CN114457029A - Pluripotent stem cell expressing VEGF-A blocking substance or derivative thereof and application - Google Patents

Abstract

The invention discloses a pluripotent stem cell or a derivative thereof expressing a VEGF-A blocker, and an application thereof. The genome of the pluripotent stem cell or its derivative is introduced with the expression sequence of the VEGF-A blocker, and the VEGF-A blocker is introduced into the genome of the pluripotent stem cell or its derivative. The A blocker is an anti-VEGF-A antibody. The pluripotent stem cells or derivatives thereof expressing VEGF-A blocker provided by the present invention can be used for autologous cells to induce iPSCs or differentiate into low-immunogenic cells such as MSCs, and they can continuously express VEGF-A blocker in vivo. Drugs for the treatment of macular degeneration and/or tumors and related diseases.

Description

Pluripotent stem cell expressing VEGF-A 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 a VEGF-A blocker or a derivative thereof and application thereof.

Background

VEGF-A (vascular endothelial growth factor A) is a highly conserved 27kDa dimeric glycoprotein, belonging to the VEGF family with VEGF-B, VEGF-C, VEGF-D and placental growth factor (PIGF). It encodes a heparin-binding protein in the form of disulfide-linked homodimers. VEGF-A is secreted by endothelial cells and tumor cells, has effects in angiogenesis, angiogenesis and endothelial cell growth, can induce endothelial cell proliferation, promote cell migration, inhibit apoptosis and induce vascular permeability, is proved to be an endothelial growth factor and a regulator of vascular permeability, and is necessary for physiological and pathological angiogenesis. Since VEGFA is a critical regulator of angiogenesis during the growth of solid tumors, VEGF-a-targeted therapeutic regimens have become innovative treatments for oncology and macular degeneration. Therefore, VEGF-A becomes a new target for treating tumor and macular degeneration, and VEGF-A blocker (anti-VEGF-A antibody) can be used as a therapeutic drug for tumor and macular degeneration. At present, ranibizumab and the like are commercially available as VEGF-A antibodies. However, anti-VEGF-A antibodies have a short duration of action, require long-term injection, and are costly to 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 such 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 important to develop a pluripotent stem cell or a derivative thereof that can express a VEGF-A blocker in humans.

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 2019, over 20000 HLA system alleles have been identified and named, and only 5000 allele factors of classical HLA-A, B, C are respectively exceeded, and various possible random combinations of these classical HLA-I/II alleles will be astronomical numbers, and as the number of combinations found for new alleles increases, there is a great obstacle to tissue matching and donor selection before organ, tissue and cell transplantation, and also great difficulty in constructing a PSCs cell library covering the immune match of the human 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, cells have been reported to overexpress CTLA4-Ig, PD-L1 and thereby inhibit allogeneic immune rejection. Recently, it has been reported that when B2M and CIITA are knocked out, CD47 is knocked in, so that cells obtain escape specific immune response, and have immune tolerance or escape natural immune response of cells such as NK cells, so that the cells have more comprehensive and stronger immune compatibility characteristics. 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-derived 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 matching 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 inherent immune response of NK cells can be inhibited through 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 presented antigen 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 species are very different, and the part of the area can only account for 70 percent by verification and calculation, while the HLA data of large sample size which is not authoritative currently in China, Indian and other big countries is displayed, so that the prepared general PSCs are still subjected to huge match vacancy tests; thirdly, the method can go through repeated gene editing for a plurality of times, at least two rounds of single cell isolation culture meters are needed 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 of 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 object of the first aspect of the present invention is to provide a VEGF-a blocker-expressing pluripotent stem cell or a derivative thereof, comprising at least one of a VEGF-a blocker-expressing non-immune compatible pluripotent stem cell or a derivative thereof, a VEGF-a blocker-expressing immune compatible reversible pluripotent stem cell or a derivative thereof; wherein, the immune compatible pluripotent stem cell or the derivative thereof expressing the VEGF-A blocker can be realized by the following scheme: knocking out B2M and/or CIITA gene in the genome of the pluripotent stem cell or the derivative thereof and/or introducing an expression sequence of an immune compatible molecule into the genome of the pluripotent stem cell or the derivative thereof; an immune compatible reversible pluripotent stem cell or derivative thereof expressing a VEGF-A blocker is achieved by the following scheme: introducing immune compatible molecules 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 molecules introduced into the genome of the pluripotent stem cells or the derivatives thereof is regulated by the inducible gene expression system, and the on and off of the inducible gene expression system are 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 in the preparation of a medicament for treating macular degeneration and/or tumor.

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 a VEGF-a blocker, wherein an expression sequence of the VEGF-a blocker is introduced into the genome of the pluripotent stem cell or the derivative thereof, and the VEGF-a blocker is an anti-VEGF-a antibody.

The sequence of the anti-VEGF-A antibody is shown in SEQ ID NO. 1.

The introduction site of the expression sequence of the VEGF-A blocker is a genome safe 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 gene of the pluripotent stem cell or the derivative thereof is knocked out, so that an immune compatible pluripotent stem cell expressing a VEGF-A blocker 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 a VEGF-A 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 genome safe site comprises one or more of an AAVS1 safe site, an eGSH safe site and an H11 safe site.

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 that includes at least one of B2M and CIITA.

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;

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

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: replacing a target sequence in the microRNA-30 or the 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 to obtain the gene.

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.

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 expression VEGF-A blocker pluripotent stem cell 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 genome safe site comprises one or more of an AAVS1 safe site, an eGSH safe site and an H11 safe site.

The introduction of the expression sequence of the VEGF-A blocker, the expression sequence of an immune compatible molecule, the shRNA and/or miRNA processing complex related gene, the anti-interferon effector molecule and the 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 for the preparation of a medicament for the treatment of macular degeneration and/or tumor.

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 VEGF-A blocker can be used for inducing iPSCs (induced pluripotent stem cells) or differentiating into MSCs (mesenchymal stem cells) which are low in immunogenicity to use by autologous cells, can continuously express the VEGF-A blocker in vivo, and is used for treating macular degeneration and/or tumors 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 pluripotent stem cells or the derivatives thereof have low immunogenicity, and when the pluripotent stem cells or the derivatives thereof are transplanted into a recipient, the problem of allogeneic immune rejection between donor cells and the recipient can be overcome, so that the donor cells can continuously express the VEGF-A blocker in the recipient for a long time.

The genome of the immune compatible reversible pluripotent stem cell or the derivative thereof for expressing the VEGF-A blocker 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 continuous 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 VEGF-a blocker in the recipient for a prolonged period of time. When the donor cell is diseased, the expression of the immune compatible molecules can be closed by induction of an exogenous inducer, so that the HLA class I molecules can be reversibly re-expressed on the surface of the donor cell, the antigen presenting capability of the donor cell is recovered, and the diseased cell can be eliminated by a receptor, thereby improving the clinical safety of the general pluripotent stem cell or the derivative thereof, and greatly expanding the value of the general pluripotent stem cell 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, even if the HLA class I molecules with unmatched HLA class I molecule expression on the surface 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 part 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 surface expression of HLA class i molecules after the recipient has become fully tolerant.

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

1.1VEGF-A blockers

The sequence of the anti-VEGF-A antibody is shown in SEQ ID NO. 1.

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 the third generation highly efficient and safe epismal-iPSCs induction system (6F/BM1-4C), pE3.1-OG- -KS and pE3.1-L-Myc- -hmiR302 cluster which are built by us 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 PD 54032901 is cultured for 2 days, iPSCs clones can be picked up after being cultured for 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. The specific construction method is as follows: 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% KSR, NEAA, diabody, glutamine, beta-mercaptoethanol, 10ng/mL bFGF and SB-431542) is cultured, fluid 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, 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 mesoderm 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 also includes adult stem cells, each germ layer cell or tissue, organ 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 verified "safe harbor" site that ensures the desired 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 number 22 chromosome of a human, 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 insertion of a foreign gene 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 sites.

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 very useful in situations where it is desirable to maintain the transgene in a sustained 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 safety 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 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-rapamycin associated 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 can only occur 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 are shRNA or shRNA-miR immune compatible molecules constructed by 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 still 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 its 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, which in turn affects 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, during IFN induction, double-stranded RNA-dependent Protein Kinase (PKR), which is a key factor of the whole cell signal transduction pathway, and 2 ', 5' Oligoadenylate Synthetase (2,5-Oligoadenylate Synthetase,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, which results in nonspecific activation of RNase, RNaseL, degradation of all mRNA in cells and cell death. The specificity of induction of type I interferons is achieved by members of the IRF transcription factor family, which are not inducible to be secreted 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 were all anti-interferon effector molecules 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 for Immunocompatible molecules, shRNA or shRNA-miR of anti-Interferon Effector molecules

The general framework sequences of the shRNA or shRNA-miR of the immune compatible molecules and the anti-interferon effector molecules are as follows:

(1) the constitutive expression framework of shRNA is:

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGCTAGCGCCACC(SEQ ID NO.164)N₁...N₂₁TTCAAGAGA(SEQ ID NO.165)

N₂₂...N₄₂TTTTTT；

wherein:

a、N₁...N₂₁shRNA target sequence for the corresponding Gene, N₂₂...N₄₂shRNA target sequence of corresponding geneThe reverse complement of (3);

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:

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGCTCGGTACCCGGGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTGCTAGCGCCACC(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 complementary 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.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 gene is obtained by replacing a target sequence in microRNA-30 with a shRNA-miR target sequence, and the specific sequence is as follows:

GAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTAAAGAAGGTATATTGCTGTTGACAGTGAGCG(SEQ ID NO.167)M₁N₁...N₂₁TAGTGAAGCCACAGATGTA(SEQ ID NO.168)N₂₂...N₄₂M₂TGCCTACTGCCTCGGACTTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAAT(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 test 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 which were 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 the two 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 Homologous 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. This fragment was obtained by digesting KI (Knock-in, the same applies hereinafter) Vector plasmid and recovering it.

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 is ordered from Addge (Plasmid 41824, Addge), then the DNA sequence is input in the website (URL: https:// ccttop. cos. uni-heidelberg. de) to design the target sequence, and finally different target sequences are respectively put into the blank sgRNA Plasmid 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 pUC18 plasmid as a template;

acquisition of aavs1 or eGSH recombination arms: extracting genome DNA of human cells and designing corresponding primers, and then amplifying and recovering the fragments 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 NuoZanza, P505-d1) through a PCR method;

d. assembling 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 that only the right and left recombination arms are different, and the other elements are the same. 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 (the Matrigel working solution is added into a cell culture bottle dish and shaken up before the cells are passaged or revived to ensure that the Matrigel completely sinks to the bottom of the culture bottle dish, and any Matrigel cannot be dried at any position before the cells are used. in order to ensure that the cells can be attached to the culture bottle dish and survive better, the Matrigel is put into a 37 ℃ culture box for the time of 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 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. In advance ofThe Matrigel in the coated cell culture flask dish was aspirated away, and the 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. after centrifugation, 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 to a petrigel-coated bottle dish prepared 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) Preparing a Matrigel-coated cell bottle dish in advance, sucking out the Matrigel before recovering the cells, adding a proper amount of BioCISO into the cell bottle dish, placing at 37 ℃ and 5% CO₂Incubating 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 changed to 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 (the 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 only difference 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 determining 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 method for determining the expression of VEGF-A blockers by pluripotent Stem cells or derivatives thereof

The anti-VEGF-a antibodies expressed by pluripotent stem cells or derivatives thereof are detected using ELISA (double antigen sandwich). Collecting culture supernatant of the pluripotent stem cells or the derivatives thereof expressing the anti-VEGF-A antibody, diluting the sample dilution by 5 times for later use, loading the sample on an ELISA plate coated with the VEGF-A antigen, adding 40uL of the sample dilution into a sample hole to be detected, then adding 10uL of the sample to be detected, adding culture supernatant of the pluripotent stem cells or the derivatives thereof not expressing the anti-VEGF-A antibody into a control group, and gently mixing the culture supernatants. Sealing the plate, placing at 37 deg.C, incubating for 30min, washing for 5 times, adding 50uL enzyme-labeled VEGF-A antigen reagent, sealing the plate, placing at 37 deg.C, incubating for 30min, washing for 5 times, adding color developing solution, developing for 15min, adding 50uL stop solution, reading, and measuring absorbance value at 450 nm. (the expression level of anti-VEGF-A antibody is positively correlated with the shade of color).

1.10Transwell assay to determine the Effect of VEGF-A blockers on tumor cell migration

Collecting culture supernatant of pluripotent stem cells expressing anti-VEGF-A antibody and derivatives thereof, mixing with serum-free DMEM basal medium at a volume ratio of 1:1, starving and culturing tumor cells hepG2 for 12 hours, digesting, and counting the cells by resuspension using the culture supernatant of pluripotent stem cells expressing anti-VEGF-A antibody and derivatives thereof, adjusting the cell number to 10⁶cells/mL, 100uL of cells were seeded in the upper chamber of a 24-well 8.0um Transwell chamber, 500uL of DMEM basal medium containing 15% FBS was added to the lower chamber, 5% CO at 37 ℃%₂And culturing for 24 h. The control group was cultured using a culture supernatant of pluripotent stem cells or derivatives thereof that did not express anti-VEGF-A antibody, and the rest of the procedure was the same as that of the experimental group. The cells on the top of the filter were then wiped off with a cotton swab and the filter was fixed with methanol for 5 min. Stain with Giemsa dye for 15 min. The number of cells passing through the membrane was selected from 5 different fields of view among the upper, lower, left and right under a 10-fold objective lens, and the average was calculated.

1.11 methods of treatment of mouse tumor models

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 IL-4 ra 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 levels of Th2 cytokines (IL-4, IL-5, IL-13).

1.12 methods of treatment of mouse macular degeneration model

In humanized NSG mice (The Jackson Laboratory (JAX)), human immune cells from The same donor are injected to reconstitute The immune system of The mice. After 2 weeks, macular degeneration mice were established by laser retinal injury to Choroidal Neovascularization (CNV). The specific method comprises the following steps: intraperitoneal injection of 0.3% sodium pentobarbital is performed, 45mg of anesthesia is performed per kg of injection, and 0.5% tolbicamide and 0.5% phenylephrine hydrochloride are used for eye mydriasis. Under a slit lamp, a 532nm time Nd: YAG laser (the spot diameter is 75lrm, the exposure time is 100ms, the energy is 180mW) is used for a front lens, the distance from the optic disc is about 1-1.5PD, the retina is shot around the optic papilla, each eye shoots 8-10 points, and the retinal blood vessels are prevented from being shot directly. Followed by tail vein injection of 200uL PBS (containing 10)⁶A pluripotent stem cell derivative expressing a VEGF-a blocker, the pluripotent stem cell derivative being derived from the same donor as the human immune cell) to perform a macular degeneration disease. The detection method adopts fundus angiography (FFA), 20% fluorescein sodium is diluted into 2% fluorescein sodium by using injection water, 0.3mL is injected into an abdominal cavity, and the angiography process is recorded by a Heidelberg Fundus Fluorescence Angiography (FFA) instrument. The treatment effect was judged by the CNV formation rate. The rate of CNV formation was judged from the FFA fluorescence leakage intensity integral. The judgment of the leakage intensity of fluorescein refers to a Takehana grading standard, namely 0 grade: no fluorescence leakage; level 1: slight fluorescence leakage; and 2, stage: moderate fluorescence leakage; grade 3 strong fluorescence leakage. Integral calculation of fluorescence leakage intensity: the total integral of each laser spot is set to be 3 minutes, and 0 grade is 0 minute; 1, 1 grade and 1 point; 2, 2 points are counted by a grade 2; grade 3, 3 points.

2. Experimental protocol

The experimental protocol for knocking in the gene expressing the VEGF-a 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 knocking in of the gene or nucleic acid sequence and the "-" sign indicates gene knock out.

TABLE 5 constitutive expression protocol

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

general principle:

the anti-VEGF-A antibody sequence is placed at the position of MCS2 of the corresponding plasmid (the anti-VEGF-A antibody structure is IL-2sig signal peptide (SEQ ID NO.2) + anti-VEGF-A antibody sequence (the tail end of the antibody sequence is added with a stop codon TGA)), the shRNA is placed in a shRNA expression frame of the corresponding plasmid, the shRNA-miR is placed in a 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

The VEGF-A antibody sequence was placed into MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid.

(2) A2 grouping

The VEGF-A antibody sequence was placed into MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid. 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 linked using EMCV IRESWt (SEQ ID NO. 178)).

(3) A3 grouping

The VEGF-A antibody sequence was placed into MCS2 of AAVS1 KI Vector (shRNA-miR, constitutive) plasmid. 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 VEGF-A antibody sequence was placed in MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid, the sgRNA target sequence of B2M was placed in the target sequence of sgRNA clone B2M plasmid (SEQ ID NO.179 and SEQ ID NO.180), and the sgRNA target sequence of CIITA was placed in the target sequence of sgRNA clone CIITA plasmid (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

Methods grouped with a 3.

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

(1) B1 grouping:

the VEGF-A antibody sequence was placed into MCS2 of AAVS1 KI Vector (shRNA, inducible) plasmid. 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 VEGF-A antibody sequence was placed into MCS2 of AAVS1 KI Vector (shRNA-miR, inducible) plasmid. 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 VEGF-A 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 for later use, loading on an ELISA plate coated with VEGF-A antigen, loading sample diluent 40uL into sample wells to be detected, then loading 10uL into samples to be detected, adding culture supernatant of pluripotent stem cells or derivatives thereof which do not express anti-VEGF-A antibody into control groups, and mixing gently. Placing the sealing plate at 37 ℃ for incubation for 30min, washing for 5 times, adding 50uL of enzyme-labeled VEGF-A antigen reagent, placing the sealing plate at 37 ℃ for incubation for 30min, washing for 5 times, adding color development liquid for color development for 15min, adding 50uL of stop solution, and reading to measure the absorbance value of 450 nm. The results of the tests of the respective experimental groups are shown in Table 7.

TABLE 7 blocking Effect of VEGF-A antibodies expressed in each experimental group on VEGF-A

As can be seen from the above table, the pluripotent stem cells or derivatives thereof of the present invention are capable of efficiently expressing VEGF-A antibodies. Moreover, the expression level is relatively constant in each group, so that the VEGF-A antibody expressed by the pluripotent stem cells or the derivatives thereof is not influenced by cell differentiation morphology and other exogenous genes (immune compatibility modification).

3.2Transwell assay to determine the Effect of VEGF-A blockers on tumor cell migration

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 culture box, collecting culture medium supernatant, mixing with serum-free DMEM basal medium at volume ratio of 1:1, starving for 12 hr to tumor cell hepG2, digesting, and culturing with culture medium containing pluripotent stem cells expressing anti-VEGF-A antibody and derivatives thereofCounting the supernatant and the suspension, adjusting the cell number to 10⁶cells/mL, 100uL of cells were seeded in the upper chamber of a 24-well 8.0um Transwell chamber, 500uL of DMEM basal medium containing 15% FBS was added to the lower chamber, 5% CO at 37 ℃%₂And culturing for 24 h. The control group was cultured using a culture supernatant of pluripotent stem cells or derivatives thereof that did not express anti-VEGF-A antibody, and the rest of the procedure was the same as that of the experimental group. The cells on the top of the filter were then wiped off with a cotton swab and the filter was fixed with methanol for 5 min. Stain with Giemsa dye for 15 min. The number of cells passing through the membrane was selected for 5 different fields of view from top to bottom and from left to right under a 10-fold objective lens, and the average was calculated. The results of the tests of the respective experimental groups are shown in Table 8.

TABLE 8 migration Effect of VEGF-A blockers expressed in each experimental group on tumor cells

As can be seen from the above table, the VEGF-A blocker expressed by the pluripotent stem cells or the derivatives thereof prepared by the invention can effectively block the migration of tumor cells.

3.3 antitumor Effect of pluripotent Stem cells expressing VEGF-A blockers or derivatives thereof

We selected cells (iPSCs, MSCs, EBs, NSCs) expressing the blocker protocol panel (a4) for testing. In a humanized NSG mouse tumor model, hPSCs and hPSCs derived derivatives (iPSCs, hPSCs-MSCs, hPSCs-NSCs and hPSCs-EBs) capable of expressing VEGF-A antibodies are injected into the humanized NSG mouse tumor model to observe the tumor treatment effect of RCC renal carcinoma, MC colon cancer and NIC lung cancer. Note that in order to avoid the problem of immune compatibility, the immune cells and the hPSCs and the derivatives of hPSCs are all from the same person, and an immune compatibility scheme of B2M and CIITA gene knockout is adopted. The results of the experiment are shown in FIG. 9.

TABLE 9 tumor treatment Effect of pluripotent Stem cells expressing VEGF-A antibody or derivatives thereof

Note: the control group refers to NSG mouse tumor models that were not injected with hPSCs and hPSCs-derived derivatives expressing VEGF-A antibodies.

Through the experiments, the stem cells expressing the VEGF-A blocker prepared by the invention or the derivatives thereof can be proved to be capable of effectively blocking VEGF-A to play an anti-tumor role.

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

Through the above embodiments, hPSCs and hPSCs derived derivatives expressing VEGF-A blockers can effectively block VEGF-A to treat tumors. 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, in a humanized NSG mouse disease (tumor) model, the MSCs are injected with hPSCs source immune compatible capable of expressing VEGF-A blockers (anti-VEGF-A antibodies), and the effect of treating tumors (NIC lung cancer) is observed. Note that the immunocytes used were derived from a non-identical human as the hPSCs-derived MSCs.

The control group refers to the NSG mouse disease (tumor) model without MSCs cell injection.

The process of adding the Dox group is: mice were fed with 0.5mg/mL Dox in the mouse diet, and the mice were used from the time of injection of the expression blocker cells until the end of the experiment. The results are shown in Table 10.

TABLE 10 reversible expression test results for immune-compatible molecule-inducible expression sets

The above experiments show that: in treating tumors, only blocking agents expressing MSCs (group 2), which have low immunogenicity and can exist in foreign body for a certain period of 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 than MSCs that have not been immuno-compatibly engineered, exist in vivo for a longer period of time (or can coexist for a longer period of time), which exert better therapeutic effects, whereas group 5 is the B2M and CIITA gene knock-out groups, which completely eliminate the effects of HLA-I and HLA-II molecules, and thus have 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 will be abolished from their immune compatibility by the use of Dox inducer (always used) simultaneously with the injection of the expression blocker cells into the mice, and will be present in vivo for a time period comparable to that of the MSCs without immune compatibility engineering, and for a therapeutic effect comparable to that of the MSCs without immune compatibility engineering.

3.5 Effect of pluripotent Stem cells expressing VEGF-A blockers or derivatives thereof in the treatment of macular degeneration

We selected cells (iPSCs, MSCs, EBs, NSCs) expressing the blocker protocol panel (a4) for testing. In a humanized NSG mouse macular degeneration model, hPSCs and hPSCs derivatives (iPSCs, hPSCs-MSCs, hPSCs-NSCs and hPSCs-EBs) capable of expressing VEGF-A antibodies are injected into the mouse, and Choroidal Neovascularization (CNV) is observed to judge the effect of treating macular degeneration. Note that in order to avoid the problem of immune compatibility, the immune cells and the hPSCs and the derivatives of hPSCs are all from the same person, and an immune compatibility scheme of B2M and CIITA gene knockout is adopted. The results of the experiment are shown in FIG. 11.

TABLE 11 therapeutic effect of macular degeneration of pluripotent stem cells expressing VEGF-A antibody or derivatives thereof

Note: the control group refers to NSG mouse macular degeneration model of hPSCs and hPSCs derived derivatives that are not injected with VEGF-A expressing antibody.

Through the experiment, the stem cell expressing the VEGF-A blocker or the derivative thereof prepared by the invention can effectively block VEGF-A to play a role in treating macular degeneration diseases.

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

Through the above embodiments, hPSCs and hPSCs derived derivatives expressing VEGF-A blockers not only can effectively block VEGF-A to play a role in treating tumors, but also can play a role in treating macular degeneration diseases. We must also consider the issue of immune compatibility of hPSCs and derivatives of hPSCs in the treatment of macular degeneration. Therefore we chose a suitable combination to test for immune compatibility.

By utilizing the characteristic of low immunogenicity of the MSCs, the MSCs are injected with hPSCs (human platelet-derived stem cells) source immune compatible MSCs capable of expressing VEGF-A blockers (anti-VEGF-A antibodies) in a humanized NSG mouse disease (macular degeneration) model, and the effect of macular degeneration treatment is observed. Note that the immunocytes used were derived from a non-identical human as the hPSCs-derived MSCs.

The control group refers to the NSG mouse disease (macular degeneration) model without MSCs cell injection.

The process of adding the Dox group is: mice were fed with 0.5mg/mL Dox in the mouse diet, and the mice were used from the time of injection of the expression blocker cells until the end of the experiment. The results are shown in Table 12.

TABLE 12 reversible expression test results for immune-compatible molecule-inducible expression sets

The above experiments show that: in the treatment of macular degeneration, only blocking agents expressing MSCs (group 2), which have low immunogenicity and can exist in foreign body for a certain period of 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 period of time (or can coexist for a long period of time) than MSCs that are not immuno-compatibly engineered, and exert better therapeutic effects, whereas group 5 is the B2M and CIITA gene knock-out group, which 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 will be abolished from their immune compatibility by the use of Dox inducer (always used) simultaneously with the injection of the expression blocker cells into the mice, and will be present in vivo for a time period comparable to that of the MSCs without immune compatibility engineering, and will be treated with a therapeutic effect comparable to that of the MSCs without immune compatibility engineering.

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 VEGF-A blocking substance or derivative thereof and application

<130>

<160> 182

<170> PatentIn version 3.5

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<400> 100

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gctagagtga ctccatctta a 21

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gcagaatatt taaggccata c 21

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<400> 131

gcccacttaa aggcagcatt a 21

<210> 132

<211> 21

<212> DNA

<213> human

<400> 132

ggtcatcaat accactgtta a 21

<210> 133

<211> 21

<212> DNA

<213> human

<400> 133

gcattcctcc ttctcctttc t 21

<210> 134

<211> 21

<212> DNA

<213> human

<400> 134

ggaggaactt tgtgaacatt c 21

<210> 135

<211> 21

<212> DNA

<213> human

<400> 135

gctgtaagaa ggatgctttc a 21

<210> 136

<211> 21

<212> DNA

<213> human

<400> 136

gctgcaggca ggattgtttc a 21

<210> 137

<211> 21

<212> DNA

<213> human

<400> 137

gcagttcgag gtcaagtttg a 21

<210> 138

<211> 21

<212> DNA

<213> human

<400> 138

gccaattagc tgagaagaat t 21

<210> 139

<211> 21

<212> DNA

<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

Claims (20)

1. A pluripotent stem cell or a derivative thereof expressing a VEGF-A blocker, characterized in that: the genome of the pluripotent stem cell or its derivative is introduced with an expression sequence of the VEGF-A blocker, and the VEGF-A blocker is introduced into the genome of the pluripotent stem cell or its derivative. -A blocker is an anti-VEGF-A antibody. 2 . The pluripotent stem cell or its derivative according to claim 1 , wherein the B2M and/or CIITA gene of the pluripotent stem cell or its derivative is knocked out. 3 . 3 . The pluripotent stem cell or its derivative according to claim 1 , wherein the genome of the pluripotent stem cell or its derivative is further introduced into one or more expression sequences of immune-compatible molecules, and the immune-compatible molecule For regulating the expression of genes related to immune response in pluripotent stem cells or their derivatives. 4. The pluripotent stem cell or derivative thereof according to claim 3, wherein the gene related to the immune response comprises: (1) Major histocompatibility complex genes, including HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA - at least one of DQB1, HLA-DPAl and HLA-DPB1; (2) Major histocompatibility complex-related genes, including at least one of B2M and CIITA. 5. The pluripotent stem cell or derivative thereof according to claim 3, wherein the immune compatible molecule comprises any one or more of the following: (1) Immune tolerance-related genes, including CD47 or HLA-G; (2) HLA-C class molecules, including HLA-C multiple alleles with a proportion of more than 90% in the population, or fusion protein genes composed of more than 90% of HLA-C multiple alleles and B2M; (3) shRNA and/or shRNA-miR of major histocompatibility complex genes, the major histocompatibility complex genes include HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1 , at least one of HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1; (4) shRNA and/or shRNA-miR of a major histocompatibility complex-related gene, the major histocompatibility complex-related gene including at least one of B2M and CIITA. 6. The pluripotent stem cell or its derivative according to claim 5, wherein: The target sequence of the B2M shRNA and/or shRNA-miR is at least one of SEQ ID NO.3 to 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 to SEQ ID NO.15; The target sequence of the HLA-A shRNA and/or shRNA-miR is at least one of SEQ ID NO.16 to SEQ ID NO.18; The target sequence of the HLA-B shRNA and/or shRNA-miR is at least one of SEQ ID NO.19-SEQ ID NO.24; The target sequence of the HLA-C shRNA and/or shRNA-miR 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. 7. The pluripotent stem cell or its derivative according to claim 3, characterized in that: shRNA and/or miRNA processing complex-related genes and/or anti-interference are also introduced into the genome of the pluripotent stem cell or its derivative effector molecules. 8. The pluripotent stem cell or derivative thereof according to claim 7, wherein the shRNA and/or miRNA processing complex-related genes comprise Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2) At least one of , PACT (PRKRA) and DGCR8; 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 its derivative according to claim 8, wherein: 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 2-5As shRNA and/or shRNA-miR 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 IRF-7 is at least one of SEQ ID NO.154-SEQ ID NO.163. 10. The pluripotent stem cell or its derivative according to claim 6 or 9, wherein the major histocompatibility complex gene, major histocompatibility complex-related gene, PKR, 2-5As, The expression framework of shRNA and/or shRNA-miR for IRF-3 or IRF-7 is shown below: (1) shRNA expression framework: from 5' to 3', it sequentially includes the shRNA target sequence described in claim 6 or 9, the stem-loop sequence, the reverse complement of the shRNA target sequence described in claim 6 or 9, and Poly T; two A reverse complementary target sequence is separated by a stem-loop sequence in the middle to form a hairpin structure, and finally Poly T is connected as the transcription terminator of RNA polymerase III; (2) shRNA-miR expression framework: obtained by replacing the target sequence in microRNA-30 or microRNA-155 with the shRNA-miR target sequence described in claim 6 or 9. The pluripotent stem cell or its derivative according to claim 10, wherein the length of the stem-loop sequence in the shRNA expression framework is 3-9 bases; the length of the Poly T is 5-6 bases base. 12 . The pluripotent stem cell or its derivative according to claim 3 or 7 , wherein an inducible gene expression system is further introduced into the genome of the pluripotent stem cell or its derivative. 13 . 13 . The pluripotent stem cell or its derivative according to claim 12 , wherein the inducible gene expression system is at least one of a Tet-Off system and a dimer inducible expression system. 14 . 14. The pluripotent stem cell or its derivative according to claim 12, wherein: The expression sequence of the VEGF-A blocker, the expression sequence of the immune compatible molecule, shRNA and/or miRNA processing complex-related genes, anti-interferon effector molecules, and inducible gene expression systems are introduced using viral vector interference, non-viral Methods of vector transfection or gene editing. 15. The pluripotent stem cell or its derivative according to claim 14, characterized in that: The expression sequence of the VEGF-A blocker, the expression sequence of the immune compatible molecule, the shRNA and/or miRNA processing complex-related gene, the anti-interferon effector molecule, and the introduction site of the inducible gene expression system are pluripotent stem cells or Genome-safe sites for its derivatives. 16. The pluripotent stem cell or derivative thereof according to claim 15, wherein the genomic safety site comprises one or more of the AAVS1 safety site, the eGSH safety site, and the H11 safety site. 17. The pluripotent stem cell or its derivative according to any one of claims 1 to 9, 11, 13 to 16, wherein the pluripotent stem cell comprises embryonic stem cells, embryonic germ cells, embryonic cancer cells, Or induced pluripotent stem cells; The pluripotent stem cell derivatives include adult stem cells, germ layer cells or tissues differentiated by pluripotent stem cells; The adult stem cells include mesenchymal stem cells and neural stem cells. 18. The pluripotent stem cell or its derivative according to claim 17, wherein the sequence of the anti-VEGF-A antibody is shown in SEQ ID NO.1. 19. Use of the pluripotent stem cells or their derivatives according to any one of claims 1 to 18 in the preparation of macular degeneration and/or tumor therapeutic drugs. 20. A preparation comprising the pluripotent stem cell or a derivative thereof according to any one of claims 1 to 18.

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