CN114457030A

CN114457030A - Pluripotent stem cell expressing IgE blocking substance or derivative thereof and application

Info

Publication number: CN114457030A
Application number: CN202011190294.0A
Authority: CN
Inventors: 王淋立; 陈月花; 杨建国; 莫健
Original assignee: Future Intelligent Regenerative Medicine Research Institute Guangzhou Co ltd
Current assignee: Future Intelligent Regenerative Medicine Research Institute Guangzhou Co ltd
Priority date: 2020-10-30
Filing date: 2020-10-30
Publication date: 2022-05-10

Abstract

The invention discloses a pluripotent stem cell expressing an IgE blocking substance or a derivative thereof and application thereof. The pluripotent stem cells or the derivatives thereof expressing the IgE blocking substances provided by the invention can be used for inducing iPSCs (induced pluripotent stem cells) from autologous cells or differentiating into low-immunogenicity cells such as MSCs (mesenchymal stem cells) for application, can continuously express the IgE blocking substances in vivo, and can be used for treating antiallergic and/or asthmatic diseases and related diseases.

Description

Pluripotent stem cell expressing IgE blocking substance or derivative thereof and application

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a pluripotent stem cell expressing an IgE blocking substance or a derivative thereof and application thereof.

Background

IgE (immunoglobulin E) is a secreted immunoglobulin consisting of two light chains and two heavy chains. It is produced by plasma cells of the lamina propria at the nasopharynx, tonsils, bronchi, gastrointestinal mucosa, etc., and is the main antibody causing type I allergy. Allergic constitution or hypersensitivity patient, the IgE in serum is obviously higher than that of normal person, and exogenous asthma patient is several times higher than that of normal person. Therefore, the excessive content of IgE in serum often indicates the existence of genetic allergic constitution or type I allergy. Therefore, IgE becomes a new target for treating allergy and asthma, and an IgE blocking substance (an anti-IgE antibody) can be used as a medicament for treating the allergy and the asthma. Currently, omalizumab is available as IgE on the market. However, anti-IgE antibodies have a short duration of action, require long-term injections, and are costly for patients.

Stem cells are "seed" cells with self-renewal ability and differentiation ability into specific functional somatic cells, have the potential to regenerate into various tissues, organs and human bodies, and play a central and irreplaceable role in immune response, aging, tumorigenesis and other important biological activities. Stem cells are mainly classified into: totipotent stem cells (Totipotent stem cells), Pluripotent Stem Cells (PSCs), and adult stem cells (adult stem cells). The typical PSCs mainly include Embryonic Stem Cells (ESCs), Embryonic Germ Cells (EGCs), Embryonic Carcinoma Cells (ECCs), Induced Pluripotent Stem Cells (iPSCs), and the like, and these cells have a very deep and wide application prospect due to their powerful functions and can be restricted to some extent by ethics.

Therefore, it is of great interest to develop a pluripotent stem cell or a derivative thereof that can express an IgE blocker in a human.

However, the conception or establishment of the autologous iPSCs cell bank or the immune matched PSCs cell bank requires great expenditure of money, material resources and manpower. The molecular immunological basis for allogeneic recipient organ, tissue or cell transplantation is based primarily on the matching of the classical major histocompatibility complexes MHC-I and MHC-II (human HLA-I, HLA-II). By 6 months of 2019, over 20000 HLA system alleles have been identified and named, and only 5000 alleles of classical HLA-A, B, C are available, each possible random combination of these classical HLA-I/II alleles will be an astronomical number, and as the number of new allele discovery combinations increases, there is a great obstacle to tissue matching and donor selection before organ, tissue and cell transplantation, and it also presents great difficulty in constructing a pool of immunocompromised PSCs covering the population.

Thus, the construction of allogeneic immune-compatible, universal PSCs is imminent. In recent years, a plurality of reports have been provided that the deletion expression of genes on the cell surfaces of HLA-I and HLA-II or the genes thereof is realized by knocking out genes such as B2M, CIITA and the like, so that the cells have immune tolerance or escape T/B cell specific immune response, and universal PSCs with immune compatibility are generated, thereby laying an important foundation for the application of wider universal PSCs source cells, tissues and organs. Also, there are reports of over-expression of CTLA4-Ig, PD-L1 by cells to inhibit allogeneic immune rejection. Recently, it has been reported that when B2M and CIITA are knocked out, CD47 is knocked in, so that cells have immune tolerance or innate immune response of cells such as NK in addition to specific immune response, thereby providing cells with more comprehensive and stronger immune compatibility. However, these approaches are either not fully immune compatible, and still allow for immunological rejection of the allogens by other routes; or completely eliminate the allogeneic immune rejection response, but simultaneously make the cells of the donor source transplant lose the antigen presenting capability, which brings great risk of diseases such as tumorigenicity and virus infection to the recipient.

Therefore, it is also reported that, when the B2M is not directly knocked out, the HLA-A, HLA-B is knocked out or the CIITA is knocked out together, the HLA-C is kept, 12 HLA-C immune match antigens covering more than 90% of people are constructed, so that the transplanted cells still have a certain degree of antigen presenting function, and the innate immune response of NK cells can be inhibited by the HLA-C. However, in the cells, the antigen type presented by HLA-I antigen is reduced by more than two thirds, the integrity of the antigen capable of being presented is reduced irreversibly, the presenting of various tumor, virus and other disease antigens has great bias, the risk of diseases such as tumor and virus infection is still kept to a certain extent, and the pathogenic risk is higher under the condition that CIITA is knocked out simultaneously; secondly, 12 high-frequency immune match HLA-C antigen families are very different, and the calculated part of the area can only account for 70 percent through verification, while large sample size HLA data display which is not authoritative in China, Indian and other big-mouth countries currently exists, so that the prepared general PSCs are still subjected to huge match vacancy test; thirdly, the method can go through repeated gene editing for a plurality of times, at least two rounds of single cell isolation culture are needed in the whole process according to each gene editing, the whole process needs at least more than six rounds of single cell isolation culture, and the processes are inevitable and cause various unpredictable mutations of cells due to multiple times of gene editing off-target or unstable chromatin or due to passage proliferation of a large number of single cells, thereby further inducing various problems such as carcinogenesis, metabolic diseases and the like. It follows that such immuno-compatible schemes are also a matter of convenience in the "transition period", and many problems remain that are not better solved.

In addition, inducing killing of the suicide gene after donor tissue and cell disease has been induced, which results in serious tissue necrosis, cytokine storm and other unpredictable disease risk problems, and it is a big problem that proper donor cells, tissues and organs do not exist after the cell death of the design.

Disclosure of Invention

In order to overcome the disadvantages of the prior art, the first aspect of the present invention aims to provide a pluripotent stem cell expressing an IgE blocker or a derivative thereof, comprising at least one of a non-immune compatible pluripotent stem cell expressing an IgE blocker or a derivative thereof, an immune compatible pluripotent stem cell expressing an IgE blocker or a derivative thereof, and an immune compatible reversible pluripotent stem cell expressing an IgE blocker or a derivative thereof; wherein the IgE blocker expressing immune compatible pluripotent stem cells or derivatives thereof can be realized by the following scheme: knocking out B2M and/or CIITA genes in the genome of the pluripotent stem cells or the derivatives thereof and/or introducing an expression sequence of an immune compatible molecule into the genome of the pluripotent stem cells or the derivatives thereof; an IgE blocker expressing immune compatible reversible pluripotent stem cell or derivative thereof is achieved by the following scheme: introducing an immune compatible molecule and an inducible gene expression system into the genome of the pluripotent stem cells or the derivatives thereof, wherein the expression of the immune compatible molecule introduced into the genome of the pluripotent stem cells or the derivatives thereof is regulated by the inducible gene expression system, and the opening and closing of the inducible gene expression system is regulated by an exogenous inducer; when the immune compatible molecule is normally expressed, the expression of genes related to immune response in the pluripotent stem cell or the derivative thereof is inhibited or overexpressed, so that the allogeneic immune rejection response between the donor cell and the recipient can be eliminated or reduced; when the donor cell is diseased, the expression of the immune compatible molecules can be switched off through the induction of an exogenous inducer, and the antigen presenting capability of the donor cell is recovered, so that the diseased donor cell can be eliminated by a receptor.

The second aspect of the present invention is to provide the use of the pluripotent stem cells or derivatives thereof for preparing anti-allergic and/or anti-asthmatic drugs.

In a third aspect, the present invention provides a preparation comprising the pluripotent stem cells or derivatives thereof.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided a pluripotent stem cell or a derivative thereof expressing an IgE blocker, wherein an expression sequence of the IgE blocker is introduced into the genome of the pluripotent stem cell or the derivative thereof, and the IgE blocker is an anti-IgE antibody.

The heavy chain sequence of the anti-IgE antibody is shown as SEQ ID NO.1, and the light chain sequence is shown as SEQ ID NO. 2.

The anti-IgE antibody is preferably a secreted antibody.

The introduction site of the expression sequence of the IgE blocker is a genome safety site of the pluripotent stem cell or the derivative thereof.

The genome safe site comprises one or more of an AAVS1 safe site, an eGSH safe site and an H11 safe site.

As another technical scheme of the invention: the B2M and/or CIITA genes of the pluripotent stem cell or the derivative thereof are knocked out, so that an IgE blocker expressing immune compatible pluripotent stem cell or the derivative thereof is obtained.

As another technical scheme of the invention: the genome of the pluripotent stem cell or the derivative thereof is further introduced with one or more immune compatible molecule expression sequences for regulating the expression of genes related to immune response (allogeneic immune rejection) in the pluripotent stem cell or the derivative thereof, thereby obtaining an immune compatible pluripotent stem cell or the derivative thereof expressing an IgE blocker.

The genes associated with the immune response include:

(1) major histocompatibility complex genes including at least one of HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB 1;

(2) major histocompatibility complex-associated genes including at least one of B2M and CIITA.

The introduction site of the expression sequence of the immune compatible molecule is a genome safety site of the pluripotent stem cell or the derivative thereof.

The immune-compatible molecule includes any one or more of:

(1) immune tolerance related genes including CD47 or HLA-G;

(2) HLA-C molecules, including HLA-C multiple alleles of which the proportion in the population is over 90 percent in total, or fusion protein genes consisting of the HLA-C multiple alleles of which the proportion is over 90 percent and B2M;

(3) shRNA and/or shRNA-miR of major histocompatibility complex genes including at least one of HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB 1;

(4) shRNA and/or shRNA-miR of a major histocompatibility complex-associated gene including at least one of B2M and CIITA.

The target sequence of the shRNA and/or shRNA-miR of the B2M is at least one of SEQ ID NO. 3-SEQ ID NO. 5;

the target sequence of the shRNA and/or shRNA-miR of the CIITA is at least one of SEQ ID NO. 6-SEQ ID NO. 15;

the target sequence of the shRNA and/or shRNA-miR of the HLA-A is at least one of SEQ ID NO. 16-SEQ ID NO. 18;

the target sequence of the shRNA and/or shRNA-miR of the HLA-B is at least one of SEQ ID NO. 19-SEQ ID NO. 24;

the target sequence of the shRNA and/or shRNA-miR of the HLA-C is at least one of SEQ ID NO. 25-SEQ ID NO. 30;

the target sequence of the shRNA and/or shRNA-miR of the HLA-DRA is at least one of SEQ ID NO. 31-SEQ ID NO. 40;

the target sequence of the shRNA and/or shRNA-miR of the HLA-DRB1 is at least one of SEQ ID NO. 41-SEQ ID NO. 45;

the target sequence of the shRNA and/or shRNA-miR of the HLA-DRB3 is at least one of SEQ ID NO. 46-SEQ ID NO. 47;

the target sequence of the shRNA and/or shRNA-miR of the HLA-DRB4 is at least one of SEQ ID NO. 48-SEQ ID NO. 57;

the target sequence of the shRNA and/or shRNA-miR of the HLA-DRB5 is at least one of SEQ ID NO. 58-SEQ ID NO. 66;

the target sequence of the shRNA and/or shRNA-miR of the HLA-DQA1 is at least one of SEQ ID NO. 67-SEQ ID NO. 73;

the target sequence of the shRNA and/or shRNA-miR of the HLA-DQB1 is at least one of SEQ ID NO. 74-SEQ ID NO. 83;

the target sequence of the shRNA and/or shRNA-miR of the HLA-DPA1 is at least one of SEQ ID NO. 84-SEQ ID NO. 93;

the target sequence of the shRNA and/or shRNA-miR of the HLA-DPB1 is at least one of SEQ ID NO. 94-SEQ ID NO. 103.

shRNA and/or miRNA processing complex related genes and/or anti-interferon effector molecules are also introduced into the genome of the pluripotent stem cell or the derivative thereof.

The shRNA and/or miRNA processing complex related gene comprises at least one of Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2), PACT (PRKRA) and DGCR 8; the anti-interferon response molecule is shRNA and/or shRNA-miR of at least one of PKR, 2-5As, IRF-3 and IRF-7.

The introduction site of the shRNA and/or miRNA processing complex related gene and/or anti-interferon effector molecule is a genome safety site of the pluripotent stem cell or the derivative thereof.

The target sequence of the shRNA and/or shRNA-miR of the PKR is at least one of SEQ ID NO. 104-SEQ ID NO. 113;

the target sequence of the shRNA and/or shRNA-miR of the 2-5As is at least one of SEQ ID NO. 114-SEQ ID NO. 143;

the target sequence of the shRNA and/or shRNA-miR of the IRF-3 is at least one of SEQ ID NO. 144-SEQ ID NO. 153;

the target sequence of the shRNA and/or shRNA-miR of the IRF-7 is at least one of SEQ ID NO. 154-SEQ ID NO. 163.

The expression frameworks of the major histocompatibility complex gene, the major histocompatibility complex related gene, PKR, 2-5As, the shRNA and/or shRNA-miR of IRF-3 or IRF-7 are As follows:

(1) shRNA expression framework: sequentially comprising an shRNA target sequence, a stem-loop sequence, a reverse complementary sequence of the shRNA target sequence and Poly T from 5 'to 3'; the two reverse complementary target sequences are separated by a middle stem-loop sequence to form a hairpin structure, and finally Poly T is connected to be used as a transcription terminator of RNA polymerase III;

(2) shRNA-miR expression framework: the gene is obtained by replacing a target sequence in microRNA-30 or microRNA-155 with the shRNA-miR target sequence of the major histocompatibility complex gene, the major histocompatibility complex related gene, PKR, 2-5As, IRF-3 or IRF-7.

The length of a stem-loop sequence in the shRNA expression frame is 3-9 bases; the length of the Poly T is 5-6 alkali groups.

The expression frame can be added with a constitutive promoter or an inducible promoter, such as a U6 promoter and an H1 promoter, and matched promoter regulatory elements at the 5' end according to requirements.

As another technical scheme of the invention: an inducible gene expression system is also introduced into the genome of the pluripotent stem cell or the derivative thereof for regulating the expression of the immune compatible molecules, so that the immune compatible reversible pluripotent stem cell expressing the IgE blocking substance or the derivative thereof is obtained.

The inducible gene expression system is at least one of a Tet-Off system and a dimer inducible expression system.

The introduction site of the inducible gene expression system is a genome safety site of the pluripotent stem cell or the derivative thereof.

The introduction of the expression sequence of the IgE blocking substance, the expression sequence of an immune compatible molecule, shRNA and/or miRNA processing complex related gene, an anti-interferon effector molecule and an inducible gene expression system adopts a method of viral vector interference, non-viral vector transfection or gene editing.

The method of gene editing comprises gene knock-in.

The pluripotent stem cells comprise embryonic stem cells, embryonic germ cells, embryonic cancer cells or induced pluripotent stem cells; the pluripotent stem cell derivative includes an adult stem cell, each germ layer cell or tissue into which the pluripotent stem cell is differentiated.

The adult stem cells comprise mesenchymal stem cells and neural stem cells.

In a second aspect of the present invention, there is provided a use of the pluripotent stem cells or derivatives thereof in the preparation of an anti-allergic and/or anti-asthmatic medicament.

In a third aspect of the invention, there is provided a formulation comprising the pluripotent stem cells or derivatives thereof.

The formulation further comprises a pharmaceutically acceptable carrier, diluent or excipient.

The invention has the beneficial effects that:

the pluripotent stem cells or the derivatives thereof expressing the IgE blocking substances provided by the invention can be used for inducing iPSCs (induced pluripotent stem cells) or differentiating into MSCs (mesenchymal stem cells) which are low in immunogenicity to be applied, can continuously express the IgE blocking substances in vivo, and can be used for treating antianaphylaxis and/or antiasthma and related diseases.

The B2M and CIITA genes in the pluripotent stem cells or the derivatives thereof are knocked out, or an immune compatible molecule expression sequence is introduced into the genome of the pluripotent stem cells or the derivatives thereof, so that the immunogenicity of the pluripotent stem cells or the derivatives thereof is low, and when the pluripotent stem cells or the derivatives thereof are transplanted into a receptor, the problem of allogeneic immune rejection between donor cells and the receptor can be overcome, so that the donor cells can continuously express the IgE blockers for a long time in the receptor.

The genome of the immune compatible reversible pluripotent stem cell or the derivative thereof for expressing the IgE blocking substance is introduced with an inducible gene expression system and an immune compatible molecule expression sequence. The inducible gene expression system is controlled by an exogenous inducer, and the opening and closing of the inducible gene expression system are controlled by adjusting the addition amount, the duration action time and the type of the exogenous inducer, so that the expression quantity of the epidemic compatible molecular expression sequence is controlled. While the immune-compatible molecule may regulate the expression of genes associated with an immune response in the pluripotent stem cell or derivative thereof. When the immune-compatible molecule is normally expressed, the expression of genes associated with the immune response in the pluripotent stem cell or derivative thereof is suppressed or overexpressed, which may eliminate or reduce the allogeneic immune rejection response between the donor cell and the recipient, allowing the donor cell to continue to express the IgE blocker in the recipient for a long period of time. When donor cells are diseased, the expression of immune compatible molecules can be closed by induction of an exogenous inducer, so that HLA class I molecules can be reversibly re-expressed on the surfaces of the donor cells, the antigen presenting capability of the donor cells is recovered, and recipients can clear diseased cells, thereby improving the clinical safety of the general pluripotent stem cells or the derivatives thereof and greatly expanding the value of the general pluripotent stem cells in clinical application.

In addition, the addition amount and the lasting action time of the exogenous inducer can be adjusted to ensure that the transplant gradually expresses low-concentration HLA molecules to stimulate the receptor, so that the receptor gradually generates tolerance on the transplant, and finally stable tolerance is achieved. At the moment, the HLA class I molecules which are not matched in surface expression of the transplanted cells can be compatible with the recipient immune system, so that after the expression of the immune compatible molecules in the transplanted cells is induced to be closed, the recipient immune system can re-identify the cells with gene mutation presented by the HLA class I molecules in the transplanted cells on one hand, and eliminate diseased cells; on the other hand, the non-mutated fraction is not cleared by the recipient immune system due to the allogeneic HLA class i molecule tolerance produced by training with the above mentioned inducers. Thus, the recipient immune system can only eliminate the graft with harmful mutation, the graft with normal function is kept, and when the harmful graft is eliminated, the mode of HLA class I molecule silencing on the cell surface of the graft can be transferred. The graft tolerance program mediated by the exogenous inducer can also be used to implant a graft that does not induce or otherwise induce the turning on or off of the expression of HLA class i molecules on the surface after the recipient has become fully tolerized.

Drawings

FIG. 1 is a plasmid map of AAVS1 KI (Knock-in, the same below) Vector (shRNA, constitutive).

FIG. 2 is an AAVS1 KI Vector (shRNA, inducible) plasmid map.

FIG. 3 is an AAVS1 KI Vector (shRNA-miR, constitutive) plasmid map.

FIG. 4 is an AAVS1 KI Vector (shRNA-miR, inducible) plasmid map.

FIG. 5 is a sgRNA clone B2M-1 plasmid map.

FIG. 6 is a sgRNA clone B2M-2 plasmid map.

Fig. 7 is a sgRNA clone CIITA-1 plasmid map.

FIG. 8 is a sgRNA clone CIITA-2 plasmid map.

Fig. 9 is a Cas9(D10A) plasmid map.

FIG. 10 is a sgRNA Clone AAVS1-1 plasmid map.

FIG. 11 is a sgRNA Clone AAVS1-2 plasmid map.

Detailed Description

The present invention will be described in further detail with reference to the following specific embodiments and accompanying drawings.

It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

1 Experimental materials and methods

1.1 IgE blockers

The Heavy Chain (HC) sequence of the anti-IgE antibody is shown as SEQ ID NO.1, and the Light Chain (LC) sequence is shown as SEQ ID NO. 2.

1.2 pluripotent Stem cells or derivatives thereof

The pluripotent stem cells can be selected from Embryonic Stem Cells (ESCs), Induced Pluripotent Stem Cells (iPSCs) and other forms of pluripotent stem cells, such as hPSCs-MSCs, NSCs, EBs cells. Wherein:

ESCs: HN4 cells were selected and purchased from Shanghai department of sciences.

And (3) iPSCs: using a third generation highly efficient and safe episomal-iPSCs induction system (6F/BM1-4C) established by us, pE3.1-OG-KS and pE3.1-L-Myc-hmiR 302 cluster are transferred into somatic cells through electricity, RM1 is cultured for 2 days, BioCISO-BM1 containing 2uM Parnate is cultured for 2 days, BioCISO-BM1 containing 2uM Parnate, 0.25mM sodium butyrate, 3uM CHIR99021 and 0.5uM PD03254901 is cultured for 2 days, iPSCs clones can be picked up after being cultured to about 17 days by using a dry cell culture medium BioCISO, and the picked iPSCs clones are purified, digested and passaged to obtain stable iPSCs. Specific construction methods are described in: stem Cell Res ther.2017nov 2; 8(1):245.

hPSCs-MSCs: iPSCs are cultured for 25 days by using a stem cell culture medium (BioCISO containing 10uM TGF beta inhibitor SB431542), during which digestion passage (2mg/mL Dispase digestion) is carried out at 80-90 confluence, passage is carried out at 1:3 into a Matrigel coated culture plate, then ESC-MSC culture medium (knockkockout DMEM culture medium containing 10% of KSR, NEAA, diabody, glutamine, beta-mercaptoethanol, 10ng/mL bFGF and SB-431542) is cultured, liquid is changed every day, passage is carried out at 80-90 confluence (passage is carried out at 1: 3), and continuous culture is carried out for 20 days. The specific construction method is as follows: proc Natl Acad Sci U S A. 2015; 112(2):530-535.

NSCs: iPSCs are cultured for 14 days by using an induction medium (a knockout DMEM medium containing 10% KSR and containing a TGF-beta inhibitor and a BMP4 inhibitor), rose annular nerve cells are picked to a low-adhesion culture plate for culture, the culture medium is cultured by using DMEM/F12 (containing 1% N2 and Invitrogen) and Neurobasal medium (containing 2% B27 and Invitrogen) in a ratio of 1:1 and also contains 20ng/ml bFGF and 20ng/ml EGF, and digestion is carried out by using Accutase for digestion and passage. The specific construction method is as follows: FASEB J.2014; 28(11):4642-4656.

EBs cells: and digesting iPSCs with the confluence of 95% for 6min by using a BioC-PDE1, scraping the cells into blocks by using a mechanical scraping method, settling and reducing cell masses, transferring the settled cell masses into a low-adhesion culture plate, culturing for 7 days by using a BioCISO-EB1, and changing the liquid every other day. After 7 days, the cells were transferred to a Matrigel-coated plate and adherent culture was continued using BioCISO, and Embryoid Bodies (EBs) having an inner, middle and outer three germ layer structure were obtained after 7 days. The specific construction method is as follows: stem Cell Res ther.2017nov 2; 8(1):245.

The pluripotent stem cell derivative further includes adult stem cells, each germ layer cell or tissue, organs into which the pluripotent stem cells are differentiated; the adult stem cells include mesenchymal stem cells or neural stem cells.

1.3 genomic safety sites

In the technical scheme of the invention, the genome safety locus for knocking-in the gene can be selected from an AAVS1 safety locus, an eGSH safety locus or other safety loci:

(1) AAVS1 safety site

The AAVS1 site (the alias "PPP 1R2C site") is located on chromosome 19 of the human genome and is a "safe harbor" site which is verified to ensure the expected function of the transferred DNA fragment. The site is an open chromosome structure, can ensure that the transgene can be normally transcribed, and has no known side effect on cells when the exogenous target segment is inserted into the site.

(2) eGSH safe site

The eGSH safe site is located on chromosome 1 of the human genome, and is another 'safe harbor' site which can ensure the expected function of the transferred DNA fragment after the paper verifies.

(3) Other safety sites

The H11 safe site (also called Hipp11) is located on the human chromosome 22, is a site between two genes Eif4enif1 and Drg1, is discovered and named in 2010 by Simon Hippenmeyer, and has little risk of influencing endogenous gene expression after the exogenous gene is inserted because the H11 site is located between the two genes. The H11 site was verified to be a safe transcription activation region between genes, a new "safe harbor" site outside the AAVS1, eGSH site.

1.4 inducible Gene expression System

The inducible gene expression system is selected from: tet-Off system or dimer-Off expression system:

(1) tet-Off system

In the absence of tetracycline, the tTA protein continues to act on the tet promoter, resulting in sustained gene expression. This system is useful in situations where it is desirable to maintain the transgene in a persistent expression state. When tetracycline is added, the tetracycline can change the structure of the tTA protein, so that the tTA protein cannot be combined with a promoter, and the expression level of a gene driven by the tTA protein is reduced. To keep the system in an "off" state, the tetracycline must be added continuously.

The invention knocks the sequence of the tet-Off system and one or more immune compatible molecules into the genome safe site of the pluripotent stem cell, and accurately turns on or Off the expression of the immune compatible molecules through the addition of tetracycline, thereby reversibly regulating and controlling the expression of major histocompatibility complex related genes in the pluripotent stem cell or the derivative thereof.

(2) Dimer-switched off expression system

Dimer-mediated gene expression regulation system: there are many ways of chemically regulating transcription of target genes, most commonly regulated using allosteric modulators that influence the activity of transcription factors. One such method is the use of dimerizing inducers or dimers to recombine active transcription factors on inactive fusion proteins. The most commonly used system is rapamycin (rapamydn), a natural product, or an analog that is biologically inactive, as the drug for dimerization. The rapamycin (or analog) sibling protein FKBP12 (the protein to which FKBP binds to FK 506) and a large serine-threonine protein kinase, known as FRAP [ FRBP-rapamycinrelated protein, mTOR (mammalian target of rapamycin) have high affinity and function to bind to both proteins, thus bringing them together as a heterologous dimer. To regulate transcription of a target gene, a DNA binding domain is fused to one or more FKBP domains and a transcription repressing domain is fused to amino acid position 93 of FRAP, designated FRB, which is sufficient to bind the FKBP-rapamycin complex. Dimerization of these two fusion proteins occurs only in the presence of rapamycin. Thus inhibiting transcription of genes having sites that bind to the DNA binding region.

1.5 immune compatible molecules

The immune compatible molecule can regulate the expression of allogeneic immune rejection related genes in the pluripotent stem cells or derivatives thereof.

The types and sequences of specific immune-compatible molecules are shown in table 1.

TABLE 1 immune compatible molecules

The target sequences of the shRNA or shRNA-miR immune compatible molecules are shown in Table 2.

TABLE 2 target sequences for shRNA or shRNA-miR

In the immune compatible molecule knock-in schemes of tables 5-6 below, the shRNA or shRNA-miR sequences of each experimental group were shRNA or shRNA-miR immune compatible molecules constructed using the target sequence 1 in table 2. Those skilled in the art will understand that: the shRNA or shRNA-miR immune compatible molecule constructed by other target sequences can also realize the technical effect of the invention and all fall into the protection scope of the claims of the invention.

1.6shRNA/miRNA processing Complex genes and anti-interferon effector molecules

The primary miRNA (pri-miRNA) in the nucleus is microprocessed through the complex Drosha-DGCR8, which cleaves the pri-miRNA into a precursor miRNA (pre-miRNA), which then forms a hairpin. Then, the pre-miRNA is transported out of the nucleus via the Exportin-5-Ran-GTP complex. The RNase Dicer enzyme, which binds to the double-stranded RNA-binding protein TRBP (TARBP2) in the cytoplasm, breaks down the pre-miRNA into mature lengths, at which point the miRNA is also in a double-stranded state. Finally, it is transported into AGO2 to form RISC (RNA-induced silencing complex). Finally, one strand of the miRNA double strand is retained in the RISC complex, and the other strand is eliminated and rapidly degraded. While DGCR8, the main binding protein of Drosha, can bind to pri-miRNA through two double-stranded RNA binding regions at the C-terminal end, recruit and guide Drosha to cut at the right position of pri-miRNA to produce pre-miRNA, which is further cut by Dicer and TRBP/PACT processing to form mature miRNA. Deletion or abnormal expression of DGCR8 affects the cleavage activity of Drosha, and thus the activity of miRNA, leading to disease. TRBP is able to recruit Dicer complex mirnas to form RISC Ago 2.

According to the invention, by using a gene knock-in technology, when the shRNA-miR expression sequences aiming at HLA class I molecules, HLA class II molecules and the like which can be induced to close expression are knocked in at a genome safety site, preferably, shRNA and/or miRNA processing machines which can be induced to close expression are knocked in at the same time, wherein the shRNA and/or miRNA processing machines comprise Drosha (access number: NM-001100412), Ago1(access number: NM-012199), Ago2(access number: NM-001164623), Dicer1(access number: NM-001195573), export-5 (access number: NM-020750), TRBP (access number: NM-134323), PACT (access number: NM-003690) and DGCR8(access number: NM-022720), so that cells do not occupy the processing of other miRNAs and influence the cell functions.

In addition, in the IFN-induced process, double-stranded RNA-dependent Protein Kinase (PKR), which is a key factor of the whole cell signal transduction pathway, and 2 ', 5' Oligoadenylate synthase (2,5-Oligoadenylate synthase, 2-5As), which are closely related to dsRNA-induced IFN, are involved. PKR can inhibit protein synthesis by phosphorylating eukaryotic cell transcription factors, arrest cells in G0/G1 and G2/M phases and induce apoptosis, while dsRNA can promote synthesis of 2-5As, and As a result, RNase, namely RNaseL, is non-specifically activated to degrade all mRNA in cells, and cause cell death. The specificity of induction of type I interferons is achieved by members of the IRF transcription factor family, which are not induced to secrete in many viral infections in the absence of IRF-3 and IRF-7 expression in cells. Lack of IFN response, in order to recover, requires the two proteins were expressed together.

According to the invention, by utilizing a gene knock-in technology, when an immune compatible molecule shRNA-miR expression sequence is knocked in at a genome safety site, shRNA and/or shRNA-miR expression sequences which can induce closed expression and aim at suppressing PKR, 2-5As, IRF-3 and IRF-7 genes are preferably knocked in at the same time, so that interferon reaction induced by dsRNA is reduced, and cytotoxicity is avoided.

The sequence of the insertion positions of the shRNA/miRNA processing complex related gene, the anti-interferon effector molecule and the immune compatible molecule at the genome safety site is not limited, and the shRNA/miRNA processing complex related gene, the anti-interferon effector molecule and the immune compatible molecule can be arranged in any sequence without mutual interference or influence on the structure and the function of other genes of the genome.

Specific target sequences for anti-interferon effector molecules are shown in table 3.

TABLE 3 target sequences for anti-interferon effector molecules

In the anti-interferon effector molecule knock-in schemes of tables 5 to 6 below, the anti-interferon effector molecules of each experimental group had target sequences that were constructed using target sequence 1 in table 3. Those skilled in the art will understand that: the anti-interferon effector molecules constructed by other target sequences can also achieve the technical effects of the invention and fall into the protection scope of the claims of the invention.

1.7 Universal framework sequences of the immune compatible molecules, the shRNA or shRNA-miR universal framework immune compatible molecules of the anti-interferon effector molecules, and the shRNA or shRNA-miR universal framework molecules of the anti-interferon effector molecules are as follows:

(1) the constitutive expression framework of shRNA is:

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAG AGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTA GAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCA TATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGA CGCTAGCGCCACC(SEQ ID NO.164)N₁...N₂₁TTCAAGAGA(SEQ ID NO.165)

N₂₂...N₄₂TTTTTT；

wherein:

a、N₁...N₂₁sh as the corresponding geneRNA target sequence, N₂₂...N₄₂Is a reverse complement sequence of the shRNA target sequence of the corresponding gene;

b. if the plasmid needs to express shRNAs of a plurality of genes, each gene corresponds to a shRNA expression frame and then is connected seamlessly;

c. constitutive shRNA plasmids with different resistance genes only have different resistance genes and have the same other sequences;

d. n represents A, T, G, C bp;

e. SEQ ID No.164 is the U6 promoter sequence;

f. SEQ ID NO.165 is a stem-loop sequence.

(2) The shRNA inducible expression framework is as follows:

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAG AGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTA GAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCA TATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGA CTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCA GTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGA AAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACT CCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAG AAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAG CTCGGTACCCGGGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGT TTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAG ACACCGGGACCGATCCAGCCTGCTAGCGCCACC(SEQ ID NO.166)N₁...N₂₁TTCAAGAGA (SEQ ID NO.165)N₂₂...N₄₂TTTTTT；

wherein:

a、N₁...N₂₁shRNA target sequence for the corresponding Gene, N₂₂...N₄₂Is a reverse complement sequence of the shRNA target sequence of the corresponding gene;

d. n represents A, T, G, C bp;

e. SEQ ID No.166 is the H1 TO promoter sequence;

f. SEQ ID NO.165 is a stem-loop sequence.

(3) The shRNA-miR constitutive or inducible expression framework is as follows:

the shRNA-miR target sequence is used for replacing a target sequence in microRNA-30 to obtain the shRNA-miR target sequence, and the specific sequence is as follows:

GAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACACTTGCTGGG ATTACTTCTTCAGGTTAACCCAACAGAAGGCTAAAGAAGGTATATTGCTGTTGACAGTGA GCG(SEQ ID NO.167)M₁N₁...N₂₁TAGTGAAGCCACAGATGTA(SEQ ID NO.168)

N₂₂...N₄₂M₂TGCCTACTGCCTCGGACTTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTA CTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTA TAAAT(SEQ ID NO.169)；

wherein:

a、N₁...N₂₁shRNA-miR target sequence, N, as a corresponding gene₂₂...N₄₂Is a reverse complementary sequence of shRNA-miR target sequence of a corresponding gene;

b. if the plasmid needs to express shRNA-miR of a plurality of genes, each gene corresponds to a shRNA-miR expression frame and is then connected seamlessly;

c. constitutive shRNA-miR plasmids with different resistance genes only have different resistance genes and have the same other sequences;

d. m is A or C, N is A, T, G, C;

e. if N is present₁Is a G base, then M₁Is A base; otherwise M₁Is a C base;

f、M₁base and M₂And (3) base complementation.

1.8 Gene editing System, Gene editing method and inspection method

1.8.1 Gene editing System

The gene editing technology of the patent adopts a CRISPR-Cas9 gene editing system. The Cas9 protein used was Cas9(D10A), Cas9(D10A) bound to sgrnas responsible for specific recognition of the target sequence (genomic DNA) which was then single-stranded cleaved by Cas9 (D10A). Double Strand breaks in genomic DNA (DSB) must occur, and two Cas 9(D10A)/sgRNA must cleave both strands of genomic DNA separately, and not too far apart. The Cas 9(D10A)/sgRNA scheme has the advantage of higher specificity and lower probability of off-target compared to the Cas 9/sgRNA scheme. The plasmids or Donor fragments used in the gene editing system were: cas9(D10A) plasmid, sgRNA clone plasmid, Donor fragment.

(1) Cas9(D10A) plasmid: a plasmid expressing the Cas9(D10A) protein, specifically single-stranded cleaving genomic DNA under the direction of sgrnas.

(2) sgRNA plasmid: a plasmid for expressing sgRNA, sgRNA (small guide RNA), is a guide RNA (guide RNA, gRNA) responsible for directing targeted cleavage of the expressed Cas9(D10A) protein at gene editing.

(3) Donor fragment: the two ends contain recombination arms which are respectively positioned at the left side and the right side of the breaking position of the genome DNA, and the middle part contains genes, fragments or expression elements needing to be inserted. In the presence of the Donor fragment, the cells undergo a Homological Recombination (HR) reaction at the site of the genomic break. If the Donor fragment is not added, Non-homologous End Joining-NHEJ reaction occurs at the site of the genomic break in the cell. The fragment is obtained by recovering after the digestion of KI Vector plasmid.

1.8.2 constitutive plasmids and inducible plasmids

Constitutive plasmid: the expression function of the Donor fragment obtained from the constitutive plasmid cannot be regulated after knocking in the genomic DNA.

Inducible plasmids: after knocking in the genomic DNA, the expression function of the Donor fragment obtained from the inducible plasmid can be controlled by adding an inducer, which is equivalent to adding a switch for turning on or off the expression function.

1.8.3 plasmid construction method

(1) Cas9(D10A) plasmid: this Plasmid no longer needs to be constructed and is ordered directly from Addgene (Plasmid 41816, Addgene).

(2) sgRNA plasmid: the original blank Plasmid was ordered from Addge (Plasmid 41824, Addge), then the DNA sequence was imported in the website (URL: https:// ccttop. cos. uni-heidelberg. de) to design the target sequence, and finally the different target sequences were put into blank sgRNA plasmids to complete the construction.

(3) KI Vector plasmid:

acquisition of Amp (R) -pUC origin fragment: designing PCR primers, and amplifying and recovering the fragment by using a high fidelity enzyme (Nanjing Nozaki organism, P505-d1) through a PCR method by using a pUC19 plasmid as a template;

acquisition of aavs1 or eGSH recombination arms: extracting the genome DNA of the human cell and designing a corresponding primer, and then amplifying and recovering the fragment by using the human genome DNA as a template and using a high fidelity enzyme (Nanjing Novozam organism, P505-d1) through a PCR method;

c. acquisition of the individual plasmid elements: designing PCR amplification primers of each element, and then respectively amplifying and recovering each plasmid element by using a plasmid containing the element as a template and using a high fidelity enzyme (Nanjing Nuozhuang organism, P505-d1) through a PCR method;

d. assembly into a complete plasmid: the fragments obtained in the previous step were ligated together using a multi-fragment recombinase (Nanjing Nozam, C113-02) to form a complete plasmid.

1.8.4 Gene editing Process

One, single cell cloning operation step of AAVS1 gene knock-in

(1) Electric transfer program:

donor cell preparation: human pluripotent stem cells.

The kit comprises: human Stem Cell

Kit 1。

The instrument comprises the following steps: an electrotransformation instrument.

Culture medium: BioCISO.

Induction of plasmid: cas9D10A, sgRNA clone AAVS1-1, sgRNA clone AAVS1-2, AAVS1 neo Vector I, and AAVS1 neo Vector II.

Note: induction plasmid used for the knock-in of the eGSH gene: cas9D10A, sgRNA clone eGSH-1, sgRNA clone eGSH-2, eGSH-neo/eGSH-puro (donor) comparison of the donor plasmid with AAVS1 shows only the difference between the right and left recombination arms and the difference between the other elements. Since the gene editing process of eGSH is the same as that of AAVS1, the following description will not be repeated.

(2) The transformed human pluripotent stem cells are screened in a double antibiotic medium containing G418 and puro.

(3) And (4) carrying out single cell clone screening and culture to obtain a single cell clone strain.

Second, AAVS1 gene knock-in single cell clone strain culture reagent

(1) Culture medium: BioCISO + 300. mu.g/mL G418+ 0.5. mu.g/mL puro (should be placed at room temperature in advance, protected from light for 30-60 minutes until room temperature is restored. Note that BioCISO should not be placed at 37 ℃ for preheating to avoid reduction of the activity of the biomolecule.).

(2) Matrix glue: hESC grade Matrigel (before cell passage or recovery, the Matrigel working solution is added into a cell culture bottle dish and is shaken up to ensure that the Matrigel completely sinks to the bottom of the culture bottle dish and any Matrigel cannot be dried before use. to ensure that the cells can be attached to the wall and survive better, the Matrigel is put into a 37 ℃ culture box for 1:100X Matrigel cannot be less than 0.5 hour and 1:200X Matrigel cannot be less than 2 hours.).

(3) Digestion solution: EDTA was dissolved using DPBS to a final concentration of 0.5mM, pH7.4 (note: EDTA cannot be diluted with water, otherwise the cells would die due to reduced osmotic pressure).

(4) Freezing and storing liquid: 60% BioCISO + 30% ESCs grade FBS + 10% DMSO (frozen stock is preferably ready for use).

Thirdly, the conventional maintenance subculture process

(1) Optimal time of passage and passage ratio

a. The best passage time: the overall confluency of the cells reaches 80 to 90 percent;

b. the optimal ratio of passage: the optimal confluence degree of the passage is maintained at 20-30% in the next day after passage of 1: 4-1: 7.

(2) Passage process

a. The Matrigel in the coated cell culture flask dishes was previously aspirated away, and an appropriate amount of medium (BioCISO + 300. mu.g/mL G418+ 0.5. mu.g/mL puro) was added to the flask and placed at 37 ℃ in 5% CO₂Incubation in an incubator;

b. when the cells meet the requirement of passage, sucking the supernatant of the culture medium, and adding a proper amount of 0.5mM EDTA digestive solution into a cell bottle dish;

c. the cells were incubated at 37 ℃ with 5% CO₂Incubating in an incubator for 5-10 minutes (digesting until most cells are observed to shrink and become round under a microscope but not float, gently blowing the cells to separate the cells from the wall, sucking the cell suspension into a centrifugal tube, and centrifuging for 5 minutes at 200 g;

d. centrifuging, discarding the supernatant, suspending the cells by using a culture medium, gently and repeatedly blowing the cells for several times until the cells are uniformly mixed, and then transferring the cells into a bottle dish prepared to be coated with Matrigel in advance;

e. after the cells were transferred to the cell flask, the cells were horizontally shaken up all around, observed under a mirror to be free from abnormality, and then shaken up and placed at 37 ℃ with 5% CO₂Culturing in an incubator;

f. observing the adherent survival state of the cells the next day, and normally and regularly changing the culture medium every day by sucking off the culture medium.

Fourthly, freezing and storing cells

(1) According to the conventional passage operation steps, digesting the cells by using 0.5mM EDTA until most cells shrink and become round but do not float, gently blowing and beating the cells, collecting cell suspension, centrifuging for 5 minutes at 200g, removing supernatant, adding a proper amount of freezing medium to resuspend the cells, and transferring the cells to a freezing tube (suggesting that one frozen cell with 80% confluence degree of a six-well plate and 0.5 mL/cell of freezing medium is frozen);

(2) placing the freezing tube in a programmed cooling box, and immediately placing the freezing tube at-80 ℃ overnight (ensuring that the temperature of the freezing tube is reduced by 1 ℃ per minute);

(3) the next day the cells were immediately transferred into liquid nitrogen.

Fifth, cell recovery

(1) Prepare in advance the Matrigel coated detailsCell bottle, before cell recovery, the Matrigel was aspirated, an appropriate amount of BioCISO was added to the cell bottle, and the mixture was incubated at 37 ℃ with 5% CO₂Incubation in an incubator;

(2) taking out the cryopreservation tube from liquid nitrogen quickly, immediately putting the tube into a 37 ℃ water bath kettle for quick shaking to quickly melt the cells, carefully observing, stopping shaking after the ice crystals completely disappear, and transferring the cells to a biological safety cabinet;

(3) adding 10mL of DMEM/F12(1:1) basal medium into a 15mL centrifuge tube in advance, balancing to room temperature, sucking 1mL of DMEM/F12(1:1) by using a Pasteur pipette, slowly adding the DMEM/F12(1:1) into a freezing tube, gently mixing, transferring the cell suspension into a prepared 15mL centrifuge tube containing DMEM/F12(1:1), and centrifuging for 5 minutes at 200 g;

(4) carefully removing supernatant, adding appropriate amount of BioCISO, gently mixing cells, seeding into a cell bottle dish prepared in advance, shaking up horizontally, and observing under the mirror, shaking up, and standing at 37 deg.C and 5% CO₂Culturing in an incubator;

(5) the adherent survival state of the cells is observed the next day, and the liquid is normally changed on time every day. If the adherence is good, the BioCISO is replaced with BioCISO + 300. mu.g/mL G418+ 0.5. mu.g/mL puro.

1.8.5AAVS1 gene knock-in detection method

First, single cell clone AAVS1 gene knock-in detection

(1) AAVS1 Gene knock-in assay

a. The purpose of the test is as follows: detecting the cells subjected to the gene knock-in treatment by PCR, and testing whether the cells are homozygotes; since the two Donor segments only have the difference in the sequences of the resistance genes, it is necessary to determine whether the cell is homozygous (two chromosomes knock in the Donor segments of different resistance genes, respectively), and it is only possible that the double-knocked-in cell is the correct homozygous by detecting whether the genome of the cell contains the Donor segments of the two resistance genes;

b. firstly, designing a primer in the Donor plasmid (non-recombinant arm part), and then designing another primer in the genome PPP1R12C (non-recombinant arm part); if the Donor fragment can be correctly inserted into the genome, a target band appears, otherwise, no target band appears);

c. test protocol primer sequences and PCR protocols are shown in Table 4.

TABLE 4 test protocol primer sequences and PCR protocol

Second, the detection method of eGSH gene knock-in is the same as the detection principle and method of AAVS1 gene knock-in, and will not be described here.

1.8.6 inspection method of knock-in Gene method at genomic safety site

(1) The purpose of the test is as follows: the cells treated by knock-in were tested for homozygote by PCR. Since the two Donor fragments have difference only in the sequences of the resistance genes, it is necessary to determine whether the cell is homozygous (the two chromosomes knock in the Donor fragments of different resistance genes), and it is only possible that the double-knocked-in cell is the correct homozygous by detecting whether the genome of the cell contains the Donor fragments of the two resistance genes.

(2) The test method comprises the following steps: first, one primer was designed inside the Donor plasmid (non-recombinant arm portion), and then the other primer was designed in the genome (non-recombinant arm portion). If the Donor fragment is inserted correctly in the genome, the target band will appear, otherwise no target band will appear.

1.9 methods for determining the expression of IgE blockers by pluripotent Stem cells or derivatives thereof

The anti-IgE antibodies expressed by the pluripotent stem cells or derivatives thereof are detected using ELISA (competition assay). Collecting culture supernatant of the pluripotent stem cells or the derivatives thereof expressing the anti-IgE antibodies, diluting the culture supernatant by 5 times by using sample diluent, mixing the dilution with the enzyme-labeled anti-IgE antibodies (1:1), loading the sample on an enzyme-labeled plate coated with the IgE antigens, adding the culture supernatant of the pluripotent stem cells or the derivatives thereof not expressing the anti-IgE antibodies to a control group, and gently mixing the culture supernatants. After the plate is sealed, the plate is placed at 37 ℃ for incubation for 30min, after 5 times of washing, developing solution is added for developing for 15min, and stop solution is added for 50uL reading to measure the absorbance value at 450nm (the expression quantity of the anti-IgE antibody is in negative correlation with the color depth).

1.10 methods of treatment of mouse model of allergic asthma

In humanized NSG mice (The Jackson Laboratory (JAX)), human immune cells of The same donor were injected to reconstitute The immune system of The mice, and a mouse allergic asthma model induced by aluminum hydroxide gel adjuvant and chicken ovalbumin was established after 2 weeks. After the IgE of the mice is obviously increased, a treatment test is carried out, and 200uLPBS (containing human immune cells and 1X 10) is injected into tail vein⁶The IgE blocker-expressing pluripotent stem cell derivative) for allergic asthma treatment, wherein only the group containing human immune cells was injected as a control group. The increase of Th2 cytokine level is one of the important features of asthma, so we can judge the treatment effect of asthma by detecting the level of Th2 cytokine (IL-4, IL-5, IL-13).

1.11 treatment of mouse model of allergic rhinitis

In humanized NSG mice (The Jackson Laboratory (JAX)), The immune system of The mice was reconstituted by injecting human immune cells from The same donor, 2 weeks later, on day 1 of The experiment, antigen adjuvant suspension was injected (one point per hind limb plantar, three points in The abdominal cavity, 5 points total, 0.2mL per point); on day 7 of the experiment, antigen adjuvant suspension was injected (one point on each hind limb metatarsal and three points in the abdominal cavity, 5 points were injected in total, and 0.2mL was injected at each point); on the 14 th to 27 th days of the test, the egg protein solution is dripped once a day (both nostrils are dripped, and each side is 50 mu L); on day 14 of the experiment, 200uL PBS (containing 10) was injected into the tail vein⁶The IgE blocker-expressing pluripotent stem cell derivative of (1), the pluripotent stem cell derivative being derived from the same donor as the human immune cell) for treating allergic rhinitis. And (4) observing results: the egg protein solution is dripped into the nose (both nostrils are dripped, 50uL is dripped on each side), the observation is carried out for 10 minutes, and the nasal spraying times and the nasal grasping times are counted.

1.12 method for treating allergic dermatitis model in mice

In humanized NSG mice (The Jackson Laboratory (JAX)), human immune cells of The same donor were injected to reconstitute The immune system of The mice, and 2 weeks later, each group of mice was injected with 50uL of a 15g/L dinitrofluorobenzene solution subcutaneously into The back, and at The same time, 200uL of PBS (containing 10 uL of PBS) was injected into The tail vein⁶The IgE blocker-expressing pluripotent stem cell derivative of (1), the pluripotent stem cell derivative being derived from the same donor as the human immune cells) for allergic dermatitis treatment. And (3) smearing a 10g/L dinitrofluorobenzene solution 30 mu L with the right ear of each group of mice on the 9 th day, killing the mice after 2h by cutting off the necks, respectively taking the left and right ears of the mice, preparing into ear pieces with the diameters of 9mm, respectively weighing, and calculating the swelling rate.

Wherein the swelling ratio (%) is (right ear weight-left ear weight)/left ear weight × 100%.

2. Experimental protocol

The protocol for knocking in the gene expressing the IgE blocker, one or more immune compatible molecules, shRNA and/or miRNA processing complex related genes, anti-interferon effector molecules into a safe site in the genome of pluripotent stem cells is shown in tables 5-6, wherein the "+" sign indicates knock-in of the gene or nucleic acid sequence and the "-" sign indicates knock-out of the gene.

TABLE 5 constitutive expression protocol

The plasmids selected and the specific knock-in positions were as follows:

general principle:

anti-IgE antibodies are secreted antibodies, which have the structure: IL-2sig signal peptide (SEQ ID No.183) + light chain (end of light chain sequence with stop codon TGA) + EMCV IRESKT (SEQ ID No.178) + IL-2sig signal peptide + heavy chain (end of heavy chain sequence with stop codon TGA).

The sequence of the anti-IgE antibody is placed at the position of MCS2 of the corresponding plasmid (IL-2 sig signal peptide is added in the front of an LC light chain and an HC heavy chain of the antibody, the LC light chain and the HC heavy chain of the antibody are connected by using EMCV IRESWT in sequence, the sequence is as follows, the LC light chain, the EMCV IRESWT and the HC heavy chain are placed in an shRNA expression frame of the corresponding plasmid, shRNA-miR is placed in an shRNA-miR expression frame of the corresponding plasmid, and other genes are placed at the position of MCS1 of the corresponding plasmid. The maps of the plasmids are shown in FIGS. 1 to 11.

Note: the sgRNA clone B2M plasmid comprises the sgRNA clone B2M-1 and sgRNA clone B2M-2 plasmids. The sgRNA clone CIITA plasmid comprises sgRNA clone CIITA-1 and sgRNA clone CIITA-2 plasmids.

(1) A1 grouping

MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid placed IgE antibody sequences.

(2) A2 grouping

MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid placed IgE antibody sequences. The shRNA expression framework places the shRNA target sequence (if multiple shrnas are present, they are seamlessly joined). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).

(3) A3 grouping

MCS2 of AAVS1 KI Vector (shRNA-miR, constitutive) plasmid places IgE antibody sequences. The shRNA-miR expression framework is put into a shRNA-miR target sequence (if a plurality of shRNA-miR exist, the shRNA-miR target sequences are connected seamlessly). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).

(4) A4 grouping

The MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid places the IgE antibody sequence, the target sequence of sgRNA clone B2M plasmid places the sgRNA target sequence of B2M (SEQ ID No.179 and SEQ ID No.180), and the target sequence of sgRNA clone CIITA plasmid places the sgRNA target sequence of CIITA (SEQ ID No.181 and SEQ ID No. 182). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).

(5) A5 grouping

Methods grouped with a 2.

(6) A6 grouping

Method of grouping with a 3.

TABLE 6 Experimental protocol for inducible expression (immuno-compatible reversible)

(1) B1 grouping:

MCS2 of AAVS1 KI Vector (shRNA, inducible) plasmid incorporates IgE antibody sequences. The shRNA expression framework places the shRNA target sequence (if multiple shrnas are present, they are seamlessly joined). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).

(2) B2 grouping:

the MCS2 of AAVS1 KI Vector (shRNA-miR, inducible) plasmid places IgE antibody sequences. The shRNA-miR expression framework is put into a shRNA-miR target sequence (if a plurality of shRNA-miR exist, the shRNA-miR target sequences are connected seamlessly). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).

(3) B3 grouping:

methods grouped with B1.

(4) B4 grouping:

methods grouped with B2.

3. Results of the experiment

3.1 detection of blocking Effect of IgE blockers expressed by Stem cells or derivatives thereof

The experimental group protocols in tables 5 and 6 were knocked into the genome safety site AAVS1 of iPSCs, MSCs, EBs and NSCs at 37 ℃ and 0.5% CO₂Culturing in an incubator, collecting culture medium supernatant, diluting with sample diluent by 5 times, mixing with enzyme-labeled anti-IgE antibody (1:1) (adding culture supernatant of pluripotent stem cells or derivatives thereof which do not express IgE antibody in control group, mixing gently), and loading on enzyme-labeled plate coated with IgE antigen. Sealing the plate, incubating at 37 deg.C for 30min, washing for 5 times, adding color developing solution, developing for 15min,stop solution 50uL was added and the reading was taken to measure absorbance at 450 nm. The results of the tests of the respective experimental groups are shown in Table 7.

TABLE 7 blocking Effect of IgE antibodies expressed in each experimental group on IgE

As can be seen from the above table, the IgE antibody expressed by the pluripotent stem cell or the derivative thereof can effectively block the binding of the enzyme-labeled IgE antibody and the cell surface IgE. And the expression level is relatively constant in each group, so that the IgE antibody expressed by the pluripotent stem cells or the derivatives thereof is not influenced by the cell differentiation form and other exogenous genes (immune compatibility modification).

3.2 Effect of pluripotent Stem cells expressing IgE blockers or derivatives thereof in the treatment of allergic asthma

We selected cells (iPSCs, MSCs, EBs, NSCs) expressing the blocker protocol panel (a1) for testing. In the humanized NSG mouse allergic asthma model, hPSCs and hPSCs source derivatives (iPSCs, hPSCs-MSCs, hPSCs-NSCs and hPSCs-EBs) capable of expressing IgE antibodies are injected, and the effect of treating allergic asthma is observed by detecting the level (pg/ml) of cytokines in bronchoalveolar lavage fluid. Note that in order to avoid the problem of immune compatibility, the immunocytes used by I are derived from the same person as the hPSCs and the derivatives derived from the hPSCs. The results of the experiment are shown in FIG. 8.

TABLE 8 therapeutic Effect of allergic asthma by IgE antibody-expressing pluripotent Stem cells or derivatives thereof

Note: the control group refers to the NSG mouse model of allergic asthma without injection of hPSCs and hPSCs derived derivatives expressing IgE antibodies.

Through the above experiments, it can be proved that the level of Th2 cytokines (IL-4, IL-5, IL-13) is reduced in mice for treating allergic asthma model by the stem cells expressing IgE blocking substances or the derivatives thereof prepared by the invention. Therefore, the injection of hPSCs and hPSCs derivatives expressing IgE blockers can play a role in treating allergic asthma.

3.3 reversible expression assay for immune-compatible molecule-inducible expression sets

Through the above examples, hPSCs and hPSCs derived derivatives expressing IgE blockers are effective in blocking IgE and thus have therapeutic effects on allergic asthma. We must also consider the issue of immune compatibility of hPSCs and derivatives of hPSCs origin. Therefore we chose a suitable combination to test for immune compatibility.

By utilizing the characteristic of low immunogenicity of the MSCs, hPSCs capable of expressing IgE blockers (anti-IgE antibodies) are injected into a humanized NSG mouse allergic asthma model to be immune-compatible with the MSCs, and the effect of the MSCs in treating the allergic asthma is observed by detecting the level (pg/ml) of cytokines in bronchoalveolar lavage fluid. Note that the immunocytes used were derived from a non-identical human as the hPSCs-derived MSCs.

The control group refers to the model of allergic asthma in NSG mice that were not injected with MSCs cells.

The process of adding the Dox group is: mice were fed with 0.5mg/mL Dox in their diets and used from the injection of expression blocker cells until the end of the experiment. The results are shown in Table 9.

TABLE 9 reversible expression test results for immune-compatible molecule-inducible expression sets

The above experiments show that: in the treatment of allergic asthma, only blocking agent expressing MSCs (group 2), which have low immunogenicity and can exist in foreign body for a certain time, can exert a certain therapeutic effect, while those that are immuno-compatibly engineered (groups 3-11, including constitutive and reversible inducible immuno-compatibility), which have better immuno-compatibility effects, are present in vivo for a longer time (or can coexist for a long time) than MSCs that are not immuno-compatibly engineered, and exert better therapeutic effects, whereas group 5, which is a B2M and CIITA gene knock-out group, completely eliminates the effects of HLA-I and HLA-II molecules, and thus has the best therapeutic effects. However, there are group 8-15 protocols set up due to their constitutive immune compatible modifications (knock-in/knock-out) which cannot be cleared when the graft becomes mutated or otherwise unwanted. In groups 12-15, the mice injected with the expression blocker cells were treated with Dox inducer (used all the time) at the same time as the injection of the expression blocker cells into the mice, and the immune compatibility of the mice injected with the expression blocker cells was abolished, and the cells existed in vivo for a period of time comparable to that of the MSCs without immune compatibility modification, and the therapeutic effect of the cells was comparable to that of the MSCs without immune compatibility modification.

3.4 Effect of pluripotent Stem cells expressing IgE blockers or derivatives thereof in the treatment of other allergic diseases (allergic rhinitis, allergic dermatitis)

Cells (iPSCs, MSCs, EBs, NSCs) expressing the blocker protocol panel (a1) were selected for testing. In other allergic disease models (allergic rhinitis and allergic dermatitis) of humanized NSG mice, hPSCs and hPSCs derivatives (iPSCs, hPSCs-MSCs, hPSCs-NSCs and hPSCs-EBs) capable of expressing IgE antibodies are injected for treatment. Note that to avoid the immune compatibility problem, we used immunocytes derived from the same person as hPSCs and hPSCs derived derivatives. The experimental results are shown in 10 and 11.

TABLE 10 therapeutic Effect of IgE antibody-expressing pluripotent Stem cells or derivatives thereof on allergic rhinitis

Note: the control group refers to the NSG mouse allergic rhinitis model without injection of hPSCs expressing IgE antibodies and hPSCs derived derivatives.

TABLE 11 therapeutic Effect of IgE antibody-expressing pluripotent Stem cells or derivatives thereof on allergic dermatitis

Note: the control group refers to the NSG mouse model of allergic dermatitis without injection of IgE antibody-expressing hPSCs and hPSCs-derived derivatives.

Through the experiment, the stem cells expressing the IgE blocking substance or the derivatives thereof prepared by the invention have obvious effects on treating the mice of allergic rhinitis models and allergic dermatitis models. Therefore, IgE blockers can treat well allergy-related diseases (asthma, rhinitis, dermatitis, etc.).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> future Chile regenerative medicine research institute (Guangzhou) Inc.; king shower stand

<120> pluripotent stem cell expressing IgE blocking substance or derivative thereof and application

<130>

<160> 183

<170> PatentIn version 3.5

<210> 1

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<212> DNA

<213> human

<400> 1

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gggagcagag aattctctta t 21

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ggagcagaga attctcttat c 21

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gagcagagaa ttctcttatc c 21

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gctacctgga gcttcttaac a 21

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ggagcttctt aacagcgatg c 21

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gggtctccag tatattcatc t 21

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gcctcctgat gcacatgtac t 21

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ggaagacctg ggaaagcttg t 21

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ggctaagctt gtacaataac t 21

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gcggaatgaa ccacatcttg c 21

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ggtacccact gctctggtta t 21

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gctcccactc catgaggtat t 21

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ggtatttctt cacatccgtg t 21

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aggagacacg gaatgtgaag g 21

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gctcccactc catgaggtat t 21

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ggtatttcta cacctccgtg t 21

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ggaccggaac acacagatct a 21

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accggaacac acagatctac a 21

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ggaacacaca gatctacaag g 21

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gaacacacag atctacaagg c 21

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ttcttacttc cctaatgaag t 21

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aagttaagaa cctgaatata a 21

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aacctgaata taaatttgtg t 21

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acctgaatat aaatttgtgt t 21

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aagcgttgat ggattaatta a 21

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agcgttgatg gattaattaa a 21

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gggtctggtg ggcatcatta t 21

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ggtctggtgg gcatcattat t 21

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gcatcattat tgggaccatc t 21

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gcacatggag gtgatggtgt t 21

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ggaggtgatg gtgtttctta g 21

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gagaagatca ctgaagaaac t 21

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gctttaatgg ctttacaaag c 21

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ggctttacaa agctggcaat a 21

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<400> 39

gctttacaaa gctggcaata t 21

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<400> 40

gctccgtact ctaacatcta g 21

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gatgaccaca ttcaaggaag a 21

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<400> 42

gaccacattc aaggaagaac t 21

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<400> 43

gctttcctgc ttggcagtta t 21

<210> 44

<211> 21

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<213> human

<400> 44

ggcagttatt cttccacaag a 21

<210> 45

<211> 21

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<213> human

<400> 45

gcagttattc ttccacaaga g 21

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<211> 21

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<400> 46

gcgtaagtct gagtgtcatt t 21

<210> 47

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<400> 47

gacaatttaa ggaagaatct t 21

<210> 48

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<400> 48

ggccatagtt ctccctgatt g 21

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gccatagttc tccctgattg a 21

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gcagatgacc acattcaagg a 21

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gatgaccaca ttcaaggaag a 21

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gaccacattc aaggaagaac c 21

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gctttgtcag gaccaggttg t 21

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gaccaggttg ttactggttc a 21

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<400> 55

gaagcctcac agctttgatg g 21

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gatggcagtg cctcatcttc a 21

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<400> 57

ggcagtgcct catcttcaac t 21

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<400> 58

gcagcaggat aagtatgagt g 21

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gcaggataag tatgagtgtc a 21

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<400> 60

ggttcctgca cagagacatc t 21

<210> 61

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<400> 61

gcacagagac atctataacc a 21

<210> 62

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<400> 62

gagacatcta taaccaagag g 21

<210> 63

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<400> 63

gagtactgga acagccagaa g 21

<210> 64

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<400> 64

gctttcctgc ttggctctta t 21

<210> 65

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<400> 65

ggctcttatt cttccacaag a 21

<210> 66

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<400> 66

gctcttattc ttccacaaga g 21

<210> 67

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<400> 67

ggatgtggaa cccacagata c 21

<210> 68

<211> 21

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<400> 68

gatgtggaac ccacagatac a 21

<210> 69

<211> 21

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<213> human

<400> 69

gtggaaccca cagatacaga g 21

<210> 70

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<213> human

<400> 70

ggaacccaca gatacagaga g 21

<210> 71

<211> 21

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<213> human

<400> 71

gagccaactg tattgcctat t 21

<210> 72

<211> 21

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<213> human

<400> 72

agccaactgt attgcctatt t 21

<210> 73

<211> 21

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<213> human

<400> 73

gccaactgta ttgcctattt g 21

<210> 74

<211> 21

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<213> human

<400> 74

gggtagcaac tgtcaccttg a 21

<210> 75

<211> 21

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<213> human

<400> 75

ggatttcgtg ttccagttta a 21

<210> 76

<211> 21

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<213> human

<400> 76

gcatgtgcta cttcaccaac g 21

<210> 77

<211> 21

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<213> human

<400> 77

gcgtcttgtg accagataca t 21

<210> 78

<211> 21

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<213> human

<400> 78

gcttatgcct gcccagaatt c 21

<210> 79

<211> 21

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<213> human

<400> 79

gcaggaaatc actgcagaat g 21

<210> 80

<211> 21

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<213> human

<400> 80

gctcagtgca ttggccttag a 21

<210> 81

<211> 21

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<213> human

<400> 81

ggtgagtgct gtgtaaataa g 21

<210> 82

<211> 21

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<213> human

<400> 82

gacatatata gtgatccttg g 21

<210> 83

<211> 21

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<400> 83

ggaaagtcac atcgatcaag a 21

<210> 84

<211> 21

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<213> human

<400> 84

gctcacagtc atcaattata g 21

<210> 85

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<400> 85

gccctgaaga cagaatgttc c 21

<210> 86

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<400> 86

gcggaccatg tgtcaactta t 21

<210> 87

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<400> 87

ggaccatgtg tcaacttatg c 21

<210> 88

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<400> 88

gcgtttgtac agacgcatag a 21

<210> 89

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<400> 89

ggctggctaa cattgctata t 21

<210> 90

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<400> 90

gctggctaac attgctatat t 21

<210> 91

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<400> 91

ggaccaggtc acatgtgaat a 21

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<400> 92

ggaaaggtct gaggatattg a 21

<210> 93

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<400> 93

ggcagattag gattccattc a 21

<210> 94

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<400> 94

gcctgatagg acccatattc c 21

<210> 95

<211> 21

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<400> 95

gcatccaata gacgtcattt g 21

<210> 96

<211> 21

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<400> 96

gcgtcactgg cacagatata a 21

<210> 97

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<400> 97

gctgtcacat aataagctaa g 21

<210> 98

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<400> 98

gctaaggaag acagtatata g 21

<210> 99

<211> 21

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<400> 99

gggatttcta aggaaggatg c 21

<210> 100

<211> 21

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<400> 100

ggagttgaag agcagagatt c 21

<210> 101

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<400> 101

gccagtgaac acttaccata g 21

<210> 102

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<400> 102

gcttctctga agtctcattg a 21

<210> 103

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<400> 103

ggctgcaact aacttcaaat a 21

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<400> 104

ggatggattt gattatgatc c 21

<210> 105

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<400> 105

ggaccttgga acaatggatt g 21

<210> 106

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<400> 106

gctaattctt gctgaacttc t 21

<210> 107

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<400> 107

gctgaacttc ttcatgtatg t 21

<210> 108

<211> 21

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<400> 108

gcctcatctc tttgttctaa a 21

<210> 109

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<400> 109

gctctggaga agatatattt g 21

<210> 110

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<400> 110

gctcttgagg gaactaatag a 21

<210> 111

<211> 21

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<400> 111

gggacggcat taatgtattc a 21

<210> 112

<211> 21

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<400> 112

ggacaaacat gcaaactata g 21

<210> 113

<211> 21

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<400> 113

gcagcaacca gctaccattc t 21

<210> 114

<211> 21

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<400> 114

gcagttctgt tgccactctc t 21

<210> 115

<211> 21

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<400> 115

gggagagttc atccaggaaa t 21

<210> 116

<211> 21

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<400> 116

ggagagttca tccaggaaat t 21

<210> 117

<211> 21

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<400> 117

gagagttcat ccaggaaatt a 21

<210> 118

<211> 21

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<213> human

<400> 118

gcctgtcaaa gagagagagc a 21

<210> 119

<211> 21

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<400> 119

gctcagcttc gtactgagtt c 21

<210> 120

<211> 21

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<400> 120

gcttcacaga actacagaga g 21

<210> 121

<211> 21

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<400> 121

gcatctactg gacaaagtat t 21

<210> 122

<211> 21

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<400> 122

ggctgaatta cccatgcttt a 21

<210> 123

<211> 21

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<400> 123

gctgaattac ccatgcttta a 21

<210> 124

<211> 21

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<400> 124

gggttggttt atccaggaat a 21

<210> 125

<211> 21

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<400> 125

ggatcagaag agaagccaac g 21

<210> 126

<211> 21

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<400> 126

ggttcaccat ccaggtgttc a 21

<210> 127

<211> 21

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<400> 127

gctctcttct ctggaactaa c 21

<210> 128

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<400> 128

gctagagtga ctccatctta a 21

<210> 129

<211> 21

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<400> 129

gctgaccacc aattataatt g 21

<210> 130

<211> 21

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<400> 130

gcagaatatt taaggccata c 21

<210> 131

<211> 21

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<400> 131

gcccacttaa aggcagcatt a 21

<210> 132

<211> 21

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<400> 132

ggtcatcaat accactgtta a 21

<210> 133

<211> 21

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<213> human

<400> 133

gcattcctcc ttctcctttc t 21

<210> 134

<211> 21

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<213> human

<400> 134

ggaggaactt tgtgaacatt c 21

<210> 135

<211> 21

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<400> 135

gctgtaagaa ggatgctttc a 21

<210> 136

<211> 21

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<213> human

<400> 136

gctgcaggca ggattgtttc a 21

<210> 137

<211> 21

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<400> 137

gcagttcgag gtcaagtttg a 21

<210> 138

<211> 21

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<400> 138

gccaattagc tgagaagaat t 21

<210> 139

<211> 21

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<213> human

<400> 139

gcaggtttac agtgtatatg t 21

<210> 140

<211> 21

<212> DNA

<213> human

<400> 140

gcctacagag actagagtag g 21

<210> 141

<211> 21

<212> DNA

<213> human

<400> 141

gcagttgggt accttccatt c 21

<210> 142

<211> 21

<212> DNA

<213> human

<400> 142

gcaactcagg tgcatgatac a 21

<210> 143

<211> 21

<212> DNA

<213> human

<400> 143

gcatggcgct ggtacgtaaa t 21

<210> 144

<211> 19

<212> DNA

<213> human

<400> 144

gcctcgagtt tgagagcta 19

<210> 145

<211> 19

<212> DNA

<213> human

<400> 145

agacattctg gatgagtta 19

<210> 146

<211> 19

<212> DNA

<213> human

<400> 146

gggtctgtta cccaaagaa 19

<210> 147

<211> 19

<212> DNA

<213> human

<400> 147

ggtctgttac ccaaagaat 19

<210> 148

<211> 19

<212> DNA

<213> human

<400> 148

ggaaggaagc ggacgctca 19

<210> 149

<211> 19

<212> DNA

<213> human

<400> 149

ggaggcagta cttctgata 19

<210> 150

<211> 19

<212> DNA

<213> human

<400> 150

cgctctagag ctcagctga 19

<210> 151

<211> 19

<212> DNA

<213> human

<400> 151

ccaccacctc aaccaataa 19

<210> 152

<211> 19

<212> DNA

<213> human

<400> 152

atttcaagaa gtcgatcaa 19

<210> 153

<211> 19

<212> DNA

<213> human

<400> 153

gaagatctga ttaccttca 19

<210> 154

<211> 21

<212> DNA

<213> human

<400> 154

ggacactggt tcaacacctg t 21

<210> 155

<211> 21

<212> DNA

<213> human

<400> 155

ggttcaacac ctgtgacttc a 21

<210> 156

<211> 21

<212> DNA

<213> human

<400> 156

acctgtgact tcatgtgtgc g 21

<210> 157

<211> 21

<212> DNA

<213> human

<400> 157

gctggacgtg accatcatgt a 21

<210> 158

<211> 21

<212> DNA

<213> human

<400> 158

ggacgtgacc atcatgtaca a 21

<210> 159

<211> 21

<212> DNA

<213> human

<400> 159

gacgtgacca tcatgtacaa g 21

<210> 160

<211> 21

<212> DNA

<213> human

<400> 160

acgtgaccat catgtacaag g 21

<210> 161

<211> 21

<212> DNA

<213> human

<400> 161

acgctatacc atctacctgg g 21

<210> 162

<211> 21

<212> DNA

<213> human

<400> 162

gcctctatga cgacatcgag t 21

<210> 163

<211> 21

<212> DNA

<213> human

<400> 163

gacatcgagt gcttccttat g 21

<210> 164

<211> 253

<212> DNA

<213> Artificial sequence

<400> 164

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgctagcgcc acc 253

<210> 165

<211> 9

<212> DNA

<213> Artificial sequence

<400> 165

ttcaagaga 9

<210> 166

<211> 686

<212> DNA

<213> Artificial sequence

<400> 166

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

ctttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca 300

gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa 360

agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc 420

ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa 480

agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagctcgg 540

tacccgggtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg 600

aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg 660

gaccgatcca gcctgctagc gccacc 686

<210> 167

<211> 119

<212> DNA

<213> Artificial sequence

<400> 167

gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta 60

cttcttcagg ttaacccaac agaaggctaa agaaggtata ttgctgttga cagtgagcg 119

<210> 168

<211> 19

<212> DNA

<213> Artificial sequence

<400> 168

tagtgaagcc acagatgta 19

<210> 169

<211> 119

<212> DNA

<213> Artificial sequence

<400> 169

tgcctactgc ctcggacttc aaggggctac tttaggagca attatcttgt ttactaaaac 60

tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat ggtataaat 119

<210> 170

<211> 22

<212> DNA

<213> Artificial sequence

<400> 170

ccatagctca gtctggtcta tc 22

<210> 171

<211> 22

<212> DNA

<213> Artificial sequence

<400> 171

tcaggatgat ctggacgaag ag 22

<210> 172

<211> 20

<212> DNA

<213> Artificial sequence

<400> 172

ccggtcctgg actttgtctc 20

<210> 173

<211> 20

<212> DNA

<213> Artificial sequence

<400> 173

ctcgacatcg gcaaggtgtg 20

<210> 174

<211> 20

<212> DNA

<213> Artificial sequence

<400> 174

cgcattggag tcgctttaac 20

<210> 175

<211> 24

<212> DNA

<213> Artificial sequence

<400> 175

cgagctgcaa gaactcttcc tcac 24

<210> 176

<211> 23

<212> DNA

<213> Artificial sequence

<400> 176

cacggcactt acctgtgttc tgg 23

<210> 177

<211> 23

<212> DNA

<213> Artificial sequence

<400> 177

cagtacaggc atccctgtga aag 23

<210> 178

<211> 590

<212> DNA

<213> Artificial sequence

<400> 178

cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 60

tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 120

gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 180

aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 240

aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 300

ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 360

cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 420

ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 480

cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 540

ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg 590

<210> 179

<211> 23

<212> DNA

<213> Artificial sequence

<400> 179

cgcgagcaca gctaaggcca cgg 23

<210> 180

<211> 23

<212> DNA

<213> Artificial sequence

<400> 180

actctctctt tctggcctgg agg 23

<210> 181

<211> 23

<212> DNA

<213> Artificial sequence

<400> 181

acccagcagg gcgtggagcc agg 23

<210> 182

<211> 23

<212> DNA

<213> Artificial sequence

<400> 182

gtcagagccc caaggtaaaa agg 23

<210> 183

<211> 60

<212> DNA

<213> Artificial sequence

<400> 183

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60

Claims

1. A pluripotent stem cell expressing an IgE blocker or a derivative thereof, wherein: the genome of the pluripotent stem cell or the derivative thereof is introduced with an expression sequence of an IgE blocker, and the IgE blocker is an anti-IgE antibody.

2. The pluripotent stem cell or derivative thereof according to claim 1, wherein: the B2M and/or CIITA gene of the pluripotent stem cell or the derivative thereof is knocked out.

3. The pluripotent stem cell or derivative thereof according to claim 1, wherein: the genome of the pluripotent stem cell or the derivative thereof is further introduced with one or more immune compatible molecule expression sequences for regulating the expression of genes associated with an immune response in the pluripotent stem cell or the derivative thereof.

4. The pluripotent stem cell or derivative thereof according to claim 3, wherein: the genes associated with the immune response include:

5. The pluripotent stem cell or derivative thereof according to claim 3, wherein: the immune-compatible molecule includes any one or more of:

(1) immune tolerance related genes including CD47 or HLA-G;

(4) shRNA and/or shRNA-miR of a major histocompatibility complex-associated gene that includes at least one of B2M and CIITA.

6. The pluripotent stem cell or derivative thereof according to claim 5, wherein:

the target sequence of the shRNA and/or shRNA-miR of B2M is at least one of SEQ ID NO. 3-SEQ ID NO. 5;

7. The pluripotent stem cell or derivative thereof according to claim 3, wherein: shRNA and/or miRNA processing complex related genes and/or anti-interferon effector molecules are also introduced into the genome of the pluripotent stem cell or the derivative thereof.

8. The pluripotent stem cell or derivative thereof according to claim 7, wherein: the shRNA and/or miRNA processing complex related gene comprises at least one of Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2), PACT (PRKRA) and DGCR 8; the anti-interferon effector molecule is shRNA and/or shRNA-miR of at least one of PKR, 2-5As, IRF-3 and IRF-7.

9. The pluripotent stem cell or derivative thereof according to claim 8, wherein:

10. The pluripotent stem cell or the derivative thereof according to claim 6 or 9, wherein: the expression frameworks of the major histocompatibility complex gene, the major histocompatibility complex related gene, PKR, 2-5As, the shRNA and/or shRNA-miR of IRF-3 or IRF-7 are As follows:

(1) shRNA expression framework: 5 'to 3' sequentially comprises the shRNA target sequence of claim 6 or 9, a stem-loop sequence, a reverse complement of the shRNA target sequence of claim 6 or 9, and Poly T; the two reverse complementary target sequences are separated by a middle stem-loop sequence to form a hairpin structure, and finally Poly T is connected to be used as a transcription terminator of RNA polymerase III;

(2) shRNA-miR expression framework: the shRNA-miR target sequence of claim 6 or 9 is used for replacing a target sequence in microRNA-30 or microRNA-155.

11. The pluripotent stem cell or the derivative thereof according to claim 10, wherein: the length of a stem-loop sequence in the shRNA expression frame is 3-9 bases; the length of the Poly T is 5-6 bases.

12. The pluripotent stem cell or the derivative thereof according to claim 3 or 7, wherein: an inducible gene expression system is also introduced into the genome of the pluripotent stem cell or the derivative thereof.

13. The pluripotent stem cell or derivative thereof of claim 12, wherein: the inducible gene expression system is at least one of a Tet-Off system and a dimer inducible expression system.

14. The pluripotent stem cell or the derivative thereof according to claim 12, wherein:

15. The pluripotent stem cell or the derivative thereof according to claim 14, wherein:

the introduction sites of the expression sequence of the IgE blocking substance, the expression sequence of the immune compatible molecule, the shRNA and/or miRNA processing complex related gene, the anti-interferon effector molecule and the inducible gene expression system are genome safety sites of the pluripotent stem cells or derivatives thereof.

16. The pluripotent stem cell or the derivative thereof of claim 15, wherein: the genome safe site comprises one or more of an AAVS1 safe site, an eGSH safe site and an H11 safe site.

17. The pluripotent stem cell or the derivative thereof according to any one of claims 1 to 9, 11 and 13 to 16, wherein: the pluripotent stem cells comprise embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, or induced pluripotent stem cells; the pluripotent stem cell derivative includes an adult stem cell, each germ layer cell or tissue into which the pluripotent stem cell is differentiated; the adult stem cells include mesenchymal stem cells or neural stem cells.

18. The pluripotent stem cell or derivative thereof of claim 17, wherein: the heavy chain sequence of the anti-IgE antibody is shown as SEQ ID NO.1, and the light chain sequence is shown as SEQ ID NO. 2.

19. Use of a pluripotent stem cell or a derivative thereof according to any of claims 1 to 18 in the manufacture of a medicament for the treatment of allergy and/or asthma.

20. A formulation, characterized by: comprising the pluripotent stem cell of any one of claims 1 to 18 or a derivative thereof.