x 10⁶、375x 10⁶、380x 10⁶、385x 10⁶、390x 10⁶、395x 10⁶、400x 10⁶、405x 10⁶、410x 10⁶、415x 10⁶、420x 10⁶、425x 10⁶、430x 10⁶、435x 10⁶、440x 10⁶、445x 10⁶、450x 10⁶、455x 10⁶、460x 10⁶、465x 10⁶、470x 10⁶、475x 10⁶、480x 10⁶、485x 10⁶、490x 10⁶、495x 10⁶ or 500x 10 ⁶) live BCMA-specific CAR-T cells per kg body weight. In some embodiments, a single dose of any BCMA-specific CAR-T cell described herein comprises about 2.5x 10 ⁸ to about 5.0x10 ⁸ (e.g., about 0.2x 10⁸、0.4x 10⁸、0.5x 10⁸、0.6x 10⁸、0.8x 10⁸、0.9x 10⁸、1.0x 10⁸、1.2x 10⁸、1.4x 10⁸、1.5x 10⁸、1.6x 10⁸、1.8x 10⁸、1.9x 10⁸、2.0x 10⁸、2.2x 10⁸、2.4x 10⁸、2.5x 10⁸、2.6x 10⁸、2.8x 10⁸、2.9x 10⁸、3.0x 10⁸、3.2x 10⁸、3.4x 10⁸、3.5x 10⁸、3.6x 10⁸、3.8x 10⁸、3.9x 10⁸、4.0x 10⁸、4.2x 10⁸、4.4x 10⁸、4.5x 10⁸、4.6x 10⁸、4.8x 10⁸、4.9x 10⁸ or 5.0x10 ⁸) live BCMA-specific CAR-T cells per kg body weight. In some embodiments, a single dose of any BCMA-specific CAR-T cell described herein comprises about 2.5x 10 ⁸ to about 5.0x10 ⁸ (e.g., about 0.2x 10⁸、0.4x 10⁸、0.5x 10⁸、0.6x 10⁸、0.8x 10⁸、0.9x 10⁸、1.0x 10⁸、1.2x 10⁸、1.4x 10⁸、1.5x 10⁸、1.6x 10⁸、1.8x 10⁸、1.9x 10⁸、2.0x 10⁸、2.2x 10⁸、2.4x 10⁸、2.5x 10⁸、2.6x 10⁸、2.8x 10⁸、2.9x 10⁸、3.0x 10⁸、3.2x 10⁸、3.4x 10⁸、3.5x 10⁸、3.6x 10⁸、3.8x 10⁸、3.9x 10⁸、4.0x 10⁸、4.2x 10⁸、4.4x 10⁸、4.5x 10⁸、4.6x 10⁸、4.8x 10⁸、4.9x 10⁸ or 5.0x10 ⁸) live BCMA-specific CAR-T cells. In some embodiments, a single infusion bag of any BCMA-specific CAR-T cell described herein comprises about 2.5x10 ⁸ to about 5.0x10 ⁸ (e.g., about 0.2x 10⁸、0.4x 10⁸、0.5x 10⁸、0.6x 10⁸、0.8x 10⁸、0.9x 10⁸、1.0x 10⁸、1.2x 10⁸、1.4x 10⁸、1.5x 10⁸、1.6x 10⁸、1.8x 10⁸、1.9x 10⁸、2.0x 10⁸、2.2x 10⁸、2.4x 10⁸、2.5x 10⁸、2.6x 10⁸、2.8x 10⁸、2.9x 10⁸、3.0x 10⁸、3.2x 10⁸、3.4x 10⁸、3.5x 10⁸、3.6x 10⁸、3.8x 10⁸、3.9x 10⁸、4.0x 10⁸、4.2x 10⁸、4.4x 10⁸、4.5x 10⁸、4.6x 10⁸、4.8x 10⁸、4.9x 10⁸ or 5.0x10 ⁸) live BCMA-specific CAR-T cells in about 10mL to about 500mL of cell suspension. In some embodiments, the cell suspension is about 50mL, 250mL, or about 500mL. In some embodiments, the dose is a therapeutically effective amount of live BCMA-specific CAR-T cells. In other embodiments, the dose is a clinically effective amount of live BCMA-specific CAR-T cells. In some embodiments, the BCMA specific CAR of the cell is a conjugate with Ai Jiwei @ lameeThe same BCMA specific CAR, a structural equivalent thereof, or a functional equivalent thereof.

BB. methods for administering low-immunogenicity cells (including T cells)

As described in further detail herein, provided herein are methods of treating a patient suffering from a condition, disorder or disorder by administering a low-immunogenicity cell (particularly a low-immunogenicity T cell). It will be appreciated that for all of the various embodiments described herein relating to the scheduling and/or combination of therapies, 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 directly infused, implanted or transplanted to a desired site, or administered by any suitable route that results in delivery to a desired location in a subject where at least a portion of the implanted cells or cellular components remain active.

Provided herein are methods for treating a patient having a condition, disorder, or disorder, comprising administering to a subject (e.g., a human patient) a population of low-immunogenic cells (e.g., primary T cells, T cells differentiated from low-immunogenic induced pluripotent stem cells, or other cells differentiated from low-immunogenic induced pluripotent stem cells described herein). For example, a population of low-immunogenicity primary T cells, such as, but not limited to, 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, congenital memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cells, is administered to a patient to treat a condition, disorder or disorder. In some embodiments, the immunosuppressant and/or immunomodulatory agent (such as, but not limited to, a lymphocyte scavenger) is not administered to the patient prior to administration of the population of hypoimmunogenic cells. 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 administration of the cells. In various embodiments, the immunosuppressant and/or immunomodulator is not administered to the patient after administration of the cells, or is administered at least 1, 2, 3, 4, 5,6, 7, 8,9, 10, 11, 12, 13, 14 days 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 administration of the cells. In some embodiments, wherein the immunosuppressant and/or immunomodulatory agent is administered to the patient prior to or after administration of the cells, the dosage administered is less than the dosage required for cells having one or more MHC I and/or MHC II molecules expressed and no exogenous CD47 expressed.

Non-limiting examples of immunosuppressants and/or immunomodulators such as, but not limited to lymphocyte depleting agents, include cyclosporin (cyclosporine), azathioprine (azathioprine), mycophenolic acid (mycophenolic acid), mycophenolic acid ester (mycophenolate mofetil), corticosteroids such as prednisone (prednisone), methotrexate, gold salts, sulfasalazine (sulfasalazine), antimalarials, buconas (brequinar), leflunomide (leflunomide), mizoribine, 15-deoxyspergualin, 6-mercaptopurine, cyclophosphamide, rapamycin (rapamycin), tacrolimus (tacrolimus) (FK-506), OKT3, anti-thymocyte globulin, thymopentapeptides, 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 antibodies that bind to any of its ligands. In some embodiments, 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, wherein the immunosuppressant and/or immunomodulatory agent is administered to the patient prior to or after administration of the cells, the dosage administered is less than the dosage required for cells having one or more MHC I and/or MHC II molecule expression, TCR expression, and no exogenous CD47 expression. In some embodiments, wherein the immunosuppressant and/or immunomodulatory agent is administered to the patient prior to or after the first administration of cells, the dose administered is lower than the dose required for cells having one or more MHC I and/or MHC II molecule expression, TCR expression, and no exogenous CD47 expression.

In some embodiments, the cells are co-administered with a therapeutic agent that binds to and/or interacts with one or more receptors selected from the group consisting of: CD94, KIR2DL4, PD-1, inhibitory NK cell receptor and activated NK receptor. In some cases, the therapeutic agent binds to a receptor on the surface of NK cells (including one or more subsets of NK cells). In some embodiments, the therapeutic agent is selected from the group consisting of: antibodies, fragments and variants thereof, antibody mimics, small molecules, blocking peptides and receptor antagonists.

In some embodiments, the cellular taboos described herein are used in patients known to have type I hypersensitivity or anaphylaxis to murine proteins, chinese Hamster Ovary (CHO) cellular proteins, or any component of the compositions described herein. In some embodiments, the cell contraindications described herein are for patients suffering from or having suffered from Progressive Multifocal Leukoencephalopathy (PML). In some embodiments, the cells described herein are not recommended for use in patients with severe, active infections.

In some embodiments, the cells described herein are administered to a previously used rituximabA subject treated with an autoimmune disease/disorder and/or an inflammatory disease/disorder. In some embodiments, the cells described herein are administered to a previously used rituximabSubjects with autoimmune diseases/disorders and/or inflammatory diseases/disorders who have failed and/or are unresponsive to rituximab therapy are treated. In some embodiments, the patient has Rheumatoid Arthritis (RA). In some embodiments, the patient has RA and rituximab treatment is combined with methotrexate. In some embodiments, the patient is an adult patient with moderate to severe active RA. In some embodiments, the patient is an adult patient with moderate to severe active RA, and rituximab treatment is combined with methotrexate. In some embodiments, the patient is an adult patient with moderate to severe active RA who is under-responsive to one or more TNF antagonist therapies, and rituximab therapy is combined with methotrexate. In some embodiments, the rituximab dose for RA in combination with methotrexate is 1000mg intravenous infusion every 24 weeks and/or based on clinical evaluation but not less than 2 weeks (a course of treatment) every 16 weeks. In some embodiments, it is recommended to administer 100mg of methylprednisolone or equivalent glucocorticoid intravenously 30 minutes prior to each infusion.

In some embodiments, the cells described herein are administered to a previously used rituximabA subject treated with an autoimmune disease/disorder and/or an inflammatory disease/disorder. In some embodiments, the cells described herein are administered to a previously used rituximabSubjects with autoimmune diseases/disorders and/or inflammatory diseases/disorders who have failed and/or are unresponsive to rituximab therapy are treated. In some embodiments, the patent has Granulomatous Polyangiitis (GPA) (wegener granulomatous). In some embodiments, the patent has Microscopic Polyangiitis (MPA) in adult patients in combination with a glucocorticoid. In some embodiments, the rituximab dose for GPA and MPA in combination with a glucocorticoid is 375mg/m ², once a week for 4 weeks. In some embodiments, rituximab is administered as a 100mg/10mL solution in a disposable vial. In some embodiments, rituximab is administered as a 500mg/50mL solution in a disposable vial.

CC method for detecting the presence of antibodies

In some embodiments, a biological sample from a patient is assayed to determine if the sample contains antibodies to one or more Y chromosome genes.

Methods for detecting antibodies in biological samples are well known in the art. In some embodiments, the anti-Y-linked tropocadherin 11 antibody and/or the anti-Y-linked fibronectin 4 antibody is detected from a biological sample obtained from a patient using any method mature in the art, such as an enzyme-linked immunosorbent assay (ELISA), nucleic acid and test, western blot, and the like.

In some embodiments, the biological sample is a plasma sample, a peripheral blood sample, a urine sample, a sputum/saliva sample, or a serum sample obtained from a patient.

In some embodiments, the methods are used to determine an appropriate cell-based therapy to administer to a patient suffering from a disease or condition that would benefit from cell-based therapy. In some embodiments, the methods are used to identify patients suffering from a disease or condition that would benefit from cell-based therapies that include reduced expression of one or more Y chromosome genes. In some embodiments, the methods are used to determine whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to NK-mediated cytotoxicity after administration to a patient. In some embodiments, the methods are used to determine whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to lysis by mature NK cells after administration to a patient. In some embodiments, the methods are used to determine whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to phagocytosis by macrophages after administration to a patient. In some embodiments, the methods are used to determine whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to an induced immune response after administration to a patient. In some embodiments, the methods are used to determine whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to an induced antibody-based immune response after administration to a patient. In some embodiments, the methods are used in methods of treating patients suffering from diseases or conditions that would benefit from cell-based therapies.

In some embodiments, the method comprises: (a) Determining whether a biological sample from a patient comprises antibodies to one or more Y chromosome genes by: (i) obtaining or having obtained a biological sample from a patient; (ii) An assay is performed or has been performed to determine whether an antibody to Y-linked tropocadherin 11 is present in the biological sample; and (iii) performing or having performed an assay to determine whether an antibody to Y-linked fibronectin 4 is present in the biological sample; and (b) administering the engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, or glial progenitor cell population of any of claims 1-50 to the patient, wherein: (i) If antibodies to Y-linked tropocadherin 11 are present in the biological sample, the population of cells comprises reduced expression of Y-linked tropocadherin 11; (ii) If antibodies to Y-linked fibronectin 4 are present in the biological sample, the population of cells comprises reduced expression of Y-linked fibronectin 4; (iii) If antibodies to Y-linked tropocadherin 11 and antibodies to Y-linked fibronectin 4 are present in the biological sample, the cell population comprises reduced expression of Y-linked tropocadherin 11 and Y-linked fibronectin 4; (iv) If there is neither an antibody to Y-linked tropocadherin 11 nor an antibody to Y-linked fibronectin 4 in the biological sample, the cell population does not comprise reduced expression of Y-linked tropocadherin 11 or Y-linked fibronectin 4.

IV. examples

Example 1: expression of tropocadherin-Y and fibronectin-Y on iPSC and T cells

iPSC

To determine if troponin-Y and/or fibronectin-Y were expressed on ipscs, troponin-Y and fibronectin-Y expression from ipscs derived from male rhesus monkeys were analyzed using standard techniques. Ipscs were analyzed by flow cytometry (using standard methods). Ipscs from human females served as controls.

Cells were blocked with anti-Fc receptor antibody and stained with either an antigen cadherin-Y antibody or an anti-fibronectin-Y antibody matched to the isotype control concentration. As shown in FIGS. 1A and 1B, tropocadherin-Y and fibronectin-Y were both expressed on iPSC from male donors. Both tropocadherin-Y and fibronectin-Y were not expressed on ipscs derived from female donors.

Without being bound by theory, the surprising discovery that tropocadherin-Y and fibronectin-Y are expressed on ipscs suggests that the antigen encoded by the Y chromosome can be recognized as a foreign antigen in female receptors, resulting in T cell and B cell activation.

T cell

To determine if tropocadherin-Y and/or fibronectin-Y were expressed on T cells, T cells from 5 male donors were sorted for CD3 expression to generate a cd3+ population, and the tropocadherin-Y and fibronectin-Y expression of cd3+ T cells were analyzed using standard techniques. T cells were analyzed by flow cytometry (using standard methods) after thawing. Cd3+ T cells from two female donors served as controls.

Cells were blocked with anti-Fc receptor antibody and stained with either an antigen cadherin-Y antibody or an anti-fibronectin-Y antibody matched to the isotype control concentration. As shown in fig. 2A, 2B and 2C, tropocadherin-Y and fibronectin-Y were both expressed on T cells derived from male donors. Both tropocadherin-Y and fibronectin-Y are not expressed on T cells derived from female donors.

Without being bound by theory, the surprising discovery that tropocadherin-Y and fibronectin-Y are expressed on T cells suggests that the antigen encoded by the Y chromosome may be recognized as a foreign antigen in female receptors, resulting in T cell and B cell activation.

ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity)

Male HIPT cells (=HLA-I/II KO; CD47 KI) were incubated with serum from men, women after girls and women after boys. Killing of HIP T cells by CDC and ADCC mechanisms was analyzed using Xcelligence cell killing assay.

As shown in fig. 3 to 5, HIP T cells of male origin are killed by NK cells and CDC in serum from females with anti-H-Y antibodies (i.e. females sensitized by prior boys but not girls).

Claims

1. An engineered cell comprising reduced expression of one or more Y chromosome genes and a Major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigen molecule relative to an unmodified wild-type or control cell and a first exogenous polynucleotide encoding CD47, wherein the engineered cell is propagated by a primary T cell or progeny thereof, or derived from an Induced Pluripotent Stem Cell (iPSC) or progeny thereof.

2. A low immunogenicity T cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified or wild-type or control cell, wherein the low immunogenicity T cell is propagated by a primary T cell or progeny thereof, or derived from an iPSC or progeny thereof.

3. A non-activated T cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified or wild-type or control cell, wherein the non-activated T cell is propagated by a primary T cell or progeny thereof, or derived from an iPSC or progeny thereof.

4. An islet cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified wild-type or control cell, wherein the islet cell is derived from an iPSC or progeny thereof.

5. A cardiomyocyte comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified wild-type or control cell, wherein the cardiomyocyte is derived from an iPSC or a progeny thereof.

6. A glial progenitor cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules relative to an unmodified wild-type or control cell and a first exogenous polynucleotide encoding CD47, wherein the cardiomyocyte is derived from iPSC or a progeny thereof.

7. An NK cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified wild-type or control cell, wherein the cardiomyocyte is derived from an iPSC or progeny thereof.

8. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-7, wherein the Y chromosome gene is a Y chromosome-linked antigen or a secondary histocompatibility antigen associated with the Y chromosome.

9. The engineered cell, hypo-immunogenic T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 8, wherein the one or more Y chromosome-linked antigens are Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

10. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-9, wherein the cell has reduced expression of Y-linked tropocadherin 11.

11. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-10, wherein the cell has reduced expression of Y-linked fibronectin 4.

12. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-11, wherein the cell has reduced expression of Y-linked tropocadherin 11 and reduced expression of Y-linked fibronectin 4.

13. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-12, wherein the cell is genetically engineered to have reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

14. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-13, wherein the cell does not express Y-linked tropocadherin 11.

15. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-14, wherein the cell does not express Y-linked fibronectin 4.

16. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-15, wherein the cell does not express Y-linked tropocadherin 11 and does not express Y-linked fibronectin 4.

17. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-16, wherein the cell is genetically engineered to not express Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

18. The engineered cell, hypoimmunogenic T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell of any one of claims 1-17 that performs NK cell, wherein reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is caused by knockout of PCDH11Y and/or NLGN4Y gene, respectively.

19. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-18, wherein the cell is derived from a human cell or an animal cell.

20. The engineered cell, hypo-immunogenic T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 19, wherein the human cell or animal cell is from a donor subject that does not have a Y chromosome.

21. The engineered cell, hypoimmunogenic T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 19, wherein the human cell or animal cell is from a donor subject having a Y chromosome, and wherein the cell is genetically engineered to have reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

22. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 21, wherein the cell is genetically engineered to not express Y-linked tropocadherin 11.

23. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 21, wherein the cell is genetically engineered to not express Y-linked fibronectin 4.

24. The engineered cell, hypoimmunogenic T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 21, wherein the cell is genetically engineered to not express Y-linked tropocadherin 11 and not express Y-linked fibronectin 4.

25. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-24, wherein the cell is propagated from or derived from a cell pool isolated from one or more donor subjects different from the patient, wherein the one or more donor subjects optionally comprise one or more subjects with a Y chromosome; one or more subjects without a Y chromosome; or a mixture of subjects with a Y chromosome and subjects without a Y chromosome.

26. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-25, wherein the cell is genetically engineered to have reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 using CRISPR/Cas gene editing.

27. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 26, wherein the CRISPR/Cas gene editing is performed using one or more guide RNAs comprising any of the sequences of tables 2-5.

28. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-27, wherein the CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, cas12a, and Cas12 b.

29. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 28, wherein the CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of:

a. Optionally selected from the group consisting of Cas3, cas8a, cas5, cas8b, cas8c, cas10d, cse1, cse2, csy1, csy2, csy3, and GSU 0054;

b. optionally selected from the group consisting of Cas9, csn2 and Cas 4;

c. optionally selected from the group consisting of Cas10, csm2, cmr5, cas10, csx11, and Csx 10;

d. Optionally Csf1;

e. Optionally selected from the group consisting of Cas12a, cas12b, cas12C, C2C4, C2C8, C2C5, C2C10, C2C9, casX (Cas 12 e) and CasY (Cas 12 d); and

F. optionally selected from the group consisting of Cas13, cas13a, C2, cas13b, cas13C, and Cas13 d.

30. The engineered cell, low-immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 26-29, wherein the CRISPR/Cas gene editing is performed ex vivo from a donor subject.

31. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 30, wherein the CRISPR/Cas gene editing is performed using a lentiviral vector.

32. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-31, wherein the cell comprises reduced expression of β -2-microglobulin (B2M) and/or MHC class II transactivator (CIITA) relative to an unmodified wild-type or control cell.

33. The engineered cell, hypo-immunogenic T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 32, wherein the cell does not express B2M and/or CIITA.

34. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-33, wherein the cell comprises reduced expression of RHD.

35. The engineered cell, hypo-immunogenic T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 34, wherein the cell does not express RHD.

36. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-35, wherein the cell is a differentiated cell derived from an induced pluripotent stem cell or a progeny thereof.

37. The engineered cell of claim 36, wherein the differentiated cell is selected from the group consisting of: t cells, NK cells, endothelial cells, islet cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, hepatocytes, glial progenitor cells, dopaminergic neurons, retinal pigment epithelial cells, and thyroid cells.

38. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-37, wherein the cell is a primary immune cell or a progeny thereof.

39. The engineered cell of claim 38, wherein the primary immune cell or progeny thereof is a T cell or NK cell.

40. An engineered cell, a low immunogenicity T cell, or a non-activated T cell according to any one of claims 1-39, wherein the cell comprises reduced expression of TCR- α and/or TCR- β.

41. The engineered cell, low immunogenicity T cell, or non-activated T cell of claim 40, wherein the cell does not express TCR- α and/or TCR- β.

42. The engineered cell, low immunogenicity T cell, non-activated T cell, or NK cell of any one of claims 1-41, wherein the cell further comprises a second exogenous polynucleotide encoding one or more Chimeric Antigen Receptors (CARs), wherein the one or more CARs comprise an extracellular ligand binding domain, hinge domain, transmembrane domain, costimulatory domain, and intracellular signaling domain specific for CD19, CD20, CD22, or BCMA.

43. The engineered cell, low immunogenicity T cell, non-activated T cell, or NK cell of claim 42, wherein the one or more CARs comprise a CD 8a hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

44. The engineered cell, low immunogenicity T cell, non-activated T cell, or NK cell of claim 43, wherein the one or more CARs comprise a CD 8a hinge domain having the amino acid sequence of SEQ ID No. 9.

45. The engineered cell, low immunogenicity T cell, non-activated T cell, or NK cell of claim 43, wherein the one or more CARs comprise a CD28 hinge domain having the amino acid sequence of SEQ ID No.10 or 113.

46. The engineered cell, low immunogenicity T cell, non-activated T cell, or NK cell of claim 43, wherein the one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID No. 11 or 12.

47. The engineered cell, low-immunogenicity T cell, non-activated T cell, or NK cell of any one of claims 42-46, wherein the one or more CARs comprise a CD8 a transmembrane domain or a CD28 transmembrane domain.

48. The engineered cell, low immunogenicity T cell, non-activated T cell, or NK cell of claim 47, wherein the one or more CARs comprise a CD8 a transmembrane domain having the amino acid sequence of SEQ ID No. 14.

49. The engineered cell, low immunogenicity T cell, non-activated T cell, or NK cell of claim 47, wherein the one or more CARs comprise a CD28 transmembrane domain having the amino acid sequence of SEQ ID No. 15 or 114.

50. The engineered cell, low-immunogenicity T cell, non-activated T cell, or NK cell of any one of claims 42-49, wherein the one or more CARs comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3 zeta signaling domain.

51. The engineered cell, low immunogenicity T cell, non-activated T cell, or NK cell of claim 50, wherein the one or more CARs comprise a 4-1BB co-stimulatory domain having the amino acid sequence of SEQ ID No. 16.

52. The engineered cell, low immunogenicity T cell, non-activated T cell, or NK cell of claim 50, wherein the one or more CARs comprise a CD28 co-stimulatory domain having the amino acid sequence of SEQ ID No. 17.

53. The engineered cell, low immunogenicity T cell, non-activated T cell, or NK cell of claim 50, wherein the one or more CARs comprise a CD3 zeta signaling domain having the amino acid sequence of SEQ id No. 18 or 115.

54. The engineered cell, low immunogenicity T cell, non-activated T cell, or NK cell of any one of claims 42-53, wherein the one or more CARs comprise an extracellular ligand binding domain comprising an scFv sequence of any one of SEQ ID NOs 19, 37, 45, 54, 63, 72, 81, or 118, or wherein the CAR has an scFv sequence comprising the heavy and light chain sequences of any one of SEQ ID NOs 20, 25, 38, 42, 46, 50, 64, 68, 73, 77, 119, or 123.

55. The engineered cell, low-immunogenicity T cell, non-activated T cell, or NK cell of any one of claims 42-54, wherein the one or more CARs have the sequence of any one of SEQ ID NOs 32, 34, 36, 117, or 128.

56. The engineered cell, low-immunogenicity T cell, non-activated T cell, or NK cell of any one of claims 42-55, wherein the one or more CARs comprise the amino acid sequence set forth in SEQ ID No. 117 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 117, the one or more CARs having the following components: CD8 a signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD8 a hinge domain, CD8 a transmembrane domain, 4-1BB co-stimulatory domain and CD3 zeta signaling domain.

57. The engineered cell, low-immunogenicity T cell, non-activated T cell, or NK cell of any one of claims 42-55, wherein the one or more CARs comprise the amino acid sequence set forth in SEQ ID No. 45 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 45.

58. The engineered cell, low immunogenicity T cell, NK cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any of claims 1-57, wherein one or more of the first and/or second exogenous polynucleotides is inserted into a first and/or second specific locus of at least one allele of the cell.

59. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 58, wherein the first and/or second specific locus is selected from the group consisting of: safe harbor or target loci, RHD loci, B2M loci, CIITA loci, TRAC loci, and TRB loci.

60. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 59, wherein the safe harbor or target locus is selected from the group consisting of: CCR5 locus, CXCR4 locus, PPP1R12C locus, ALB locus, SHS231 locus, CLYBL locus, rosa locus, F3 (CD 142) locus, MICA locus, MICB locus, LRP1 (CD 91) locus, HMGB1 locus, ABO locus, FUT1 locus and KDM5D locus.

61. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-60, wherein the first and/or second exogenous polynucleotide is introduced into the cell using a gene therapy vector or a transposase system selected from the group consisting of a transposase, a PiggyBac transposon, a sleeping beauty (SB 11) transposon, a Mos1 transposon, and a Tol2 transposon.

62. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 61, wherein the gene therapy vector is a retrovirus or a fusion.

63. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 62, wherein the retrovirus is a lentiviral vector.

64. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any of claims 1-63, wherein the first and/or second exogenous polynucleotide is introduced into the cell using CRISPR/Cas gene editing.

65. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-64, wherein the CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, cas12a, and Cas12 b.

66. The engineered cell, low-immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 65, wherein the CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of:

b. optionally selected from the group consisting of Cas9, csn2 and Cas 4;

d. Optionally Csf1;

67. The engineered cell, low-immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any of claims 64-66, wherein the CRISPR/Cas gene editing is performed ex vivo from a donor subject.

68. The engineered cell, low-immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of claim 67, wherein the CRISPR/Cas gene editing is performed using a lentiviral vector.

69. The engineered cell, low-immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-68, wherein the cell or progeny thereof, upon administration to a patient, evades NK cell-mediated cytotoxicity.

70. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-69, wherein the cell or progeny thereof is protected from cell lysis by mature NK cells after administration to a patient.

71. The engineered cell, low-immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-70, wherein the cell or progeny thereof evades macrophage phagocytosis upon administration to a patient.

72. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-71, wherein the cell or progeny thereof does not induce an immune response against the cell upon administration to a patient.

73. The engineered cell, low-immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any of claims 1-72, wherein the cell or progeny thereof does not induce an antibody-based immune response against the cell upon administration to a patient.

74. The engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell of any one of claims 1-73, wherein the wild-type cell or the control cell is a starting material.

75. A pharmaceutical composition comprising the engineered cell, low immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell population of any of claims 1-74, and a pharmaceutically acceptable additive, carrier, diluent, or excipient.

76. The pharmaceutical composition of claim 75, wherein the composition comprises one or more cell populations selected from the group consisting of a low-immunogenicity T cell population, a non-activated T cell population, a low-immunogenicity CD19 CAR T cell population, and a low-immunogenicity CD22 CAR T cell population, and a pharmaceutically acceptable additive, carrier, diluent, or excipient.

77. A method of treating a patient suffering from a disease or condition that would benefit from cell-based therapy, the method comprising administering to the patient the engineered cells, low-immunogenicity T cells, non-activated T cells, islet cells, cardiomyocytes, glial progenitor cells, or NK cell population of any of claims 1-76.

78. The method of claim 77, wherein the patient does not have a Y chromosome.

79. The method of claim 77 or 78, wherein the patient is insensitive to Y chromosome genes.

80. The method of claim 77 or 78, wherein the patient is sensitive to a Y chromosome gene.

81. The method of claim 80, wherein the patient has previously received cell therapy derived from a donor subject having a Y chromosome or cell therapy that otherwise expresses one or more Y chromosome genes.

82. The method of claim 80 or 81, wherein the patient is a female patient who previously had a male child.

83. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient the primary immune cell population of any one of claims 1-3 or 7-74.

84. The method of claim 83, wherein the primary immune cells are selected from the group consisting of T cells and NK cells.

85. The method of claim 83 or 84, wherein the patient does not have a Y chromosome.

86. The method of any one of claims 83-85, wherein the patient is insensitive to Y chromosome genes.

87. The method of any one of claims 83-85, wherein the patient is sensitive to a Y chromosome gene.

88. The method of claim 87, wherein the patient has previously received cell therapy derived from a donor subject having a Y chromosome or cell therapy that otherwise expresses one or more Y chromosome genes.

89. The method of claim 87 or 88, wherein the patient is a female patient who previously had a male child.

90. A method of determining an appropriate cell-based therapy to administer to a patient having a disease or condition that would benefit from a cell-based therapy, the method comprising:

(a) Determining whether a biological sample from the patient comprises antibodies to one or more Y chromosome genes by:

(i) Obtaining or having obtained a biological sample from the patient;

(ii) Performing or having performed an assay to determine whether an antibody to Y-linked tropocadherin 11 is present in the biological sample; and

(Iii) Performing or having performed an assay to determine whether an antibody to Y-linked fibronectin 4 is present in the biological sample; and

(B) Administering to the patient the engineered cell, low-immunogenicity T cell, non-activated T cell, islet cell, cardiomyocyte, glial progenitor cell, or NK cell population of any of claims 1-74, wherein:

(i) If antibodies to Y-linked tropocadherin 11 are present in the biological sample, the population of cells comprises reduced expression of Y-linked tropocadherin 11;

(ii) If antibodies to Y-linked fibronectin 4 are present in the biological sample, the population of cells comprises reduced expression of Y-linked fibronectin 4;

(iii) If antibodies to Y-linked tropocadherin 11 and antibodies to Y-linked fibronectin 4 are present in the biological sample, the population of cells comprises reduced expression of Y-linked tropocadherin 11 and Y-linked fibronectin 4;

(iv) If there is neither an antibody to Y-linked tropocadherin 11 nor an antibody to Y-linked fibronectin 4 in the biological sample, the cell population does not comprise reduced expression of Y-linked tropocadherin 11 or Y-linked fibronectin 4.

91. A method of identifying a patient having a disease or condition that would benefit from a cell-based therapy comprising reduced expression of one or more Y chromosome genes, the method comprising:

(i) Obtaining or having obtained a biological sample from the patient;

92. A method for identifying a patient having a disease or condition that would benefit from a cell-based therapy comprising reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4, the method comprising:

(a) Determining whether a biological sample from said patient comprises antibodies to Y-linked tropocadherin 11 and/or antibodies to Y-linked fibronectin 4 by:

(i) Obtaining or having obtained a biological sample from the patient;

93. A method of determining whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to NK-mediated cytotoxicity after administration to a patient, the method comprising:

(i) Obtaining or having obtained a biological sample from the patient;

94. A method of determining whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to lysis by mature NK cells after administration to a patient, the method comprising:

(i) Obtaining or having obtained a biological sample from the patient;

95. A method of determining whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to phagocytosis by macrophages after administration to a patient, the method comprising:

(i) Obtaining or having obtained a biological sample from the patient;

96. A method of determining whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to an induced immune response after administration to a patient, the method comprising:

(i) Obtaining or having obtained a biological sample from the patient;

97. A method of determining whether a cell-based therapy that does not include reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is susceptible to an induced antibody-based immune response after administration to a patient, the method comprising:

(i) Obtaining or having obtained a biological sample from the patient;

98. A method of treating a patient suffering from a disease or condition that would benefit from cell-based therapy, the method comprising:

(i) Obtaining or having obtained a biological sample from the patient;

99. The method of any one of claims 77-98, wherein the Y chromosome gene is a Y chromosome-linked antigen or a minor histocompatibility antigen associated with the Y chromosome.

100. The method of claim 99, wherein the one or more Y chromosome-linked antigens are Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

101. The method of any one of claims 77-100, wherein the cell has reduced expression of Y-linked tropocadherin 11.

102. The method of any one of claims 77-101, wherein the cell has reduced expression of Y-linked fibronectin 4.

103. The method of any one of claims 77-102, wherein the cell has reduced expression of Y-linked tropocadherin 11 and reduced expression of Y-linked fibronectin 4.

104. The method of any one of claims 77-103, wherein the cell is genetically engineered to have reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

105. The method of any one of claims 77-104, wherein the cell does not express Y-linked tropocadherin 11.

106. The method of any one of claims 77-105, wherein the cell does not express Y-linked fibronectin 4.

107. The method of any one of claims 77-106, wherein the cell does not express Y-linked tropocadherin 11 and does not express Y-linked fibronectin 4.

108. The method of any one of claims 77-107, wherein the cell is genetically engineered to not express Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

109. The method of any one of claims 77-108, wherein reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is caused by knockout of PCDH11Y and/or NLGN4Y genes, respectively.

110. The method of any one of claims 77-109, wherein the cells are derived from human cells or animal cells.

111. The method of claim 110, wherein the human or animal cell is from a donor subject that does not have a Y chromosome.

112. The method of claim 110, wherein the human or animal cell is from a donor subject having a Y chromosome, and wherein the cell is genetically engineered to have reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

113. The method of claim 112, wherein the cells are genetically engineered to not express Y-linked tropocadherin 11.

114. The method of claim 112, wherein the cell is genetically engineered to not express Y-linked fibronectin 4.

115. The method of claim 112, wherein the cell is genetically engineered to not express Y-linked tropocadherin 11 and not express Y-linked fibronectin 4.

116. The method of any one of claims 77-115, wherein the cells are propagated from or derived from a cell pool isolated from one or more donor subjects different from the patient, wherein the one or more donor subjects optionally comprise one or more subjects having a Y chromosome; one or more subjects without a Y chromosome; or a mixture of subjects with a Y chromosome and subjects without a Y chromosome.

117. The method of any one of claims 77-116, wherein the cells are genetically engineered to have reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 using CRISPR/Cas gene editing.

118. The method of claim 117, wherein the CRISPR/Cas gene editing is performed using one or more guide RNAs comprising any of the sequences of tables 2-5.

119. The method of any one of claims 117-118, wherein the CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, cas12a, and Cas12 b.

120. The method of claim 119, wherein the CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of:

b. optionally selected from the group consisting of Cas9, csn2 and Cas 4;

d. Optionally Csf1;

121. The method of any one of claims 117-120, wherein the CRISPR/Cas gene editing is performed ex vivo from a donor subject.

122. The method of claim 121, wherein the CRISPR/Cas gene editing is performed using a lentiviral vector.

123. The method of any one of claims 77-122, wherein the cell comprises reduced expression of B2M and/or CIITA relative to an unmodified or wild-type or control cell.

124. The method of claim 123, wherein the cell does not express B2M and/or CIITA.

125. The method of any one of claims 77-124, wherein said cell comprises reduced expression of RHD.

126. The method of claim 125, wherein the cell does not express RHD.

127. The method of any one of claims 77-126, wherein the cell is a differentiated cell derived from an induced pluripotent stem cell or progeny thereof.

128. The method of claim 127, wherein the differentiated cell is selected from the group consisting of: t cells, NK cells, endothelial cells, islet cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, hepatocytes, glial progenitor cells, dopaminergic neurons, retinal pigment epithelial cells, and thyroid cells.

129. The method of any one of claims 77-126, wherein the cell is a primary immune cell or a progeny thereof.

130. The engineered cell of claim 129, wherein the primary immune cell or progeny thereof is a T cell or NK cell.

131. The method of any one of claims 77-130, wherein the cell comprises reduced expression of TCR-a and/or TCR- β.

132. The method of claim 131, wherein the cell does not express TCR-a and/or TCR- β.

133. The method of any one of claims 77-132, wherein the cell further comprises a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain specific for CD19, CD20, CD22, or BCMA.

134. The method of claim 133, wherein the one or more CARs comprise a CD8 a hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

135. The method of claim 134, wherein the one or more CARs comprise a CD8 a hinge domain having the amino acid sequence of SEQ ID No. 9.

136. The method of claim 134, wherein the one or more CARs comprise a CD28 hinge domain having the amino acid sequence of SEQ ID No. 10 or 113.

137. The method of claim 134, wherein the one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID No. 11 or 12.

138. The method of any one of claims 133-137, wherein the one or more CARs comprise a CD8 a transmembrane domain or a CD28 transmembrane domain.

139. The method of claim 138, wherein the one or more CARs comprise a CD8 a transmembrane domain having the amino acid sequence of SEQ ID No. 14.

140. The method of claim 138, wherein the one or more CARs comprise a CD28 transmembrane domain having the amino acid sequence of SEQ ID No. 15 or 114.

141. The method of any one of claims 133-140, wherein the one or more CARs comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3 zeta signaling domain.

142. The method of claim 141, wherein the one or more CARs comprise a 4-1BB co-stimulatory domain having the amino acid sequence of SEQ ID No. 16.

143. The method of claim 141, wherein the one or more CARs comprise a CD28 co-stimulatory domain having the amino acid sequence of SEQ ID No. 17.

144. The method of claim 141, wherein the one or more CARs comprise a CD3 zeta signaling domain having the amino acid sequence of SEQ ID No. 18 or 115.

145. The method of any one of claims 133-144, wherein the one or more CARs comprise an extracellular ligand binding domain comprising an scFv sequence of any one of SEQ ID NOs 19, 37, 45, 54, 63, 72, 81, or 118, or wherein the CAR has an scFv sequence comprising the heavy and light chain sequences of any one of SEQ ID NOs 20, 25, 38, 42, 46, 50, 64, 68, 73, 77, 119, or 123.

146. The method of any of claims 133-145, wherein the one or more CARs have the sequence of any of SEQ ID NOs 32, 34, 36, 117, or 128.

147. The method of any one of claims 133-146, wherein the one or more CARs comprise the amino acid sequence set forth in SEQ ID No. 117 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in the SEQ ID No. 117, the one or more CARs having the following components: CD8 a signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD8 a hinge domain, CD8 a transmembrane domain, 4-1BB co-stimulatory domain and CD3 zeta signaling domain.

148. The method of any one of claims 133-146, wherein the one or more CARs comprise the amino acid sequence set forth in SEQ ID No. 45 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 45.

149. The method of any one of claims 77-148, wherein one or more of the first and/or second exogenous polynucleotides is inserted into a first and/or second specific locus of at least one allele of the cell.

150. The method of claim 149, wherein the first and/or second specific loci are selected from the group consisting of: safe harbor or target loci, RHD loci, B2M loci, CIITA loci, TRAC loci, and TRB loci.

151. The method of claim 150, wherein the safe harbor or target locus is selected from the group consisting of: CCR5 locus, CXCR4 locus, PPP1R12C locus, ALB locus, SHS231 locus, CLYBL locus, rosa locus, F3 (CD 142) locus, MICA locus, MICB locus, LRP1 (CD 91) locus, HMGB1 locus, ABO locus, FUT1 locus and KDM5D locus.

152. The method of any one of claims 77-151, wherein said first and/or second exogenous polynucleotide is introduced into said cell using a gene therapy vector or a transposase system selected from the group consisting of a transposase, a PiggyBac transposon, a sleeping beauty (SB 11) transposon, a Mos1 transposon, and a Tol2 transposon.

153. The method of claim 152, wherein the gene therapy vector is a retrovirus or fusion.

154. The method of claim 153, wherein the retrovirus is a lentiviral vector.

155. The method of any one of claims 77-154, wherein the first and/or second exogenous polynucleotide is introduced into the cell using CRISPR/Cas gene editing.

156. The method of any one of claims 77-155, wherein the CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, cas12a, and Cas12 b.

157. The method of claim 156, wherein the CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of:

b. optionally selected from the group consisting of Cas9, csn2 and Cas 4;

d. Optionally Csf1;

158. The method of any one of claims 155-157, wherein the CRISPR/Cas gene editing is performed ex vivo from a donor subject.

159. The method of claim 158, wherein the CRISPR/Cas gene editing is performed using a lentiviral vector.

160. The method of any one of claims 77-159, wherein the cells or progeny thereof, upon administration to a patient, evade NK cell-mediated cytotoxicity.

161. The method of any one of claims 77-160, wherein the cells or progeny thereof are protected from cell lysis of mature NK cells after administration to a patient.

162. The method of any one of claims 77-161, wherein the cells or their progeny evade phagocytosis by macrophages after administration to a patient.

163. The method of any one of claims 77-162, wherein the cell or progeny thereof does not induce an immune response against the cell after administration to a patient.

164. The method of any one of claims 77-163, wherein the cell or progeny thereof does not induce an antibody-based immune response against the cell upon administration to a patient.

165. The method of any one of claims 77-164, wherein the wild-type cell or the control cell is a starting material.

166. Use of an engineered T cell population for treating a disorder or condition in a patient, wherein the engineered T cell comprises reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules relative to unmodified wild-type or control cells and a first exogenous polynucleotide encoding CD47, wherein the engineered T cell is propagated by primary T cells or progeny thereof, or derived from ipscs or progeny thereof.

167. Use of an engineered differentiated cell population for treating a disorder or condition in a patient, wherein the engineered differentiated cell comprises reduced expression of one or more Y chromosome genes and MHC class I and/or class II human leukocyte antigen molecules relative to an unmodified wild-type or control cell and a first exogenous polynucleotide encoding CD47, wherein the engineered differentiated cell is derived from an iPSC or progeny thereof.

168. The use of claim 166 or 167, wherein the Y chromosome gene is a Y chromosome-linked antigen or a minor histocompatibility antigen associated with the Y chromosome.

169. The use of claim 168, wherein the one or more Y chromosome-linked antigens are Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

170. The use of any one of claims 166-169, wherein said cell has reduced expression of Y-linked tropocadherin 11.

171. The use of any one of claims 166-170, wherein the cell has reduced expression of Y-linked fibronectin 4.

172. The use of any one of claims 166-171, wherein the cell has reduced expression of Y-linked tropocadherin 11 and reduced expression of Y-linked fibronectin 4.

173. The use of any one of claims 166-172, wherein the cell is genetically engineered to have reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

174. The use of any one of claims 166-173, wherein the cell does not express Y-linked tropocadherin 11.

175. The use of any one of claims 166-174, wherein the cell does not express Y-linked fibronectin 4.

176. The use of any one of claims 166-175, wherein the cell does not express Y-linked tropocadherin 11 and does not express Y-linked fibronectin 4.

177. The use of any one of claims 166-176, wherein the cell is genetically engineered to not express Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

178. The use of any one of claims 166-177, wherein reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 is caused by knockout of PCDH11Y and/or NLGN4Y gene, respectively.

179. The use of any one of claims 166-178, wherein the cell is derived from a human cell or an animal cell.

180. The use of claim 179, wherein the human or animal cells are from a donor subject that does not have a Y chromosome.

181. The use of claim 179, wherein the human or animal cell is from a donor subject having a Y chromosome, and wherein the cell is genetically engineered to have reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4.

182. The use of claim 181, wherein the cell is genetically engineered to not express Y-linked tropocadherin 11.

183. The use of claim 181, wherein the cell is genetically engineered to not express Y-linked fibronectin 4.

184. The use of claim 181, wherein the cell is genetically engineered to not express Y-linked tropocadherin 11 and not express Y-linked fibronectin 4.

185. The use of any one of claims 166-184, wherein the cells are propagated from or derived from a cell pool isolated from one or more donor subjects different from the patient, wherein the one or more donor subjects optionally comprise one or more subjects having a Y chromosome; one or more subjects without a Y chromosome; or a mixture of subjects with a Y chromosome and subjects without a Y chromosome.

186. The use of any one of claims 166-185, wherein the cell is genetically engineered to have reduced expression of Y-linked tropocadherin 11 and/or Y-linked fibronectin 4 using CRISPR/Cas gene editing.

187. The use of claim 186, wherein the CRISPR/Cas gene editing is performed using one or more guide RNAs comprising any of the sequences of tables 2-5.

188. The use of any one of claims 186-187, wherein the CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, cas12a, and Cas12 b.

189. The use of claim 188, wherein the CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of:

b. optionally selected from the group consisting of Cas9, csn2 and Cas 4;

d. Optionally Csf1;

190. The use of any one of claims 186-189, wherein the CRISPR/Cas gene editing is performed ex vivo from a donor subject.

191. The use of claim 144, wherein the CRISPR/Cas gene editing is performed using a lentiviral vector.

192. The use of any one of claims 166-191, wherein the cell comprises reduced expression of B2M and/or CIITA relative to an unmodified or wild-type or control cell.

193. The use of claim 192, wherein the cell does not express B2M and/or CIITA.

194. The use of any one of claims 166-193, wherein said cell comprises reduced expression of RHD.

195. The use of claim 194, wherein the cell does not express RHD.

196. The use of any one of claims 166-195, wherein the cell is a differentiated cell derived from an induced pluripotent stem cell or progeny thereof.

197. The use of claim 196, wherein the differentiated cell is selected from the group consisting of: t cells, NK cells, endothelial cells, islet cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, hepatocytes, glial progenitor cells, dopaminergic neurons, retinal pigment epithelial cells, and thyroid cells.

198. The use of any one of claims 166-195, wherein the cell is a primary immune cell or progeny thereof.

199. The use of claim 198, wherein the primary immune cell or progeny thereof is a T cell or NK cell.

200. The use of any one of claims 166-199, wherein the cell comprises reduced expression of TCR-a and/or TCR- β.

201. The use of claim 200, wherein the cell does not express TCR-a and/or TCR- β.

202. The use of any one of claims 166-201, wherein the cell further comprises a second exogenous polynucleotide encoding one or more CARs, wherein the one or more CARs comprise an extracellular ligand binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain that are specific for CD19, CD20, CD22, or BCMA.

203. The use of claim 202, wherein the one or more CARs comprise a CD8 a hinge domain, a CD28 hinge domain, or an IgG4 hinge domain.

204. The use of claim 203, wherein the one or more CARs comprise a CD8 a hinge domain having the amino acid sequence of SEQ ID NO 9.

205. The use of claim 203, wherein the one or more CARs comprise a CD28 hinge domain having the amino acid sequence of SEQ ID No. 10 or 113.

206. The use of claim 203, wherein the one or more CARs comprise an IgG4 hinge domain having the amino acid sequence of SEQ ID No. 11 or 12.

207. The use of any one of claims 202-206, wherein said one or more CARs comprise a CD8 a transmembrane domain or a CD28 transmembrane domain.

208. The use of claim 207, wherein the one or more CARs comprise a CD8 a transmembrane domain having the amino acid sequence of SEQ ID No. 14.

209. The use of claim 207, wherein the one or more CARs comprise a CD28 transmembrane domain having the amino acid sequence of SEQ ID No. 15 or 114.

210. The use of any one of claims 202-209, wherein said one or more CARs comprises a 4-1BB costimulatory domain, CD28 costimulatory domain, or CD3 zeta signaling domain.

211. The use of claim 210, wherein the one or more CARs comprise a 4-1BB co-stimulatory domain having the amino acid sequence of SEQ ID No. 16.

212. The use of claim 210, wherein the one or more CARs comprise a CD28 co-stimulatory domain having the amino acid sequence of SEQ ID No. 17.

213. The use of claim 210, wherein the one or more CARs comprise a CD3 zeta signaling domain having the amino acid sequence of SEQ ID No. 18 or 115.

214. The use of any one of claims 202-213, wherein the one or more CARs comprise an extracellular ligand binding domain comprising an scFv sequence of any one of SEQ ID NOs 19, 37, 45, 54, 63, 72, 81 or 118, or wherein the CAR has an scFv sequence comprising the heavy and light chain sequences of any one of SEQ ID NOs 20, 25, 38, 42, 46, 50, 64, 68, 73, 77, 119 or 123.

215. The use of any one of claims 202-214, wherein said one or more CARs have the sequence of any one of SEQ ID NOs 32, 34, 36, 117, or 128.

216. The use of any one of claims 202-215, wherein the one or more CARs comprise the amino acid sequence set forth in SEQ ID No. 117 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 117, the one or more CARs having the following components: CD 8a signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD 8a hinge domain, CD 8a transmembrane domain, 4-1BB co-stimulatory domain and CD3 zeta signaling domain.

217. The use of any one of claims 202-216, wherein the one or more CARs comprise the amino acid sequence set forth in SEQ ID No. 45 or an amino acid sequence having at least 80% identity (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity) to the amino acid sequence set forth in SEQ ID No. 45.

218. The use of any one of claims 166-217, wherein one or more of the first and/or second exogenous polynucleotides is inserted into a first and/or second specific locus of at least one allele of the cell.

219. The use of claim 218, wherein the first and/or second specific locus is selected from the group consisting of: safe harbor or target loci, RHD loci, B2M loci, CIITA loci, TRAC loci, and TRB loci.

220. The use of claim 219, wherein the safe harbor or target locus is selected from the group consisting of: CCR5 locus, CXCR4 locus, PPP1R12C locus, ALB locus, SHS231 locus, CLYBL locus, rosa locus, F3 (CD 142) locus, MICA locus, MICB locus, LRP1 (CD 91) locus, HMGB1 locus, ABO locus, FUT1 locus and KDM5D locus.

221. The use of any one of claims 166-220, wherein the first and/or second exogenous polynucleotide is introduced into the engineered T cell using a gene therapy vector or a transposase system selected from the group consisting of a transposase, a PiggyBac transposon, a sleeping beauty (SB 11) transposon, a Mos1 transposon, and a Tol2 transposon.

222. The use of claim 221, wherein the gene therapy vector is a retrovirus or fusion.

223. The use of claim 222, wherein the retrovirus is a lentiviral vector.

224. The use of any one of claims 166-223, wherein the first and/or second exogenous polynucleotide is introduced into the cell using CRISPR/Cas gene editing.

225. The use of any one of claims 177-224, wherein the CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of Cas9, cas12a, and Cas12 b.

226. The use of claim 225, wherein the CRISPR/Cas gene editing is performed using a Cas effector protein selected from the group consisting of:

b. optionally selected from the group consisting of Cas9, csn2 and Cas 4;

d. Optionally Csf1;

227. The use of any one of claims 224-226, wherein the CRISPR/Cas gene editing is performed ex vivo from a donor subject.

228. The use of claim 227, wherein the CRISPR/Cas gene editing is performed using a lentiviral vector.

229. The use of any one of claims 166-228, wherein the cells or progeny thereof, upon administration to a patient, evade NK cell-mediated cytotoxicity.

230. The use of any one of claims 166-229, wherein the cells or progeny thereof are protected from cell lysis of mature NK cells after administration to a patient.

231. The use of any one of claims 166-230, wherein the cells or progeny thereof, after administration to a patient, evade phagocytosis by macrophages.

232. The use of any one of claims 166-231, wherein the cell or progeny thereof does not induce an immune response against the cell after administration to a patient.

233. The use of any one of claims 166-232, wherein the cell or progeny thereof does not induce an antibody-based immune response against the cell after administration to a patient.

234. The method of any one of claims 177-233, wherein the wild-type cell or the control cell is a starting material.

235. A method for producing an engineered cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified wild-type or control cell, the method comprising:

(a) Obtaining an isolated cell;

(b) Genetically modifying the cell to reduce expression of the one or more Y chromosome genes in the cell;

(c) Genetically modifying the cell to reduce expression of MHC class I human leukocyte antigen molecules and/or MHC class II human leukocyte antigen molecules in the cell; and

(D) Introducing a polynucleotide encoding CD47 into said isolated cell, thereby producing said engineered cell.

236. A method for producing an engineered cell comprising reduced expression of one or more Y chromosome genes and MHC class I and/or II human leukocyte antigen molecules and a first exogenous polynucleotide encoding CD47 relative to an unmodified wild-type or control cell, the method comprising:

(a) Obtaining an isolated cell;

(b) Contacting the cells with a composition comprising a lentiviral vector comprising

(I) A CD4 binding agent or a CD8 binding agent,

(Ii) Polynucleotides encoding CRISPR/Cas gene editing components targeting the one or more Y chromosome loci,

(Iii) Polynucleotides encoding CRISPR/Cas gene editing components targeting the MHC class I and/or class II human leukocyte antigen loci, and

(Iv) A first exogenous polynucleotide encoding CD47, thereby producing the engineered cell.