CN114457028A

CN114457028A - Pluripotent stem cell expressing CD38 antibody, derivative and application thereof

Info

Publication number: CN114457028A
Application number: CN202011190253.1A
Authority: CN
Inventors: 王淋立; 陈月花; 莫健; 杨建国
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 a CD38 inhibitor, a derivative thereof and application thereof, wherein the genome of the pluripotent stem cell or the derivative thereof is introduced with an expression sequence of the CD38 inhibitor. The pluripotent stem cells or derivatives thereof expressing the CD38 inhibitory factor can be used for inducing iPSCs (induced pluripotent stem cells) from autologous cells or differentiating the iPSCs into MSCs (mesenchymal stem cells) which are low in immunogenicity for application, can continuously express the CD38 inhibitory factor in vivo, and can be used for treating CD38 high-expression tumors and related diseases.

Description

Pluripotent stem cell expressing CD38 antibody, derivative and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a pluripotent stem cell expressing a CD38 antibody, a derivative thereof and application thereof.

Background

CD38 is a 45kD type II transmembrane glycoprotein and is also a bifunctional extracellular enzyme. The CD38 molecule is uniformly expressed on the surface of Multiple Myeloma (MM) cells, but is expressed less on other lymphocytes or myeloid cells, and the CD38 molecule may also be expressed on some non-blood cells. CD38 also has cell adhesion, so it is important to treat MM by targeting CD38 and develop monoclonal antibody, antibody conjugate drug, bispecific antibody, CAR-T and other therapies based on this. The existing research shows that myeloma cells can be effectively killed by targeting CD38 molecules, while Daratumumab which is an antibody targeting CD38 is approved by FDA to be used for the treatment of multiple myeloma, but the existing treatment methods inevitably cause leukopenia so as to generate more serious adverse reactions, so that the practical application of the antibody targeting CD38 is greatly limited.

On the other hand, in the field of cell therapy, the problem of immunological compatibility of allogens remains a big problem. 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. 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

The present invention aims to provide a pluripotent stem cell or a derivative thereof;

it is another object of the present invention to provide another pluripotent stem cell or a derivative thereof;

the invention also aims to provide the application of the pluripotent stem cells or the derivatives thereof in preparing a medicine for treating the tumor with high expression of CD 38;

it is another object of the present invention to provide a formulation.

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 comprising a CD38 antibody sequence, wherein the CD38 antibody sequence is preferably inserted into the genome of the pluripotent stem cell or the derivative thereof.

The inventor finds that after the expression sequence of the CD38 inhibitor is introduced into the pluripotent stem cells or the derivatives thereof, the CD38 inhibitor targeting CD38 is secreted in the pluripotent stem cells or the derivatives thereof, and the inhibitor is combined with target cells to effectively eliminate tumor cells.

In a second aspect of the present invention, there is provided:

a pluripotent stem cell or a derivative thereof comprising a CD38 antibody sequence, preferably the CD38 antibody sequence being inserted into the genome of the pluripotent stem cell or the derivative thereof;

the B2M gene and/or CIITA gene of the genome of the pluripotent stem cell or the derivative thereof are knocked out.

When B2M and CIITA genes are knocked out, the influence of HLA-I and HLA-II molecules is completely eliminated, and therefore, the tumor treatment effect is optimal.

In a third aspect of the present invention, there is provided:

the pluripotent stem cells or the derivatives thereof further comprise an immune compatible molecule expression sequence, and the immune compatible molecule is used for regulating and controlling the expression of genes related to immune response in the pluripotent stem cells or the derivatives thereof; the above-mentioned sequence for expression of an immune-compatible molecule is preferably inserted into the genome of the pluripotent stem cell or a derivative thereof.

In a fourth aspect of the present invention, there is provided:

the pluripotent stem cells or the derivatives thereof further comprise an immune compatible molecule expression sequence, and the immune compatible molecule is used for regulating and controlling the expression of genes related to immune response in the pluripotent stem cells or the derivatives thereof; the above-mentioned immune compatible molecule expression sequence is preferably inserted into the genome of said pluripotent stem cell or a derivative thereof;

the pluripotent stem cell or the derivative thereof further comprises an inducible gene expression system; the inducible gene expression system is preferably inserted into the genome of the pluripotent stem cell or a derivative thereof.

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

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.

The dimer-switched-off expression system specifically refers to: dimerized inducers or dimers are used 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 the target gene, a DNA binding region is fused to one or more FKBP domains and a transcriptional repression 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.

Still further, the above-mentioned immune-compatible molecules include one or more of the following:

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

(II) HLA-C molecules comprising HLA-C alleles in a proportion of more than 90% in total in the population, or fusion protein genes consisting of more than 90% of HLA-C alleles and B2M;

(III) shRNA and/or shRNA-miR targeting the gene associated with the immune response.

Further, the above-mentioned genes related to immune response include:

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;

(II) major histocompatibility complex related genes comprising at least one of B2M and CIITA.

Furthermore, the target sequence of the shRNA and/or shRNA-miR targeting B2M is selected from one of SEQ ID NO. 3-SEQ ID NO. 5;

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

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

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

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

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

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

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

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

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

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

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

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

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

Further, at least one of a gene related to shRNA processing complex, a gene related to miRNA processing complex, and an anti-interferon effector molecule is further introduced into the genome of the pluripotent stem cell or the derivative thereof.

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

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.

Furthermore, the target sequence of the shRNA and/or shRNA-miR targeting the PKR is selected from one of SEQ ID NO. 104-SEQ ID NO. 113;

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

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

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

Further, the shRNA expression framework described above: the gene sequence sequentially comprises an shRNA target sequence, a stem-loop sequence, a reverse complementary sequence of the shRNA target sequence and Poly T from 5 'to 3';

wherein the shRNA target sequence, the stem-loop sequence and the reverse complementary sequence of the shRNA target sequence form a hairpin structure; poly T is a transcription terminator of RNA polymerase III;

shRNA-miR expression framework: the shRNA-miR target sequence is obtained by using the shRNA target sequence.

Furthermore, the length of the stem-loop sequence in the shRNA expression frame is 3-9 bases; the Poly T is 5 to 6 bases in length.

Furthermore, the CD38 antibody sequence, the immune compatible molecule expression sequence or the inducible gene expression system is inserted into the safe site of the genome of the pluripotent stem cell or the derivative thereof.

Further, the genome safety site comprises one or more of an AAVS1 safety site, an eGSH safety site and an H11 safety site.

Further, the pluripotent stem cells include embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, or induced pluripotent stem cells.

Further, the above-mentioned pluripotent stem cell derivatives include adult stem cells, each germ layer cells or tissues into which the pluripotent stem cells are differentiated;

the adult stem cells include mesenchymal stem cells or neural stem cells.

Furthermore, the heavy chain sequence of the CD38 antibody is shown as SEQ ID NO.1, and the light chain sequence is shown as SEQ ID NO. 2.

In a fifth aspect of the present invention, there is provided:

the application of the pluripotent stem cells or the derivatives thereof in preparing medicaments for treating tumors with high CD38 expression.

In a sixth aspect of the present invention, there is provided:

a preparation comprising the pluripotent stem cells or derivatives thereof.

The invention has the beneficial effects that:

1. the pluripotent stem cells or derivatives thereof expressing the CD38 inhibitory factor can be used for inducing iPSCs (induced pluripotent stem cells) from autologous cells or differentiating the iPSCs into MSCs (mesenchymal stem cells) which are low in immunogenicity for application, can continuously express the CD38 inhibitory factor in vivo, and can be used for treating CD38 high-expression tumors and related diseases.

2. 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 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 C38 inhibitor in the recipient for a long time.

3. The genome of the immune compatible reversible pluripotent stem cell or the derivative thereof expressing the CD38 inhibitor 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 immune 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 related to the immune response in the pluripotent stem cell or the derivative thereof is suppressed or overexpressed, and the allogeneic immune rejection response between the donor cell and the recipient can be eliminated or reduced, so that the donor cell can continuously express the CD38 inhibitor in the recipient for a long 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.

Drawings

FIG. 1 is a plasmid map of AAVS1 KI Vector (shRNA, Puro, constitutive);

FIG. 2 is a plasmid map of AAVS1 KI Vector (shRNA, inducible);

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

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

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;

FIG. 12 is a histogram of the inhibitory effect of expressed CD38 antibody versus MFI;

FIG. 13 shows pluripotent stem cells expressing CD38 antibody⁵¹The release rate of Cr;

FIG. 14 shows the effect of pluripotent stem cells expressing CD38 antibody on various tumor therapies;

FIG. 15 is a graph showing the effect of an immune compatibility test on pluripotent stem cells expressing the CD38 antibody.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Selection of CD38 inhibitors

CD38 inhibitor: antibodies targeting CD38 (CD38 antibodies).

The Heavy Chain (HC) sequence of the CD38 antibody is shown as SEQ ID NO.1, and the Light Chain (LC) sequence is shown as SEQ ID NO. 2.

The Heavy Chain (HC) sequence of the CD38 antibody is:

5’-GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGTGAGCGGCTTCACCTTCAACAGCTTCGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGCCATCAGCGGCAGCGGCGGCGGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTTCTGCGCCAAGGACAAGATCCTGTGGTTCGGCGAGCCCGTGTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCAGGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG-3’(SEQ ID NO.1)。

the Light Chain (LC) sequence of the CD38 antibody is:

5’-GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGGAGCAACTGGCCCCCCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC-3’(SEQ ID NO.2)。

2. selection of immune compatible molecules

The types and sequences of the immune-compatible molecules used in the examples of the present invention are shown in tables 1 and 2.

TABLE 1 types and roles of immune-compatible molecules

TABLE 2 target sequences for immune-compatible molecules

Selection of shRNA/miRNA processing Complex genes and anti-Interferon Effector molecules

(1) selection of shRNA/miRNA processing Complex genes

shRNA/miRNA processing complex genes used in the present invention include shRNA and/or miRNA processing machinery that can induce off-expression. The processor capable of inducing and closing the shRNA and/or miRNA to express specifically comprises: 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).

The specific functions of the shRNA and/or miRNA processing machine capable of inducing closed expression in cells are as follows:

the primary miRNA (pri-miRNA) in the nucleus is microprocessed through the complex Drosha-DGCR8, which cleaves the pri-miRNA to a precursor miRNA (pre-miRNA), which 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.

(2) Selection of anti-Interferon Effector molecules

The anti-interferon effector molecules used in the invention comprise shRNA and/or shRNA-miR expression sequences which can induce closed expression and aim at inhibiting PKR, 2-5As, IRF-3 and IRF-7 genes so As to reduce the interferon reaction induced by dsRNA and avoid generating cytotoxicity.

Wherein the target sequences of the anti-interferon effector molecules are shown in Table 3.

TABLE 3 target sequences for anti-interferon effector molecules

The specific functions of the interferon effector molecules PKR, 2-5As, IRF-3 and IRF-7 in the cell are As follows:

protein Kinase (PKR) and 2 ', 5' Oligoadenylate Synthetase (2,5-Oligoadenylate synthase, 2-5As) are key factors of cell signal transduction pathway in IFN-induced process, and these two enzymes are closely related to dsRNA-induced IFN. 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.

4. Construction of plasmid vectors

Plasmid vectors used in embodiments of the invention include:

(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. The fragment is obtained by recovering after the digestion of KI Vector plasmid.

The genes in the plasmids are edited by a CRISPR-Cas9 gene editing system, the used Cas9 protein is Cas9(D10A), Cas9(D10A) is combined with sgRNA which is responsible for specifically recognizing a target sequence (genome DNA of a cell vector), and then Cas9(D10A) performs single-strand cleavage on the target sequence. Double Strand breaks in genomic DNA (DSB) must occur, and two Cas 9(D10A)/sgRNA groups must cleave both strands of genomic DNA of the cell vector 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.

Wherein, the specific sequence of the sgRNA is as follows:

sgRNA-B2M-1：5’-CGCGAGCACAGCTAAGGCCACGG-3’(SEQ ID NO.164)；

sgRNA-B2M-2：5’-ACTCTCTCTTTCTGGCCTGGAGG-3’(SEQ ID NO.165)。

sgRNA-CIITA-1：5’-ACCCAGCAGGGCGTGGAGCCAGG-3’(SEQ ID NO.166)；

sgRNA-CIITA-2：5’-GTCAGAGCCCCAAGGTAAAAAGG-3’(SEQ ID NO.167)。

the specific method for constructing the plasmid vector comprises the following steps:

(1) cas9(D10A) plasmid: obtained directly from an Addgene (plasma 41816, Addgene) subscription.

(2) sgRNA plasmid: the Plasmid was constructed by putting different target sequences into original blank plasmids (Plasmid 41824, Addgene).

Wherein the target sequence put into the sgRNA plasmid comprises the sequence of the immune compatible molecule, the sequence of shRNA/miRNA processing complex gene and the sequence of anti-interferon effector molecule.

(3) Donor fragment (KI plasmid):

a) designing a PCR primer, and amplifying by using a pUC18 plasmid (Takara, Code No.3218) as a template and a high fidelity enzyme (Nanjing Nozan organism, P505-d1) through a PCR method to obtain an Amp (R) -pUC origin fragment;

b) extracting genome DNA of human cells and designing corresponding primers, and then amplifying by using the human genome DNA as a template and a PCR method by using high-fidelity enzyme to obtain an AAVS1 or eGSH recombinant arm;

c) designing PCR amplification primers of KI (Knock-in) plasmid elements, and then carrying out high-fidelity enzyme amplification by using plasmids containing the KI plasmid elements as templates to obtain KI plasmid elements (subclones);

d) the amplified Amp (R) -pUC origin fragment, AAVS1 or eGSH recombination arm and KI plasmid element are connected by using multi-fragment recombinase (Nanjing Novozam organism, C113-02) or overlap PCR to form a complete circular plasmid.

The plasmids prepared by the embodiment of the invention can be divided into constitutive plasmids and inducible plasmids according to the expression frame type in the plasmids.

The expression frame in the constitutive plasmid comprises shRNA constitutive expression frame and shRNAMIR constitutive expression frame. The expression function of the Donor fragment obtained by enzyme digestion of the constitutive plasmid cannot be regulated after knocking in the stem cell vector genome DNA.

The expression frames in the inducible plasmid comprise shRNA inducible expression frames and shRNAMIR inducible expression frames. After the Donor fragment obtained from the inducible plasmid is digested by enzyme and the stem cell vector genome DNA is knocked in, the expression function of the fragment can be regulated and controlled by adding an inducer, which is equivalent to adding a switch for turning on or off the expression function.

The specific sequence requirements and structural composition of the expression frameworks described above are as follows.

(1) The sequence composition of the shRNA constitutive expression framework is:

5’-GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACgctagcgccacc(SEQ ID NO.168)N₁...N₂₁TTCAAGAGA(SEQ ID NO.169)N₂₂...N₄₂TTTTTT(SEQ ID NO.170)-3’；

wherein the content of the first and second substances,

a)N₁...N₂₁shRNA target sequence which is the above-mentioned target sequence, N₂₂...N₄₂The reverse complementary sequence of the shRNA target sequence of the target sequence;

b) when plasmids constructed by using the shRNA constitutive expression frame need to express shRNAs of a plurality of genes, each gene corresponds to a shRNA expression frame respectively and then is connected seamlessly;

c) when the shRNA constitutive expression frame needs to carry different resistance genes, only the resistance gene sequences in the shRNA constitutive expression frame are different, and other sequences are the same;

d) n represents A or T or G or C base.

(2) The sequence composition of the shRNAmiR constitutive expression framework is as follows:

5’-GAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTAAAGAAGGTATATTGCTGTTGACAGTGAGCG(SEQ ID NO.171)M₁N₁...N₂₁TAGTGAAGCCACAGATGTA(SEQ ID NO.172)N₂₂...N₄₂M₂TGCCTACTGCCTCGGACTTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAAT(SEQ ID NO.173)-3’；

wherein the content of the first and second substances,

a)N₁...N₂₁shRNAMIR target sequence, N, being the above target sequence₂₂...N₄₂The reverse complement of the shRNAmiR target sequence to the target sequence;

b) when a plasmid constructed by using the shRNAMIR constitutive expression framework needs to express shRNAMIR of a plurality of genes, each gene corresponds to one shRNAMIR expression framework respectively and then is connected seamlessly;

c) when the shRNAmid R constitutive expression framework needs to carry different resistance genes, only the resistance gene sequences in the shRNAmid R constitutive expression framework are different, and other sequences are the same;

d) n represents A or T or G or C base, M base represents A or C base;

e) if N1 is a G base, then M₁Is A base; otherwise M₁Is a C base;

f)M₁base and M₂And (3) base complementation.

(3) The sequence composition of the shRNA inducible expression framework is:

5’-GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagctcggtacccgggtcgaggtaggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctgctagcgccacc(SEQ ID NO.174)N₁...N₂₁TTCAAGAGAN₂₂...N₄₂TTTTTT-3’；

wherein the content of the first and second substances,

d) n represents A or T or G or C base.

(4) The sequence composition of the shRNAmiR inducible expression framework is shown in the sequence composition of the shRNAmiR constitutive expression framework.

The principle and method for constructing the eGSH recombinant plasmid are the same as those of the AAVS recombinant plasmid.

The constructed plasmid frameworks are shown in FIGS. 1-11.

5. Construction of Stem cell vectors

(1) Selection of Stem cell vectors

The stem cell carrier in the invention is a pluripotent stem cell, and the pluripotent stem cell 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 and EBs cells.

Wherein, the preparation method of the pluripotent stem cells comprises the following steps:

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

And (2) iPSCs: using an episomal-iPSCs induction system (6F/BM1-4C), pE3.1-OG- -KS and pE3.1-L-Myc- -hmiR302 cluster were electrotransferred into somatic cells, RM1 medium was cultured for 2 days, 2 days with 2uM Parnate-containing BioCISO-BM1 medium, 2 days with 2uM Parnate, 0.25mM sodium butyrate, 3uM CHIR99021 and 0.5uM PD 03254901-containing BioCISO-BM1 medium, and then cultured continuously with BioCISO medium without other substances for about 17 days, iPSCs clonal cells were picked, and the picked iPSCs clonal cells were 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 were cultured for 25 days in BioCISO medium containing 10uM TGF β inhibitor SB431542 until 80-90 cell confluence (2mg/mL Dispase), 1:3 passaging was performed on Matrigel-coated plates, followed by ESC-MSC medium (knockout DMEM medium containing 10% KSR, NEAA, diabody, glutamine, β -mercaptoethanol, 10ng/mL bFGF and SB-431542), fluid was changed every day, and culture was continued for 20 days until 80-90 cell confluence (1:3 passaging), and specific construction methods were as follows: proc Natl Acad Sci U S A.2015; 112(2):530-535.

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

EBs cells: the iPSCs with cell confluency of 95% were digested with BioC-PDE1 for 6min, and the cells were scraped into clumps by mechanical scraping, and the clumps were settled. The settled cell pellet was transferred to a low adhesion culture plate and cultured for 7 days using BioCISO-EB1, with fluid changes every other day. After 7 days, the cells were transferred to a Matrigel-coated plate and subjected to adherent culture using a BioCISO medium for 7 days, thereby obtaining Embryoid Bodies (EBs) having an inner, middle and outer mesoderm structure. 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.

(2) Knock-in of safe site in genome of constructed stem cell vector

In the technical scheme of the invention, the safety sites knocked in the genome of the constructed stem cell vector comprise an AAVS1 safety site, an eGSH safety site and an H11 safety site.

The AAVS1 safe site (PPP1R2C site) is located on chromosome 19 of the human genome and is a verified "safe harbor" site which can ensure the expected function of transferred DNA fragments. 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.

The eGSH safe site is located on chromosome 1 of the human genome and is another "safe harbor" site that ensures the desired function of the transferred DNA fragment.

The H11 safe site (Hipp11), located on human chromosome 22, is a site between the two genes Eif4enif1 and Drg 1. Because the H11 locus is positioned between two genes, the risk of influencing endogenous gene expression after the exogenous gene is inserted is small.

The single-cell cloning operation of the knock-in of the AAVS1 gene comprises the following steps:

a) electric transfer program:

donor cell preparation: human pluripotent stem cells

The kit comprises: human Stem Cell

Kit 1

The instrument comprises the following steps: electric rotating instrument

Culture medium: BioCISO

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

Wherein, if the eGSH knock-in is used, the induction plasmids used are: cas9D10A, sgRNA clone eGSH-1, sgRNA clone eGSH-2, eGSH-neo/eGSH-puro (donor). Among them, when using the eGSH gene knock-in, the donor plasmid differs only in the right and left recombination arms, and the other plasmid elements are the same, compared to the AAVS1 gene knock-in. Since the gene editing process of eGSH is the same as that of AAVS1, the following description will not be repeated.

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

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

d) The obtained single-cell clone was cultured.

Wherein, the culture reagent of the single-cell clone strain comprises:

the culture medium is as follows: BioCISO medium, 300. mu.g/ml G418 and 0.5. mu.g/ml puro. The culture medium needs to be placed at room temperature in advance, placed for 30-60 minutes in a dark condition until the room temperature is recovered, but not placed at 37 ℃ for preheating, so that the activity of the biological molecules is prevented from being reduced.

Matrix glue: hESC grade Matrigel. Before passage or cell recovery, adding the Matrigel working solution into a cell culture bottle dish and shaking up to ensure that the Matrigel completely submerges the bottom of the culture bottle dish and any Matrigel cannot be dried off before use. To ensure that cells adhere better and survive, Matrigel was placed in a 37 ℃ incubator for a period of time: 1:100 Xmatrigel can not be less than 0.5 hour; the 1:200 Xmatrigel cannot be less than 2 hours.

Digestion solution: EDTA was dissolved using DPBS to a final concentration of 0.5mM, pH 7.4. The EDTA cannot be diluted with water, otherwise the cells die due to reduced osmotic pressure.

Freezing and storing liquid: 60% BioCISO, 30% ESCS grade FBS, and 10% DMSO.

The culture step of the single-cell clone adopts the conventional subculture maintaining process in the field.

Wherein, the optimal passage time is that the overall confluency of the cells reaches 80 to 90 percent.

The optimal ratio of passage is 1: 4-1: 7, and the optimal confluency of the next day after passage is maintained at 20-30%.

The specific passage operation steps in the invention are as follows:

a) discarding the Matrigel from the coated cell culture flask, adding appropriate amount of the above culture medium, and adding 5% CO at 37 deg.C₂Incubation in an incubator;

b) when the cells meet the passage requirements, removing culture medium supernatant, and adding a proper amount of 0.5mM EDTA digestive juice 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), 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 supernatant, suspending the cells with culture medium, repeatedly blowing the cells until uniformly mixing, transferring the cells to a petrigel-coated bottle dish, shaking, observing under a mirror without abnormality, shaking, and standing at 37 deg.C and 5% CO₂Culturing in an incubator;

e) observing the adherent survival state of the cells the next day, sucking off the culture medium, and changing the culture medium on time every day.

The single cell clone obtained by passage can be directly used for experiment or selection freezing storage treatment.

The cell freezing method comprises the following specific steps:

a) digesting the cells by using 0.5mM EDTA until most cells shrink and become round but do not float, blowing the cells, collecting cell suspension, centrifuging for 5 minutes at 200g, abandoning supernatant, adding a proper amount of freezing solution to resuspend the cells, and transferring the cells to a freezing tube (one six-hole plate is recommended to be frozen with 80% confluence, and the volume of the freezing solution is 0.5 ml/piece);

b) 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);

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

The cell recovery method of the cryopreserved cells comprises the following specific steps of:

a) preparing a Matrigel-coated cell flask dish (before resuscitating the cells, the Matrigel is discarded), adding an appropriate amount of BioCISO medium, and placing at 37 ℃ in 5% CO₂Incubation in an incubator;

b) 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, observing that the shaking is stopped when the ice crystals completely disappear, and transferring the cells to a biological safety cabinet;

c) preparing 10mL of DMEM/F12 (volume ratio of 1:1) basal medium, balancing to room temperature, sucking 1mL of DMEM/F12 (volume ratio of 1:1) basal medium, slowly adding the basal medium into a freezing tube, uniformly mixing, transferring the mixture into a prepared 15mL centrifuge tube containing 9mL of DMEM/F12 (volume ratio of 1:1) basal medium, and centrifuging for 5 minutes at 200 g;

d) discarding supernatant, adding appropriate amount of BioCISO culture medium, mixing cells, transferring to cell bottle dish, shaking for observation without abnormality, shaking, and placing at 37 deg.C and 5% CO₂Culturing in an incubator;

e) 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 medium is replaced with the above medium mixed with BioCISO, 300. mu.g/ml G418 and 0.5. mu.g/ml puro.

(3) Detection of AAVS1 Gene knock-in cell vectors

The detection principle 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.

The detection method comprises the following steps:

one primer was designed inside (non-recombinant arm portion) the Donor plasmid (KI plasmid), and then the other primer was designed in the genome of the cell (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.

The specific sequences of the primers and amplification conditions are shown in the following table.

TABLE 4 specific sequences of primers and amplification conditions

The detection method of the eGSH gene knock-in situation is the same as the AAVS1 gene knock-in detection principle and method.

(4) Knock-in of the selected plasmid into the stem cell vector genome using techniques conventional in the art

According to the grouping in the following tables 5 and 6, the sequence of the CD38 antibody, the sequence of the immune compatible molecule, the sequence of the shRNA/miRNA processing complex gene and the sequence of the anti-interferon effector molecule are knocked into the safe locus of the stem cell expression vector (stem cell vector) by adopting the conventional technology in the field, so as to obtain different types of constitutive stem cell expression vectors and inducible stem cell expression vectors, and detect the expression feasibility of the constitutive stem cell expression vectors and the inducible stem cell expression vectors.

TABLE 5 constitutive knock-in expression experiment grouping

Wherein a "+" symbol indicates a knock-in of a gene or nucleic acid sequence and a "-" symbol indicates a knock-out of a gene.

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

general principle: the CD38 antibody sequence is put into MCS2 (constitutive expression) of the corresponding plasmid, other molecules are put into shRNA or shRNA-miR expression frames of the corresponding plasmid according to shRNA and shRNA-miR, and other genes are put into MCS1 of the corresponding plasmid.

Wherein, IL-2sig signal peptides are added in the front of an LC light chain and an HC heavy chain of the CD38 antibody, and are connected by EMCV IRESWT in the middle, and the specific structure is as follows: the LC light chain of the IL-2sig signal peptide-CD 38 antibody (containing a stop codon, which is TGA) -EMCV IRESSwt-IL-2 sig signal peptide-HC heavy chain of the CD38 antibody (containing a stop codon, which is TGA).

The sequence of EMCV IRESWt is shown as SEQ ID NO. 183;

the sequence of the IL-2sig signal peptide is: 5'-ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG-3' (SEQ ID NO. 184).

Wherein the sgRNA clone B2M plasmid comprises 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 CD38 antibody sequences.

(2) A2 grouping:

MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid places CD38 antibody sequences, shRNA expression framework places shRNA target sequences of other molecules (seamlessly joined if multiple shrnas are present), and MCS1 places other gene sequences.

(3) A3 grouping:

the CD38 antibody sequence is placed in MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid, shRNA-miR target sequences of other molecules are placed in shRNA-miR expression framework (if a plurality of shRNAs exist, the shRNA-miR target sequences are connected in a seamless mode), and other gene sequences are placed in MCS 1.

(4) A4 grouping:

the MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid was placed into the CD38 antibody sequence, B2M and CIITA were knocked out, and MCS1 was placed into other gene sequences.

(5) A5 grouping:

(6) A6 grouping:

When immune compatibility transformation is carried out, transformation can be carried out on hPSCs, and the derivative which is transformed into the pluripotent stem cells is used after the transformation is finished; the immune compatibility can also be modified after differentiation of hPSCs into derivatives of pluripotent stem cells.

TABLE 6 inducible knock-in expression experiment grouping

(1) B1 grouping:

the CD38 antibody sequence is placed in MCS2 of AAVS1 KI Vector (shRNA, inducible) plasmid, the shRNA expression frame is placed in shRNA target sequences of other molecules (if a plurality of shRNAs exist, the shRNA target sequences are connected in a seamless mode), and the MCS1 is placed in other gene sequences and inserted into a Tet-Off system.

(2) B2 grouping:

CD38 antibody sequences are placed in MCS2 of AAVS1 KI Vector (shRNA, inducible) plasmid, shRNA-miR expression frameworks are placed in shRNA-miR target sequences of other molecules (if a plurality of shRNAs exist, the shRNA-miR target sequences are connected in a seamless mode), other gene sequences are placed in MCS1, and the Tet-Off system is inserted.

(3) B3 grouping:

(4) B4 grouping:

CD38 antibody sequences are placed in MCS2 of AAVS1 KI Vector (shRNA, inducible) plasmids, shRNA-miR target sequences of other molecules are placed in shRNA-miR expression frameworks (if a plurality of shRNAs exist, the shRNA-miR target sequences are connected in a seamless mode), MCS1 is placed in gene sequences, and the gene sequences are inserted into a Tet-Off system.

The Tet-Off system in the invention specifically comprises the following steps:

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 sequence of the Tet-Off system and one or more immune compatible molecules is knocked into a genome safety site of the pluripotent stem cell, and the expression of the immune compatible molecules is accurately turned on or Off through the addition of tetracycline, so that the expression of major histocompatibility complex related genes in the pluripotent stem cell or the derivative thereof can be reversibly regulated and controlled.

(5) Detection of expression effect of CD38 antibody expressed by pluripotent stem cell expressing CD38 antibody or derivative thereof

The experimental set protocols in tables 5 and 6 were knocked into the genome of MSCs cellsSafe site, 37 ℃, 0.5% CO₂Culturing in an incubator, and collecting culture medium supernatant. The CD 38-expressing CHO cells were resuspended and incubated at 37 ℃ for 30 minutes. The cells were washed 2 times with PBS and FITC-labeled CD38 fusion protein was added to the tubes and incubated at 37 ℃ for 30 minutes. Flow cytometric analysis was performed using a Flow Cytometer (FCM). Binding of anti-CD 38 antibody expressed by pluripotent stem cells to human CD38 transfected CHO cells was measured as Mean Fluorescence Intensity (MFI) of staining. Namely, the inhibitory effect of the anti-CD 38 antibody on CD38, which is expressed by the pluripotent stem cells expressing the CD38 inhibitor, can be shown.

The N (control) group refers to CD 38-expressing CHO cells cultured without addition of CD38 inhibitor. The method specifically comprises the following steps: untreated CHO cells expressing CD38 were incubated at 37 ℃ for 30 minutes, the cells were washed 2 times with PBS and FITC labeled CD38 fusion protein was added to the tubes and incubated at 37 ℃ for 30 minutes.

Positive for non-immune compatible pluripotent stem cell derivatives expressing anti-CD 38 antibody.

The results of the tests of the respective experimental groups are shown in table 7 and fig. 12.

TABLE 7 expression results of CD38 antibody expressed by pluripotent stem cells or derivatives thereof

Grouping	Relative MFI value	Deviation (+/-)	Independent sample T test (. p)<0.01)
				N (control)	0.971	0.076	-
A1	0.421	0.031	*
				A2	0.372	0.024	*
A3	0.407	0.024	*
				A4	0.378	0.019	*
A5	0.386	0.029	*
				A6	0.397	0.034	*
B1	0.406	0.047	*
				B2	0.407	0.023	*
B3	0.413	0.016	*
				B4	0.387	0.028	*

As can be seen from the above table, the anti-CD 38 antibody expressed by the pluripotent stem cells or derivatives thereof of the present invention can effectively bind to CD 38. And the expression level is relatively constant in each group, so that the CD38 antibody expressed by the pluripotent stem cell derivative is not influenced by cell differentiation morphology and other exogenous genes (immune compatibility modification).

(6) Effect of pluripotent Stem cells expressing CD38 antibody on T cell killing of tumor cells

By using⁵¹The Cr release method for detecting the influence of the pluripotent stem cells expressing the CD38 antibody on the killing of tumor cells by T cells comprises the following specific steps:

preparation of effector cells:

t cell isolation: human Peripheral Blood Mononuclear Cells (PBMC) were isolated using Ficoll density gradient centrifugation (Ficoll-hypaque density gradient centrifugation) followed by Dynabeads^TMCD3(Invitrogen^TMAnd the cargo number: 11151D) T cells are isolated by the kit. The cells were resuspended in RPMI1640 medium containing 10% FBS, the cells were counted by trypan blue staining, and concentrated to 1X 10⁷cells/mL.

Preparation of target cells:

tumor (MM myeloma) cells were digested and resuspended, and cells were counted by trypan blue staining to give 1X 10 cells⁷cells/mL of cell suspension.

⁵¹Cr Release test:

the principle is as follows: when tumor cells are first incubated with culture supernatants of pluripotent stem cells expressing the CD38 antibody and then contacted with T cells, T attacks the tumor cells and causes cell lysis and death. Whereas culture supernatants of pluripotent stem cells not expressing CD38 antibody incubated with tumor cells were not recognized by T cells and immune escape occurred. So by detecting in the medium⁵¹The amount of Cr reflects the ability of T cells to kill tumors.⁵¹The less the amount of Cr released into the culture medium, the more immune escape of the tumor cells will occur.

Quantitative determination of cell-mediated cytotoxicity with radioisotopes⁵¹Cr-labeled target cells, co-incubated with effector molecules or cells, released upon lysis of the target cells⁵¹The number of Cr radiation pulses (cpm) was used to determine the cytotoxic activity.

The method comprises the following specific steps:

a. target cells were treated with 100. mu. Ci (Ci, radioactivity units) of Na⁵¹CrO₄Labeling at 37 deg.C for 120min, shaking every 15 min, labeling, centrifuging with cleaning solution for 5 times, and resuspending in culture medium to obtain 1 × 10⁶cells/mL for use;

b. target cells and T cells were added to 96-well plates, and 100. mu.L of target cells (2.5X 10) per well was added³One) and 100 μ L of effector cells (E/T ═ 1:2, 1:5, 1:10, E/T is the ratio of target cells to effector cells T), while a natural release control well (100 μ L of target cells +100 μ L of medium) and a maximum release well (100 μ L of target cells +100 μ L of 2% SDS) were established; standing at 37 deg.C and 5% CO₂The culture was incubated for 4 h. Taking out, sucking out supernatant of each hole by using a pipettor, centrifuging to take 100 mu L of supernatant, and measuring cpm value by using a gamma counter;

c. and (4) calculating a result: according to the formula⁵¹Natural Cr release rate and T cell activity:

wherein: general requirements⁵¹Natural release rate of Cr<10％。

The results of the tests of the respective experimental groups are shown in table 8 and fig. 13.

TABLE 8 Effect of pluripotent Stem cells or derivatives thereof on T cell killing of tumor cells in each experimental group

Grouping	⁵¹Average value of Cr Release (%)	Deviation (+/-)	Independent sample T test (. p)<0.01)
				N (control)	29.742	0.422	-
A1	59.967	1.065	*
				A2	60.818	0.932	*
A3	60.561	1.954	*
				A4	58.840	0.929	*
A5	59.189	0.627	*
				A6	60.661	1.589	*
B1	59.578	1.169	*
				B2	58.839	0.541	*
B3	59.468	1.006	*
				B4	59.631	1.763	*

Through the experiments, the stem cells expressing the CD38 antibody and the derivatives thereof prepared by the invention can be proved to be capable of effectively blocking and activating T cells to play an anti-tumor role.

(7) Application of pluripotent stem cells expressing CD38 antibody in treatment of various tumors

Immune compatible cells (MSCs) from both B2M and CIITA gene knock-out protocol groups (a4) were selected for testing.

In the humanized NSG mouse tumor model, experimental cells (hPSCs and hPSCs-derived derivatives (hPSCs-MSCs, hPSCs-NSCs, hPSCs-EBs) capable of expressing the CD38 antibody) were injected into each group of experimental cells, and the effect of the experimental cells on the treatment of RCC renal carcinoma, MM myeloma, and NIC lung cancer was observed. To avoid the problem of immune compatibility, the immune cells used were derived from the same person as hPSCs and hPSCs derived derivatives, and an immune compatibility protocol of B2M and CIITA gene knock-out was used.

The method comprises the following specific steps:

in humanized NSG mice (The Jackson Laboratory (JAX)), The right axilla was injected subcutaneously with 5X 10⁶Tumor (RCC renal carcinoma, MC colon carcinoma, NIC lung carcinoma) cells until the tumor grows to 60mm³At size, tail vein injection of 200uL PBS (containing human immune cells and 10 mg)⁶The expressed CD38 antibody) for tumor treatment, wherein only the group containing human immune cells was injected as a control group. Mice were sacrificed after 20 days and tumor sizes were compared between groups and statistical analysis of differences was performed.

The N (control) group refers to the NSG mouse tumor model without injection of experimental cells.

The results are shown in table 9 and fig. 14.

TABLE 9 antitumor Effect of pluripotent Stem cells or derivatives thereof in Each test group

Through the experiments, the stem cells expressing the CD38 antibody or the derivatives thereof prepared by the invention can be proved to be capable of effectively combining with CD38 to play an anti-tumor role.

(8) Immune compatibility testing of pluripotent stem cells expressing CD38 antibody

By utilizing the characteristic of low immunogenicity of the MSCs, hPSCs capable of expressing CD38 antibodies are injected into humanized NSG mouse tumor models to achieve immune compatibility with the MSCs, and the effect of tumor (MM myeloma) treatment is observed. Wherein the immunocyte and the MSCs derived from hPSCs are derived from non-identical persons.

The control group is an NSG mouse tumor model without MSCs cell injection;

the Dox addition group is: mice were continuously fed with 0.5mg/mL of Dox in the mouse diet starting from injection of CD38 antibody expressing cells until the end of the experiment. The sequences (immune compatible molecules) into which Dox can be knocked in by using an inducer exhibit an effect of not expressing.

The results of the reversible expression test for the immune-compatible molecule-inducible expression panel are shown in table 10 and fig. 15.

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

The above experiments show that: MSCs expressing only suppressive factors (group 2), which have low immunogenicity and can exist in xenobiotics for a certain period of time, can exert a certain tumor therapeutic effect, while those that have been processed by immuno-compatible engineering (groups 3-11, including constitutive and reversible inducible immuno-compatibility), have better immuno-compatible effects than MSCs that have not been processed by immuno-compatible engineering, which exist in vivo for a longer period of time (or can coexist for a longer period of time), and exert a better tumor therapeutic effect. And the group 5 is a B2M and CIITA gene knockout group, which completely eliminates the influence of HLA-I and HLA-II molecules, so that the tumor treatment effect is optimal. 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 shown to have abolished the immune compatibility effect by administering Dox inducer (always used) to the mice at the same time as the injection of the expression blocker cells into the mice, and the cells existed in vivo for a period of time comparable to that of the MSCs without immune compatibility modification, and the tumor treatment effect was comparable to that of the MSCs without immune compatibility modification.

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 institute (Guangzhou) Co., Ltd

Wang Linli

<120> pluripotent stem cell expressing CD38 antibody, and derivative and application thereof

<130>

<160> 184

<170> PatentIn version 3.5

<210> 1

<211> 1356

<212> DNA

<213> Artificial sequence

<400> 1

gaggtgcagc tgctggagag cggcggcggc ctggtgcagc ccggcggcag cctgaggctg 60

agctgcgccg tgagcggctt caccttcaac agcttcgcca tgagctgggt gaggcaggcc 120

cccggcaagg gcctggagtg ggtgagcgcc atcagcggca gcggcggcgg cacctactac 180

gccgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgag ggccgaggac accgccgtgt acttctgcgc caaggacaag 300

atcctgtggt tcggcgagcc cgtgttcgac tactggggcc agggcaccct ggtgaccgtg 360

agcagcgcca gcaccaaggg ccccagcgtg ttccccctgg cccccagcag caagagcacc 420

agcggcggca ccgccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc 480

gtgagctgga acagcggcgc cctgaccagc ggcgtgcaca ccttccccgc cgtgctgcag 540

agcagcggcc tgtacagcct gagcagcgtg gtgaccgtgc ccagcagcag cctgggcacc 600

cagacctaca tctgcaacgt gaaccacaag cccagcaaca ccaaggtgga caagagggtg 660

gagcccaaga gctgcgacaa gacccacacc tgccccccct gccccgcccc cgagctgctg 720

ggcggcccca gcgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcagg 780

acccccgagg tgacctgcgt ggtggtggac gtgagccacg aggaccccga ggtgaagttc 840

aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcccag ggaggagcag 900

tacaacagca cctacagggt ggtgagcgtg ctgaccgtgc tgcaccagga ctggctgaac 960

ggcaaggagt acaagtgcaa ggtgagcaac aaggccctgc ccgcccccat cgagaagacc 1020

atcagcaagg ccaagggcca gcccagggag ccccaggtgt acaccctgcc ccccagcagg 1080

gaggagatga ccaagaacca ggtgagcctg acctgcctgg tgaagggctt ctaccccagc 1140

gacatcgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 1200

cccgtgctgg acagcgacgg cagcttcttc ctgtacagca agctgaccgt ggacaagagc 1260

aggtggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 1320

tacacccaga agagcctgag cctgagcccc ggcaag 1356

<210> 2

<211> 642

<212> DNA

<213> Artificial sequence

<400> 2

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gagggccacc 60

ctgagctgca gggccagcca gagcgtgagc agctacctgg cctggtacca gcagaagccc 120

ggccaggccc ccaggctgct gatctacgac gccagcaaca gggccaccgg catccccgcc 180

aggttcagcg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctggagccc 240

gaggacttcg ccgtgtacta ctgccagcag aggagcaact ggccccccac cttcggccag 300

ggcaccaagg tggagatcaa gaggaccgtg gccgccccca gcgtgttcat cttccccccc 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420

cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600

ctgagcagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642

<210> 3

<211> 21

<212> DNA

<213> human

<400> 3

gggagcagag aattctctta t 21

<210> 4

<211> 21

<212> DNA

<213> human

<400> 4

ggagcagaga attctcttat c 21

<210> 5

<211> 21

<212> DNA

<213> human

<400> 5

gagcagagaa ttctcttatc c 21

<210> 6

<211> 21

<212> DNA

<213> human

<400> 6

gctacctgga gcttcttaac a 21

<210> 7

<211> 21

<212> DNA

<213> human

<400> 7

ggagcttctt aacagcgatg c 21

<210> 8

<211> 21

<212> DNA

<213> human

<400> 8

gggtctccag tatattcatc t 21

<210> 9

<211> 21

<212> DNA

<213> human

<400> 9

gcctcctgat gcacatgtac t 21

<210> 10

<211> 21

<212> DNA

<213> human

<400> 10

ggaagacctg ggaaagcttg t 21

<210> 11

<211> 21

<212> DNA

<213> human

<400> 11

ggctaagctt gtacaataac t 21

<210> 12

<211> 21

<212> DNA

<213> human

<400> 12

gcggaatgaa ccacatcttg c 21

<210> 13

<211> 21

<212> DNA

<213> human

<400> 13

ggccttctct gaaggacatt g 21

<210> 14

<211> 21

<212> DNA

<213> human

<400> 14

ggactcaatg cactgacatt g 21

<210> 15

<211> 21

<212> DNA

<213> human

<400> 15

ggtacccact gctctggtta t 21

<210> 16

<211> 21

<212> DNA

<213> human

<400> 16

gctcccactc catgaggtat t 21

<210> 17

<211> 21

<212> DNA

<213> human

<400> 17

ggtatttctt cacatccgtg t 21

<210> 18

<211> 21

<212> DNA

<213> human

<400> 18

aggagacacg gaatgtgaag g 21

<210> 19

<211> 21

<212> DNA

<213> human

<400> 19

gctcccactc catgaggtat t 21

<210> 20

<211> 21

<212> DNA

<213> human

<400> 20

ggtatttcta cacctccgtg t 21

<210> 21

<211> 21

<212> DNA

<213> human

<400> 21

ggaccggaac acacagatct a 21

<210> 22

<211> 21

<212> DNA

<213> human

<400> 22

accggaacac acagatctac a 21

<210> 23

<211> 21

<212> DNA

<213> human

<400> 23

ggaacacaca gatctacaag g 21

<210> 24

<211> 21

<212> DNA

<213> human

<400> 24

gaacacacag atctacaagg c 21

<210> 25

<211> 21

<212> DNA

<213> human

<400> 25

ttcttacttc cctaatgaag t 21

<210> 26

<211> 21

<212> DNA

<213> human

<400> 26

aagttaagaa cctgaatata a 21

<210> 27

<211> 21

<212> DNA

<213> human

<400> 27

aacctgaata taaatttgtg t 21

<210> 28

<211> 21

<212> DNA

<213> human

<400> 28

acctgaatat aaatttgtgt t 21

<210> 29

<211> 21

<212> DNA

<213> human

<400> 29

aagcgttgat ggattaatta a 21

<210> 30

<211> 21

<212> DNA

<213> human

<400> 30

agcgttgatg gattaattaa a 21

<210> 31

<211> 21

<212> DNA

<213> human

<400> 31

gggtctggtg ggcatcatta t 21

<210> 32

<211> 21

<212> DNA

<213> human

<400> 32

ggtctggtgg gcatcattat t 21

<210> 33

<211> 21

<212> DNA

<213> human

<400> 33

gcatcattat tgggaccatc t 21

<210> 34

<211> 21

<212> DNA

<213> human

<400> 34

gcacatggag gtgatggtgt t 21

<210> 35

<211> 21

<212> DNA

<213> human

<400> 35

ggaggtgatg gtgtttctta g 21

<210> 36

<211> 21

<212> DNA

<213> human

<400> 36

gagaagatca ctgaagaaac t 21

<210> 37

<211> 21

<212> DNA

<213> human

<400> 37

gctttaatgg ctttacaaag c 21

<210> 38

<211> 21

<212> DNA

<213> human

<400> 38

ggctttacaa agctggcaat a 21

<210> 39

<211> 21

<212> DNA

<213> human

<400> 39

gctttacaaa gctggcaata t 21

<210> 40

<211> 21

<212> DNA

<213> human

<400> 40

gctccgtact ctaacatcta g 21

<210> 41

<211> 21

<212> DNA

<213> human

<400> 41

gatgaccaca ttcaaggaag a 21

<210> 42

<211> 21

<212> DNA

<213> human

<400> 42

gaccacattc aaggaagaac t 21

<210> 43

<211> 21

<212> DNA

<213> human

<400> 43

gctttcctgc ttggcagtta t 21

<210> 44

<211> 21

<212> DNA

<213> human

<400> 44

ggcagttatt cttccacaag a 21

<210> 45

<211> 21

<212> DNA

<213> human

<400> 45

gcagttattc ttccacaaga g 21

<210> 46

<211> 21

<212> DNA

<213> human

<400> 46

gcgtaagtct gagtgtcatt t 21

<210> 47

<211> 21

<212> DNA

<213> human

<400> 47

gacaatttaa ggaagaatct t 21

<210> 48

<211> 21

<212> DNA

<213> human

<400> 48

ggccatagtt ctccctgatt g 21

<210> 49

<211> 21

<212> DNA

<213> human

<400> 49

gccatagttc tccctgattg a 21

<210> 50

<211> 21

<212> DNA

<213> human

<400> 50

gcagatgacc acattcaagg a 21

<210> 51

<211> 21

<212> DNA

<213> human

<400> 51

gatgaccaca ttcaaggaag a 21

<210> 52

<211> 21

<212> DNA

<213> human

<400> 52

gaccacattc aaggaagaac c 21

<210> 53

<211> 21

<212> DNA

<213> human

<400> 53

gctttgtcag gaccaggttg t 21

<210> 54

<211> 21

<212> DNA

<213> human

<400> 54

gaccaggttg ttactggttc a 21

<210> 55

<211> 21

<212> DNA

<213> human

<400> 55

gaagcctcac agctttgatg g 21

<210> 56

<211> 21

<212> DNA

<213> human

<400> 56

gatggcagtg cctcatcttc a 21

<210> 57

<211> 21

<212> DNA

<213> human

<400> 57

ggcagtgcct catcttcaac t 21

<210> 58

<211> 21

<212> DNA

<213> human

<400> 58

gcagcaggat aagtatgagt g 21

<210> 59

<211> 21

<212> DNA

<213> human

<400> 59

gcaggataag tatgagtgtc a 21

<210> 60

<211> 21

<212> DNA

<213> human

<400> 60

ggttcctgca cagagacatc t 21

<210> 61

<211> 21

<212> DNA

<213> human

<400> 61

gcacagagac atctataacc a 21

<210> 62

<211> 21

<212> DNA

<213> human

<400> 62

gagacatcta taaccaagag g 21

<210> 63

<211> 21

<212> DNA

<213> human

<400> 63

gagtactgga acagccagaa g 21

<210> 64

<211> 21

<212> DNA

<213> human

<400> 64

gctttcctgc ttggctctta t 21

<210> 65

<211> 21

<212> DNA

<213> human

<400> 65

ggctcttatt cttccacaag a 21

<210> 66

<211> 21

<212> DNA

<213> human

<400> 66

gctcttattc ttccacaaga g 21

<210> 67

<211> 21

<212> DNA

<213> human

<400> 67

ggatgtggaa cccacagata c 21

<210> 68

<211> 21

<212> DNA

<213> human

<400> 68

gatgtggaac ccacagatac a 21

<210> 69

<211> 21

<212> DNA

<213> human

<400> 69

gtggaaccca cagatacaga g 21

<210> 70

<211> 21

<212> DNA

<213> human

<400> 70

ggaacccaca gatacagaga g 21

<210> 71

<211> 21

<212> DNA

<213> human

<400> 71

gagccaactg tattgcctat t 21

<210> 72

<211> 21

<212> DNA

<213> human

<400> 72

agccaactgt attgcctatt t 21

<210> 73

<211> 21

<212> DNA

<213> human

<400> 73

gccaactgta ttgcctattt g 21

<210> 74

<211> 21

<212> DNA

<213> human

<400> 74

gggtagcaac tgtcaccttg a 21

<210> 75

<211> 21

<212> DNA

<213> human

<400> 75

ggatttcgtg ttccagttta a 21

<210> 76

<211> 21

<212> DNA

<213> human

<400> 76

gcatgtgcta cttcaccaac g 21

<210> 77

<211> 21

<212> DNA

<213> human

<400> 77

gcgtcttgtg accagataca t 21

<210> 78

<211> 21

<212> DNA

<213> human

<400> 78

gcttatgcct gcccagaatt c 21

<210> 79

<211> 21

<212> DNA

<213> human

<400> 79

gcaggaaatc actgcagaat g 21

<210> 80

<211> 21

<212> DNA

<213> human

<400> 80

gctcagtgca ttggccttag a 21

<210> 81

<211> 21

<212> DNA

<213> human

<400> 81

ggtgagtgct gtgtaaataa g 21

<210> 82

<211> 21

<212> DNA

<213> human

<400> 82

gacatatata gtgatccttg g 21

<210> 83

<211> 21

<212> DNA

<213> human

<400> 83

ggaaagtcac atcgatcaag a 21

<210> 84

<211> 21

<212> DNA

<213> human

<400> 84

gctcacagtc atcaattata g 21

<210> 85

<211> 21

<212> DNA

<213> human

<400> 85

gccctgaaga cagaatgttc c 21

<210> 86

<211> 21

<212> DNA

<213> human

<400> 86

gcggaccatg tgtcaactta t 21

<210> 87

<211> 21

<212> DNA

<213> human

<400> 87

ggaccatgtg tcaacttatg c 21

<210> 88

<211> 21

<212> DNA

<213> human

<400> 88

gcgtttgtac agacgcatag a 21

<210> 89

<211> 21

<212> DNA

<213> human

<400> 89

ggctggctaa cattgctata t 21

<210> 90

<211> 21

<212> DNA

<213> human

<400> 90

gctggctaac attgctatat t 21

<210> 91

<211> 21

<212> DNA

<213> human

<400> 91

ggaccaggtc acatgtgaat a 21

<210> 92

<211> 21

<212> DNA

<213> human

<400> 92

ggaaaggtct gaggatattg a 21

<210> 93

<211> 21

<212> DNA

<213> human

<400> 93

ggcagattag gattccattc a 21

<210> 94

<211> 21

<212> DNA

<213> human

<400> 94

gcctgatagg acccatattc c 21

<210> 95

<211> 21

<212> DNA

<213> human

<400> 95

gcatccaata gacgtcattt g 21

<210> 96

<211> 21

<212> DNA

<213> human

<400> 96

gcgtcactgg cacagatata a 21

<210> 97

<211> 21

<212> DNA

<213> human

<400> 97

gctgtcacat aataagctaa g 21

<210> 98

<211> 21

<212> DNA

<213> human

<400> 98

gctaaggaag acagtatata g 21

<210> 99

<211> 21

<212> DNA

<213> human

<400> 99

gggatttcta aggaaggatg c 21

<210> 100

<211> 21

<212> DNA

<213> human

<400> 100

ggagttgaag agcagagatt c 21

<210> 101

<211> 21

<212> DNA

<213> human

<400> 101

gccagtgaac acttaccata g 21

<210> 102

<211> 21

<212> DNA

<213> human

<400> 102

gcttctctga agtctcattg a 21

<210> 103

<211> 21

<212> DNA

<213> human

<400> 103

ggctgcaact aacttcaaat a 21

<210> 104

<211> 21

<212> DNA

<213> human

<400> 104

ggatggattt gattatgatc c 21

<210> 105

<211> 21

<212> DNA

<213> human

<400> 105

ggaccttgga acaatggatt g 21

<210> 106

<211> 21

<212> DNA

<213> human

<400> 106

gctaattctt gctgaacttc t 21

<210> 107

<211> 21

<212> DNA

<213> human

<400> 107

gctgaacttc ttcatgtatg t 21

<210> 108

<211> 21

<212> DNA

<213> human

<400> 108

gcctcatctc tttgttctaa a 21

<210> 109

<211> 21

<212> DNA

<213> human

<400> 109

gctctggaga agatatattt g 21

<210> 110

<211> 21

<212> DNA

<213> human

<400> 110

gctcttgagg gaactaatag a 21

<210> 111

<211> 21

<212> DNA

<213> human

<400> 111

gggacggcat taatgtattc a 21

<210> 112

<211> 21

<212> DNA

<213> human

<400> 112

ggacaaacat gcaaactata g 21

<210> 113

<211> 21

<212> DNA

<213> human

<400> 113

gcagcaacca gctaccattc t 21

<210> 114

<211> 21

<212> DNA

<213> human

<400> 114

gcagttctgt tgccactctc t 21

<210> 115

<211> 21

<212> DNA

<213> human

<400> 115

gggagagttc atccaggaaa t 21

<210> 116

<211> 21

<212> DNA

<213> human

<400> 116

ggagagttca tccaggaaat t 21

<210> 117

<211> 21

<212> DNA

<213> human

<400> 117

gagagttcat ccaggaaatt a 21

<210> 118

<211> 21

<212> DNA

<213> human

<400> 118

gcctgtcaaa gagagagagc a 21

<210> 119

<211> 21

<212> DNA

<213> human

<400> 119

gctcagcttc gtactgagtt c 21

<210> 120

<211> 21

<212> DNA

<213> human

<400> 120

gcttcacaga actacagaga g 21

<210> 121

<211> 21

<212> DNA

<213> human

<400> 121

gcatctactg gacaaagtat t 21

<210> 122

<211> 21

<212> DNA

<213> human

<400> 122

ggctgaatta cccatgcttt a 21

<210> 123

<211> 21

<212> DNA

<213> human

<400> 123

gctgaattac ccatgcttta a 21

<210> 124

<211> 21

<212> DNA

<213> human

<400> 124

gggttggttt atccaggaat a 21

<210> 125

<211> 21

<212> DNA

<213> human

<400> 125

ggatcagaag agaagccaac g 21

<210> 126

<211> 21

<212> DNA

<213> human

<400> 126

ggttcaccat ccaggtgttc a 21

<210> 127

<211> 21

<212> DNA

<213> human

<400> 127

gctctcttct ctggaactaa c 21

<210> 128

<211> 21

<212> DNA

<213> human

<400> 128

gctagagtga ctccatctta a 21

<210> 129

<211> 21

<212> DNA

<213> human

<400> 129

gctgaccacc aattataatt g 21

<210> 130

<211> 21

<212> DNA

<213> human

<400> 130

gcagaatatt taaggccata c 21

<210> 131

<211> 21

<212> DNA

<213> human

<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> 23

<212> DNA

<213> human

<400> 164

cgcgagcaca gctaaggcca cgg 23

<210> 165

<211> 23

<212> DNA

<213> human

<400> 165

actctctctt tctggcctgg agg 23

<210> 166

<211> 23

<212> DNA

<213> human

<400> 166

acccagcagg gcgtggagcc agg 23

<210> 167

<211> 23

<212> DNA

<213> human

<400> 167

gtcagagccc caaggtaaaa agg 23

<210> 168

<211> 253

<212> DNA

<213> Artificial sequence

<400> 168

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> 169

<211> 9

<212> DNA

<213> Artificial sequence

<400> 169

ttcaagaga 9

<210> 170

<211> 6

<212> DNA

<213> Artificial sequence

<400> 170

tttttt 6

<210> 171

<211> 119

<212> DNA

<213> Artificial sequence

<400> 171

gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta 60

cttcttcagg ttaacccaac agaaggctaa agaaggtata ttgctgttga cagtgagcg 119

<210> 172

<211> 19

<212> DNA

<213> Artificial sequence

<400> 172

tagtgaagcc acagatgta 19

<210> 173

<211> 119

<212> DNA

<213> Artificial sequence

<400> 173

tgcctactgc ctcggacttc aaggggctac tttaggagca attatcttgt ttactaaaac 60

tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat ggtataaat 119

<210> 174

<211> 686

<212> DNA

<213> Artificial sequence

<400> 174

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> 175

<211> 22

<212> DNA

<213> Artificial sequence

<400> 175

ccatagctca gtctggtcta tc 22

<210> 176

<211> 22

<212> DNA

<213> Artificial sequence

<400> 176

tcaggatgat ctggacgaag ag 22

<210> 177

<211> 20

<212> DNA

<213> Artificial sequence

<400> 177

ccggtcctgg actttgtctc 20

<210> 178

<211> 20

<212> DNA

<213> Artificial sequence

<400> 178

ctcgacatcg gcaaggtgtg 20

<210> 179

<211> 20

<212> DNA

<213> Artificial sequence

<400> 179

cgcattggag tcgctttaac 20

<210> 180

<211> 24

<212> DNA

<213> Artificial sequence

<400> 180

cgagctgcaa gaactcttcc tcac 24

<210> 181

<211> 23

<212> DNA

<213> Artificial sequence

<400> 181

cacggcactt acctgtgttc tgg 23

<210> 182

<211> 23

<212> DNA

<213> Artificial sequence

<400> 182

cagtacaggc atccctgtga aag 23

<210> 183

<211> 590

<212> DNA

<213> Artificial sequence

<400> 183

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> 184

<211> 60

<212> DNA

<213> Artificial sequence

<400> 184

atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg 60

Claims

1. A pluripotent stem cell or derivative thereof comprising a CD38 antibody sequence, preferably inserted into the genome of the pluripotent stem cell or derivative thereof, wherein the CD38 antibody sequence is a CD38 antibody sequence.

2. A pluripotent stem cell or derivative thereof comprising a CD38 antibody sequence, preferably inserted into the genome of the pluripotent stem cell or derivative thereof;

the B2M gene and/or CIITA gene of the genome of the pluripotent stem cell or the derivative thereof is knocked out.

3. A pluripotent stem cell or derivative thereof comprising a CD38 antibody sequence, preferably inserted into the genome of the pluripotent stem cell or derivative thereof;

the pluripotent stem cells or the derivatives thereof also comprise an immune compatible molecule expression sequence, and the immune compatible molecule is used for regulating and controlling the expression of genes related to immune response in the pluripotent stem cells or the derivatives thereof; the immune compatible molecule expression sequence is preferably inserted in the genome of the pluripotent stem cell or a derivative thereof.

4. A pluripotent stem cell or derivative thereof comprising a CD38 antibody sequence, preferably inserted into the genome of the pluripotent stem cell or derivative thereof;

the pluripotent stem cells or the derivatives thereof also comprise an immune compatible molecule expression sequence, and the immune compatible molecule is used for regulating and controlling the expression of genes related to immune response in the pluripotent stem cells or the derivatives thereof; the immune compatible molecule expression sequence is preferably inserted into the genome of the pluripotent stem cell or a derivative thereof;

5. The pluripotent stem cell or the derivative thereof according to claim 4, wherein the inducible gene expression system is at least one of a Tet-Off system and a dimer inducible expression system.

6. The pluripotent stem cell or derivative thereof of claim 3 or 4, wherein the immune-compatible molecule comprises one or more of:

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

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

8. The pluripotent stem cell or the derivative thereof according to claim 6,

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

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

9. The pluripotent stem cell or the derivative thereof according to claim 3 or 4, wherein at least one of an shRNA processing complex-associated gene, an miRNA processing complex-associated gene, and an anti-interferon effector molecule is further introduced into the genome of the pluripotent stem cell or the derivative thereof.

10. The pluripotent stem cell or the derivative thereof according to claim 9, wherein the shRNA-processing complex-associated gene or miRNA-processing complex-associated gene comprises at least one of Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2), PACT (PRKRA), DGCR 8; the anti-interferon effector molecule is preferably shRNA and/or shRNA-miR targeting at least one of PKR, 2-5As, IRF-3 and IRF-7.

11. The pluripotent stem cell or the derivative thereof according to claim 10,

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

12. The pluripotent stem cell or the derivative thereof according to claim 6 or 9, wherein the pluripotent stem cell or the derivative thereof,

the shRNA expression framework is as follows: the gene sequence sequentially comprises an shRNA target sequence, a stem-loop sequence, a reverse complementary sequence of the shRNA target sequence and Poly T from 5 'to 3';

shRNA-miR expression framework: and replacing the shRNA target sequence with the shRNA-miR target sequence to obtain the target sequence.

13. The pluripotent stem cell or the derivative thereof according to claim 12, wherein the stem-loop sequence in the shRNA expression framework is 3 to 9 bases in length; the length of the Poly T is 5-6 bases.

14. The pluripotent stem cell or the derivative thereof according to any one of claims 1 to 4, wherein the CD38 antibody sequence, the immune-compatible molecule expression sequence or the inducible gene expression system is inserted into a safe site of the genome of the pluripotent stem cell or the derivative thereof.

15. The pluripotent stem cell or the derivative thereof of claim 14, wherein the genomic safety site comprises one or more of an AAVS1 safety site, an eGSH safety site, and an H11 safety site.

16. The pluripotent stem cell or the derivative thereof according to any one of claims 1 to 4, wherein the pluripotent stem cell comprises an embryonic stem cell, an embryonic germ cell, an embryonic carcinoma cell, or an induced pluripotent stem cell.

17. The pluripotent stem cell or the derivative thereof according to any one of claims 1 to 4, wherein the derivative of the pluripotent stem cell comprises 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 the derivative thereof according to any one of claims 1 to 4, wherein the CD38 antibody has a heavy chain sequence as shown in SEQ ID No.1 and a light chain sequence as shown in SEQ ID No. 2.

19. Use of the pluripotent stem cell or derivative thereof according to any one of claims 1 to 18 for the preparation of a medicament for treating a tumor with high expression of CD 38.

20. A formulation comprising the pluripotent stem cells or derivatives thereof of any of claims 1 to 18.