CN114657139A - Pluripotent stem cell expressing LAG-3 targeted inhibitory factor, derivative and application thereof - Google Patents

Abstract

The invention discloses a pluripotent stem cell expressing LAG-3 targeted inhibitory factor, and a derivative and application thereof. An expression sequence of a LAG-3 inhibitor is introduced into a genome of the pluripotent stem cell or a derivative thereof, wherein the LAG-3 inhibitor is at least one of shRNA and/or shRNA-miR of a target LAG-3. After the LAG-3 inhibitor expressed by the pluripotent stem cells or the derivatives thereof is encapsulated by exosomes, the exosomes carry the LAG-3 inhibitor to be combined with target cells and release the LAG-3 inhibitor contained in the exosomes, so that the expression of LAG-3 is inhibited, the immunosuppression is relieved, the immune system is activated, and tumor cells are effectively eliminated.

Description

A pluripotent stem cell expressing LAG-3 targeting inhibitor and its derivative and application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a pluripotent stem cell expressing a LAG-3 targeting inhibitory factor, a derivative thereof and an application thereof.

Background technique

Stem cells are a type of "seed" cells that have the ability to self-renew and differentiate into specific functional somatic cells. They have the potential to regenerate into various tissues and organs and the human body. core and irreplaceable role. According to the degree of stem cell characteristics, stem cells are mainly divided into: totipotent stem cells (Totipotent stem cells), pluripotent stem cells (PSCs) and adult stem cells (adult stem cells). Among them, pluripotent stem cells (PSCs) have almost unlimited self-renewal ability and the potential to develop and differentiate into organs, tissues and cells of all germ layers in the embryo under normal developmental conditions. Typical PSCs mainly include embryonic stem cells (ESCs). ), embryonic germ cells (EGCs), embryonic carcinoma cells (ECCs), and induced pluripotentstem cells (iPSCs). It can pass ethical restrictions to a certain extent, so it has a very far-reaching and wide-ranging application prospect.

Lymphocyte activation gene-3 (LAG-3, CD223) belongs to the immunoglobulin superfamily and consists of three parts: extracellular domain, transmembrane domain and cytoplasmic domain. The gene of LAG-3 is located on chromosome 12 (12P13), which is similar to the location and structure of CD4 molecule on chromosome. Mainly distributed in activated T lymphocytes, NK cells and dendritic cells, it is a negative immune regulator molecule, which has the functions of maintaining internal environment stability and participating in immune regulation, and is closely related to the occurrence and development of tumors. Inhibition of LAG-3 allows T cells to regain cytotoxicity, thereby enhancing tumor killing. Simultaneous inhibition of LAG-3 can also reduce the function of regulatory T cells to suppress immune responses. Therefore, LAG-3 is considered a more attractive target than other immune checkpoint proteins. FGL1 is a major ligand of LAG-3 immunosuppression. When FGL1 protein is used to bind the LAG-3 receptor on the surface of T cells, T cell proliferation is inhibited and immune activity is also affected. The expression of FGL1 was significantly up-regulated in various solid tumors, with the highest proportion in lung cancer.

Therefore, it is of great significance to develop a pluripotent stem cell or its derivative that can express LAG-3 inhibitory factor in humans.

However, the conception or establishment of an autologous iPSCs cell bank or an immune-matched PSCs cell bank requires enormous financial, material and human resources. The molecular immunological basis of organ, tissue or cell transplantation of allogeneic donors and recipients is mainly based on the classical major histocompatibility complex MHC-I and MHC-II (also known as HLA-I, HLA-II in humans) 's matching. As of June 2019, more than 20,000 HLA system alleles have been identified and named, and only classical HLA-A, B, and C alleles have more than 5,000 alleles. These classical HLA-I/II The number of possible random combinations of alleles will be astronomical, and the number of combinations will increase with the discovery of new alleles, bringing great importance to the tissue matching and donor selection of organs, tissues, and cells before transplantation. This is a huge obstacle, and it also brings huge difficulties to the construction of a population-covering immune-matched PSCs cell bank.

Therefore, the construction of alloimmune compatible universal PSCs is imminent. In recent years, there have been many reports that by knocking out B2M, CIITA and other genes, the expression of HLA-I and HLA-II cells surface or their own genes is lost, so that cells have immune tolerance or escape T/B cell-specific immune responses, resulting in The immune-compatible universal PSCs have laid an important foundation for the wider application of universal PSC-derived cells, tissues and organs. It has also been reported that cells overexpress CTLA4-Ig and PD-L1 to inhibit allogeneic immune rejection. Recently, it has been reported that while knocking out B2M and CIITA, CD47 is knocked in, so that the cells can escape from the specific immune response, but also have immune tolerance or escape the innate immune response of NK cells, so that the cells have More comprehensive and stronger immune compatibility features. However, these regimens are either not fully immune compatible, and allogeneic immune rejection still occurs through other means; or they completely eliminate the allogeneic immune rejection response, but the cells of the donor-derived graft lose their antigen-presenting ability at the same time. capacity, which poses a great risk to the receptor for diseases such as tumorigenicity and viral infection.

For this reason, it has also been reported that, instead of directly knocking out B2M, HLA-A, HLA-B or CIITA are knocked out together, while retaining HLA-C, and constructing 12 HLA-C immune systems that cover more than 90% of the population. Matching antigens, so that the transplanted cells still have a certain degree of antigen presentation function, and at the same time can inhibit the innate immune response of NK cells through HLA-C. However, the types of antigens presented by HLA-I antigens have been reduced by more than two-thirds, and the integrity of the antigens that can be presented has been greatly and irreversibly reduced. For various tumors, viruses and other disease antigens The presentation is highly biased, and still retains a considerable degree of risk of tumorigenicity and viral infection, and its pathogenic risk is higher when CIITA is knocked out at the same time; secondly, 12 high-frequency immune matching The ethnic differences of HLA-C antigens are very large. Through our verification and calculation, some regions can only account for 70% of the proportion. However, China, India and other populous countries currently do not have authoritative large-scale HLA data display. The use of type PSCs is still subject to the huge matching vacancy test; thirdly, this method will undergo several iterations of gene editing work. Based on at least two rounds of single-cell isolation and culture for each gene editing, the whole process requires at least more than six rounds of single-cell isolation and culture. Cell isolation and culture, these processes are inevitably and highly likely to cause various unpredictable mutations in cells due to multiple off-target gene editing or chromatin instability or due to a large number of single-cell passages and proliferations, and then induce various problems such as carcinogenesis and metabolic diseases . It can be seen that this type of immunocompatibility program is also an expedient measure in the "transition period", and there are still many problems that have not been better resolved.

Exosomes are disc-shaped vesicles with a diameter of 40-100 nm. A variety of cells can secrete exosomes under normal and pathological conditions. It is mainly derived from the multivesicular bodies formed by the invagination of intracellular lysosomal particles, and is released into the extracellular matrix after the fusion of the outer membrane of the multivesicles with the cell membrane. When they are secreted from host cells into recipient cells, exosomes can regulate the biological activity of recipient cells through the proteins, nucleic acids, lipids, etc. they carry. At present, attempts have been made to use exosomes to carry siRNA, chemical small molecule drugs, etc. for gene therapy and tumor therapy.

In addition, some people have designed suicide genes to induce killing after the donor tissue and cells become diseased. The consequences of doing so will cause severe tissue necrosis, cytokine storms and other unpredictable disease risk problems, and such designed cells Another problem is that after killing, there will be no suitable donor cells, tissues and organs.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a pluripotent stem cell or a derivative thereof.

Another object of the present invention is to provide the application of the above-mentioned pluripotent stem cells or derivatives thereof and exosomes secreted by them in the preparation of anti-tumor therapeutic preparations or medicines.

Another object of the present invention is to provide an exosome.

The technical scheme adopted by the present invention is:

A first aspect of the present invention provides:

A pluripotent stem cell or a derivative thereof, the genome of the pluripotent stem cell or its derivative is imported with an expression sequence of a LAG-3 inhibitory factor; the LAG-3 inhibitory factor is in shRNA and shRNA-miR targeting LAG-3 at least one of.

Further, the sequences of the above-mentioned shRNA targeting LAG-3 and shRNA-miR are shown in SEQ ID NO.1.

A second aspect of the present invention provides:

A pluripotent stem cell or a derivative thereof, the genome of the pluripotent stem cell or its derivative is imported with an expression sequence of a LAG-3 inhibitory factor; the LAG-3 inhibitory factor is in shRNA and shRNA-miR targeting LAG-3 at least one of.

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

When the B2M and CIITA genes were knocked out, they completely eliminated the effects of HLA-I and HLA-II molecules, so the tumor treatment effect was the best.

A third aspect of the present invention provides:

A pluripotent stem cell or a derivative thereof, the genome of the pluripotent stem cell or its derivative is imported with an expression sequence of a LAG-3 inhibitory factor; the LAG-3 inhibitory factor is in shRNA and shRNA-miR targeting LAG-3 at least one of.

A first nucleic acid molecule is introduced into the genome of the above-mentioned pluripotent stem cell or its derivative; and a second nucleic acid molecule is introduced into the 3'UTR region of the immune response-related gene in the above-mentioned pluripotent stem cell or its derivative; the above-mentioned first nucleic acid The molecule encodes a small nucleic acid molecule that mediates RNA interference, the small nucleic acid molecule specifically targets the transcription product of the second nucleic acid molecule, and the small nucleic acid molecule does not target any other mRNA or mRNA of the pluripotent stem cell or its derivative. lncRNAs.

In this technical solution, the small nucleic acid molecule encoded by the first nucleic acid molecule can specifically bind to the transcription product of the second nucleic acid molecule introduced into the 3'UTR region of the immune response-related gene, thereby starting the RNA interference program, and the immune response-related gene The mRNA is degraded or silenced, thereby blocking the expression of immune response-related genes, thereby making such cells immune-compatible, which can eliminate or reduce the allogeneic immune rejection response. Furthermore, the RNA interference program acts only on such engineered pluripotent stem cells or derivatives thereof, so that when such cells or derivatives are transplanted into the recipient, the small nucleic acid molecule encoded by the first nucleic acid molecule The RNA interference of the immune response-related gene mediated by the second nucleic acid molecule introduced at the 3'UTR of the immune response-related gene only acts on the donor cell, and does not interfere with the genome of the recipient's cell.

Further, the above-mentioned small nucleic acid molecules include short interfering nucleic acid, short interfering RNA, and double-stranded RNA, preferably at least one of miRNA, shRNA, and shRNA-miR.

Further, the above-mentioned pluripotent stem cells or derivatives thereof are derived from humans; the sequences of the above-mentioned small nucleic acid molecules are random sequences of non-human species that do not target any human mRNA or lncRNA.

The aforementioned small nucleic acid molecules are preferably derived from Caenorhabditis elegans. E.g:

5'-TTGTACTACACAAAAGTACTG-3' (SEQ ID NO. 2);

5'-TCACAACCTCCTAGAAAGAGTAGA-3' (SEQ ID NO. 101).

Further, the above-mentioned second nucleic acid molecule includes at least 3 repeating reverse complement sequences of small nucleic acid molecule sequences, preferably 6-10 repeating reverse complement sequences of small nucleic acid molecule sequences.

Further, an inducible gene expression system is also introduced into the genome of the pluripotent stem cell or its derivative for regulating the expression of the first nucleic acid molecule.

In this technical solution, the inducible gene expression system is regulated by the exogenous inducer, and the opening and closing of the inducible gene expression system is controlled by adjusting the addition amount, duration and type of the exogenous inducer, thereby controlling the small nucleic acid molecule expression.

When the inducible gene expression system is turned on, the normally expressed small nucleic acid molecule specifically binds to the transcription product of the second nucleic acid molecule introduced into the 3'UTR region of the immune response-related gene, thereby initiating the RNA interference program and converting the mRNA of the immune response-related gene Degradation or silencing, thereby blocking the expression of immune response-related genes. Therefore, when such cells or derivatives are transplanted into the recipient, the allogeneic immune rejection response can be eliminated or reduced, and the immunocompatibility between the transplant and the recipient can be improved.

When the graft becomes diseased, the inducible gene expression system can be turned off by adding an exogenous inducer, thereby turning off the expression of small nucleic acid molecules, stopping the interference of small nucleic acid molecules on the mRNA of immune response-related genes, and restoring the expression of immune response-related genes. Normal expression, thereby restoring the antigen-presenting ability of the graft cells, enabling the recipient to clear the diseased graft, thereby improving the clinical safety of such pluripotent stem cells or their derivatives, and greatly expanding their value in clinical applications .

Furthermore, the RNA interference program acts only on such engineered pluripotent stem cells or derivatives thereof, so that when such cells or derivatives are transplanted into the recipient, the small nucleic acid molecule encoded by the first nucleic acid molecule The RNA interference of the immune response-related gene mediated by the second nucleic acid molecule introduced at the 3'UTR of the immune response-related gene only acts on the donor cell, and does not interfere with the genome of the recipient's cell.

A fourth aspect of the present invention provides:

A pluripotent stem cell or a derivative thereof, the genome of the pluripotent stem cell or its derivative is imported with an expression sequence of a LAG-3 inhibitory factor; the LAG-3 inhibitory factor is in shRNA and shRNA-miR targeting LAG-3 at least one of.

The genome of the above-mentioned pluripotent stem cells or their derivatives is also introduced with an expression sequence of at least one immune-compatible molecule, and the above-mentioned immune-compatible molecules are used to regulate the expression of genes related to immune response in the pluripotent stem cells or their derivatives.

In this technical solution, the immune-compatible molecules can regulate the expression of genes related to immune response in pluripotent stem cells or their derivatives, so such pluripotent stem cells or their derivatives have low immunogenicity, and they are transplanted into recipients. It can eliminate or reduce the allogeneic immune rejection response and improve the immune compatibility between the graft and the recipient. The graft can continuously express shRNA/shRNA-miR targeting LAG-3 in the recipient. After these inhibitory factors are encapsulated by exosomes, the exosomes carry them to bind to the target cells and release them, thereby preventing the growth of LAG-3. The LAG-3 pathway is interrupted, immune suppression is relieved, the immune system is activated, and T cell activity is restored, enabling it to effectively remove tumor cells.

Further, an inducible gene expression system is also introduced into the genome of the pluripotent stem cells or their derivatives for regulating the expression of immune compatible molecules.

In this technical solution, the inducible gene expression system is regulated by the exogenous inducer, and the opening and closing of the inducible gene expression system is controlled by adjusting the addition amount, duration and type of the exogenous inducer, and the immune compatible molecules are further controlled. The expression level of the expressed sequence can be reversibly regulated by the immunocompatibility of pluripotent stem cells or their derivatives. When immune-compatible molecules are normally expressed, the expression of genes related to immune response in pluripotent stem cells or their derivatives is inhibited or overexpressed, so that allogeneic cell therapy can eliminate or reduce the allogeneic immune rejection response. , to improve the immune compatibility between donor cells and recipients. When the donor cell becomes diseased, the expression of immune-compatible molecules can be induced to shut down by exogenous inducers, thereby reversibly re-expressing HLA class I molecules on the surface of the donor cell, restoring the antigen-presenting ability of the donor cell, and then accepting the receptor. The immune system increases this risk by recognizing mismatched HLA class I molecules or mutated molecules through cross-HLA class I antigen presentation (antigen presentation/recognition between classically incompatible HLAs), enabling receptors to clear diseased cells. The clinical safety of general-purpose pluripotent stem cells or their derivatives greatly expands their value in clinical applications.

Regarding the third and fourth aspects of the present invention, further, the genes related to immune response include:

(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.

Regarding the fourth aspect of the present invention, further, the immunocompatible molecule includes at least one of the following:

(1) Immune tolerance-related genes, including at least one of CD47 and 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 targeting major histocompatibility complex genes, including HLA-A, HLA-B, HLA-C, HLA-DRA, HLA- at least one of DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB1;

(4) shRNA and/or shRNA-miR targeting major histocompatibility complex-related genes, the major histocompatibility complex-related genes including at least one of B2M and CIITA.

Further, the target sequence of the above-mentioned B2M-targeting shRNA and/or shRNA-miR is selected from one of SEQ ID NO.9 to SEQ ID NO.11;

The target sequence of shRNA and/or shRNA-miR targeting CIITA is selected from one of SEQ ID NO.12 to SEQ ID NO.14;

The target sequence of shRNA and/or shRNA-miR targeting HLA-A is selected from one of SEQ ID NO.15 to SEQ ID NO.17;

The target sequence of shRNA and/or shRNA-miR targeting HLA-B is selected from one of SEQ ID NO.18-SEQ ID NO.20;

The target sequence of shRNA and/or shRNA-miR targeting HLA-C is selected from one of SEQ ID NO.21 to SEQ ID NO.23;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DRA is selected from one of SEQ ID NO.24 to SEQ ID NO.26;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB1 is selected from one of SEQ ID NO.27-SEQ ID NO.29;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB3 is selected from one of SEQ ID NO.30 to SEQ ID NO.31;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB4 is selected from one of SEQ ID NO.32 to SEQ ID NO.34;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB5 is selected from one of SEQ ID NO.35-SEQ ID NO.37;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DQA1 is selected from one of SEQ ID NO.38 to SEQ ID NO.40;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DQB1 is selected from one of SEQ ID NO.41 to SEQ ID NO.43;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DPA1 is selected from one of SEQ ID NO.44 to SEQ ID NO.46;

The target sequence of the shRNA and/or shRNA-miR targeting HLA-DPB1 is selected from one of SEQ ID NO. 47 to SEQ ID NO. 49.

Regarding the first to fourth aspects of the present invention, further, shRNA and/or miRNA processing complex-related genes and/or anti-interferon effector molecules are introduced into the genome of the pluripotent stem cells or their derivatives, Wherein: shRNA and/or miRNA processing complex-related genes include at least one of Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2), PACT (PRKRA), and DGCR8; the above-mentioned anti-interferon effector molecule is the target shRNA and/or shRNA-miR directed to at least one of PKR, 2-5As, IRF-3, and IRF-7.

Further, the target sequence of the above-mentioned PKR-targeting shRNA and/or shRNA-miR is selected from one of SEQ ID NO.50-SEQ ID NO.52;

The target sequence of shRNA and/or shRNA-miR targeting 2-5As is selected from one of SEQ ID NO.53 to SEQ ID NO.61;

The target sequence of shRNA and/or shRNA-miR targeting IRF-3 is selected from one of SEQ ID NO.62-SEQ ID NO.64;

The target sequence of shRNA and/or shRNA-miR targeting IRF-7 is selected from one of SEQ ID NO.65-SEQ ID NO.67.

Regarding the third and fourth aspects of the present invention, the inducible gene expression system includes at least one of the Tet-Off system and the dimer inducible expression system.

When the inducible gene expression system used is the Tet-Off system, the expression of small nucleic acid molecules in cells or derivatives thereof can be controlled by adding an exogenous inducer tetracycline (Doxycycline, Dox). When the pluripotent stem cells or their derivatives are transplanted into the donor, the expression of small nucleic acid molecules can even be gradually reduced by adjusting the amount of Dox added, so that the cells can gradually express low concentrations of immune-related genes To stimulate the donor, so that the donor gradually develops tolerance to the transplanted cells or their derivatives, and finally achieves a stable tolerance. Typically, Dox is added in an amount of 0-100uM.

Regarding the first to fourth aspects of the present invention, further, the genome of the above-mentioned pluripotent stem cells or their derivatives is further introduced into exosome processing and synthesis genes, and the exosome processing and synthesis genes include STEAP3, Syndevan-4 , at least one of L-aspartate oxidase fragment, CD63-L7Ae and Cx43S368A.

The introduction of exosome processing synthetic genes into the genome of stem cells or their derivatives can improve the secretion efficiency of exosomes and their encapsulation efficiency for shRNA, shRNA-miR and processed siRNA.

The dimer-inducible expression system specifically refers to: using a dimerization inducer or dimer to reconstitute an active transcription factor on an inactive fusion protein. The most commonly used system uses the natural product rapamydn or a biologically inactive analog as the dimerized drug. Rapamycin (or analog) homoplasmic protein FKBP12 (the protein that FKBP binds to FK506) and a large serine-threonine protein kinase called FRAP (FRBP-rapamycin-associated protein, or mTOR ( The mammalian target of rapamycin)) has a high affinity and has the function of combining with these two proteins, so the two proteins are brought together as a heterodimer. To regulate target gene transcription, a DNA binding domain is fused to one or more FKBP domains, and a transcriptional repression domain is fused to amino acid 93 of FRAP, called FRB, which is sufficient to bind the FKBP-rapamycin complex. The two fusion proteins dimerized only in the presence of rapamycin. Transcription of genes with sites that bind to the DNA binding region is thus inhibited.

Regarding the first to fourth aspects of the present invention, further, the LAG-3 targeting shRNA and/or shRNA-miR, major histocompatibility complex gene, major histocompatibility complex related The expression framework of genes and anti-interferon effector molecules is as follows:

Described shRNA expression frame: from 5 ' to 3 ' sequentially comprises shRNA sequence, stem-loop sequence, the reverse complement of shRNA sequence, Poly T;

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

shRNA-miR expression framework: obtained by replacing the shRNA target sequence in the shRNA expression framework with the shRNA-miR sequence.

Regarding the first to fourth aspects of the present invention, further, the first nucleic acid molecule, the expression sequence of the LAG-3 inhibitor, the expression sequence of the immunocompatible molecule, the shRNA and/or the miRNA processing complex-related gene , Anti-interferon effector molecules, inducible gene expression systems, and exosome processing synthetic genes are introduced by viral vector interference, non-viral vector transfection or gene editing, and the gene editing method is preferably gene knock-in.

Regarding the first to fourth aspects of the present invention, further, the expression sequence of the LAG-3 inhibitor, the exosome processing synthetic gene, the expression sequence of the immune compatible molecule, the shRNA and/or the miRNA processing complex The introduction site of related genes, anti-interferon effector molecules, inducible gene expression systems, and exosome processing synthetic genes is a genome safety site, preferably one of AAVS1 safety site, eGSH safety site, and H11 safety site. one or more.

Regarding the first to fourth aspects of the present invention, further, the pluripotent stem cells include embryonic stem cells, embryonic germ cells, embryonic cancer cells, or induced pluripotent stem cells;

The pluripotent stem cell derivatives include adult stem cells differentiated from pluripotent stem cells, cells of each germ layer or tissues and organs;

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

A fifth aspect of the present invention provides:

The application of the above-mentioned pluripotent stem cells or derivatives thereof and exosomes secreted in the preparation of anti-tumor therapeutic preparations or medicines.

Further, the application of the above-mentioned pluripotent stem cells or derivatives thereof and the exosomes secreted thereof in the preparation of LAG-3 high expression tumor treatment preparations or medicines.

A sixth aspect of the present invention provides:

An exosome is secreted from the above-mentioned pluripotent stem cells or derivatives thereof.

The beneficial effects of the present invention are:

1. The pluripotent stem cells or derivatives thereof provided in the first aspect of the present invention can be applied to iPSCs or MSCs low-immunogenic cells induced by autologous cells. By introducing the expression sequence of shRNA/shRNA-miR targeting LAG-3 inhibitory factor of LAG-3 into the genome of iPSCs induced by autologous cells, iPSCs can massively express shRNA/shRNA-miR targeting LAG-3, and Encapsulated by exosomes secreted by cells. The exosomes carry these inhibitory factors and bind to target cells and release them, which can effectively inhibit the expression of LAG-3, activate the immune system, and restore the activity of T cells, enabling them to effectively remove tumor cells.

2. The pluripotent stem cells or derivatives thereof provided in the second aspect of the present invention can also be applied to allogeneic cell therapy. Since the B2M and CIITA genes in the pluripotent stem cells or their derivatives of the present invention are knocked out, the immunogenicity of such pluripotent stem cells or their derivatives is low. The RNA interference mediated by the transcription product of the small nucleic acid molecule encoded by one nucleic acid molecule and the transcription product of the second nucleic acid molecule against immune response-related genes only acts on the donor cells, and does not interfere with the genome of the recipient cells. Improve immunocompatibility between graft and recipient. The graft can continuously express shRNA/shRNA-miR targeting LAG-3 in the recipient. After these inhibitory factors are encapsulated by exosomes, the exosomes carry them to bind to target cells and then release them, which can effectively Inhibits the expression of LAG-3, activates the immune system, and restores T cell activity, enabling it to effectively clear tumor cells.

3. The pluripotent stem cells or derivatives thereof provided in the third aspect of the present invention have the characteristic of immune compatibility, and can eliminate or reduce the allogeneic immune rejection response. Moreover, the RNA interference program of the pluripotent stem cells or derivatives thereof only acts on such engineered pluripotent stem cells or derivatives thereof. Therefore, when such cells or derivatives are transplanted into recipients, the RNA interference mediated by the transcription products of the small nucleic acid molecule encoded by the first nucleic acid molecule and the transcription product of the second nucleic acid molecule against genes involved in the immune response only affects the in the donor cell without interfering with the genome of the recipient cell.

Further, further, an inducible gene expression system is also introduced into the genome of the pluripotent stem cells or their derivatives for regulating the expression of the first nucleic acid molecule, thereby realizing the expression of such pluripotent stem cells or their derivatives. Immunocompatibilities are reversible.

4. The pluripotent stem cells or derivatives thereof provided in the fourth aspect of the present invention have an immunocompatibility molecule expression sequence introduced into their genome, so such pluripotent stem cells or their derivatives have low immunogenicity, and when they are transplanted When it reaches the recipient, the RNA interference mediated by the transcription product of the small nucleic acid molecule encoded by the first nucleic acid molecule and the transcription product of the second nucleic acid molecule against immune response-related genes only acts on the donor cells and does not affect the recipient. interfere with the genome of the cell. Improve immunocompatibility between graft and recipient. The graft can continuously express shRNA or shRNA-miR targeting LAG-3 in the recipient. After these inhibitory factors are encapsulated by exosomes, the exosomes carry them to bind to target cells and then release them, which can effectively Inhibits the expression of LAG-3, activates the immune system, and restores T cell activity, enabling it to effectively clear tumor cells.

Furthermore, the genome of the pluripotent stem cells or their derivatives provided by the fourth aspect of the present invention also introduces an inducible gene expression system, and the inducible gene expression system can regulate the expression of immune-compatible molecules, while the inducible gene expression system can regulate the expression of immune-compatible molecules. The system is regulated by exogenous inducers. By adjusting the addition amount, duration, and type of exogenous inducers, the inducible gene expression system is controlled on and off, and the expression of immune-compatible molecular expression sequences is further controlled, so as to achieve multiple Reversible regulation of immunocompatibility in competent stem cells or their derivatives. When the inducible gene expression system is turned on, the normally expressed small molecule nucleic acid specifically binds to the transcription product of the second nucleic acid molecule introduced into the 3'UTR region of the immune response-related gene, thereby initiating the RNA interference program and converting the mRNA of the immune response-related gene Degradation or silencing, thereby blocking the expression of immune response-related genes. Therefore, when such cells or derivatives are transplanted into the recipient, the allogeneic immune rejection response can be eliminated or reduced, and the immunocompatibility between the transplant and the recipient can be improved. When the graft becomes diseased, the inducible gene expression system can be turned off by adding an exogenous inducer, thereby stopping the expression of small nucleic acid molecules and the interference of small nucleic acid molecules on the mRNA of immune response-related genes, and restoring the normal expression of immune-related genes. , thereby restoring the antigen-presenting ability of the graft cells, enabling the recipient to clear the diseased graft, thereby improving the clinical safety of such pluripotent stem cells or their derivatives, and greatly expanding their value in clinical applications.

5. The pluripotent stem cells or their derivatives in the present invention can also gradually reduce the expression of small nucleic acid molecules in the pluripotent stem cells or their derivatives by adjusting the amount of exogenous inducers and the duration of action, so that the donor cells It can gradually express low concentrations of immune-related genes to stimulate the donor, so that the donor gradually develops tolerance to the transplanted cells or their derivatives, and finally achieves a stable tolerance. At this time, even if the graft cell surface expresses mismatched HLA class I molecules, it can be compatible with the recipient immune system.

Description of drawings

Figure 1 shows the plasmid map of Cas9 (D10A).

Figure 2 is the plasmid map of sgRNA Clone AAVS1-1.

Figure 3 is the plasmid map of sgRNA Clone AAVS1-2.

Figure 4 is the plasmid map of sgRNA clone B2M-1.

Figure 5 is the plasmid map of sgRNA clone B2M-2.

Figure 6 is the plasmid map of sgRNA clone B2M-3.

Figure 7 is the plasmid map of sgRNA clone B2M-4.

Figure 8 is the plasmid map of sgRNA clone CIITA-1.

Figure 9 is the plasmid map of sgRNA clone CIITA-2.

Figure 10 is the plasmid map of sgRNA clone CIITA-3.

Figure 11 is the plasmid map of sgRNA clone CIITA-4.

Figure 12 is a plasmid map of AAVS1KI Vector (shRNA, constitutive).

Figure 13 is a plasmid map of AAVS1KI Vector (shRNA, inducible).

Figure 14 is a plasmid map of AAVS1KI Vector (shRNA-miR, constitutive).

Figure 15 is a plasmid map of AAVS1KI Vector (shRNA-miR, inducible).

Figure 16 is a plasmid map of B2M KI Vector.

Figure 17 is a plasmid map of CIITA KI Vector.

Detailed ways

In order to make the invention purpose, technical solutions and technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention, rather than for limiting the present invention.

The experimental materials and reagents used, unless otherwise specified, are conventional consumables and reagents that can be obtained from commercial sources.

Experimental Materials:

1. Selection of LAG-3 Inhibitors

LAG-3 inhibitor: shRNA or shRNA-miR targeting LAG-3.

The target sequences of the shRNA or shRNA-miR sequences targeting LAG-3 used in the examples of the present invention are shown in Table 1.

Table 1 Target sequences of shRNA or shRNA-miR sequences targeting LAG-3

2. Construction of small nucleic acid molecules

The sequence of the small nucleic acid molecule is a random sequence derived from a non-human species that does not target any mRNA or lncRNA in humans, preferably from Caenorhabditis elegans.

The sequence of the small nucleic acid molecule used in this example is

5'-TTGTACTACACAAAAGTACTG-3' (SEQ ID NO. 2);

The first nucleic acid molecule and the second nucleic acid molecule are designed according to the above-mentioned small nucleic acid molecules, respectively as follows:

First nucleic acid molecule (ie, shRNA expression framework or shRNA-miR expression framework for small nucleic acid molecules):

(1) The sequence composition of the shRNA expression framework of the small nucleic acid molecule is:

5'-CCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGCTCGGTACCCGGGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTGCTAGCGCCACC(SEQ ID NO.3)N ₁ ...N ₂₁ TTCAAGAGA(SEQ ID NO.4)N ₂₂ ...N ₄₂ TTTTTT-3'。

in,

_a ) N1... _N21 are the above-mentioned small nucleic acid molecule sequences, and _N22 ... _N42 are the reverse complementary sequences of the above-mentioned small nucleic acid molecule sequences;

b) If the plasmid needs to express shRNA of multiple genes, each gene corresponds to a shRNA expression frame, and then seamlessly connected;

c) Constitutive shRNA plasmids with different resistance genes, only the resistance genes are different, and other sequences are the same;

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

(2) shRNA-miR expression framework of small nucleic acid molecule: obtained by replacing the target sequence in microRNA-30 or microRNA-155 with the sequence of small nucleic acid molecule. The specific sequence is as follows:

5'-GAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTAAAGAAGGTATATTGCTGTTGACAGTGAGCG(SEQ ID NO.5)M ₁ N ₁ ...N ₂₁ TAGTGAAGCCACAGATGTA(SEQ ID NO.6)N ₂₂ ...N ₄₂ M ₂ TGCCTACTGCCTCGGACTTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAAT-3'(SEQ ID NO.7)。

in,

_a ) N1... _N21 is the sequence of the small nucleic acid molecule, and _N22 ... _N42 is the reverse complementary sequence of the sequence of the small nucleic acid molecule;

b) If the plasmid needs to express shRNA-miRs of multiple genes, each gene corresponds to one shRNA-miR expression frame, and then they are seamlessly connected;

c) Constitutive shRNA-miR plasmids with different resistance genes, only the resistance genes are different, and other sequences are the same;

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

e) if N ₁ is a G base, then M ₁ is an A base; otherwise, M ₁ is a C base;

f) _The M1 base is complementary to the _M2 base.

The second nucleic acid molecule: includes at least 3 repeats of the reverse complement of the sequence of the small nucleic acid molecule, preferably the reverse complement of the sequence of 6 to 10 repeats of the small nucleic acid molecule. Reverse complements of small nucleic acid molecule sequences can be linked by random Linker sequences.

As an embodiment of the present invention, the second nucleic acid molecule is formed by connecting the random sequence of the first 10 nt and the reverse complementary sequence of 8 repeated small nucleic acid molecule sequences through a random linker sequence (CGTA):

5'-atTCTAGATACAGTACTTTTGTGTAGTACAACGTACAGTACTTTTGTGTAGTACAACGTACAGTACTTTTGTGTAGTACAACGTACAGTACTTTTGTGTAGTACAACGTACAGTACTTTTGTGTAGTACAACGTACAGTACTTTTGTGTAGTACAACGTACAGTACTTTTGTGTAGTACAACGTACAGTACTTTTGTGTAGTACAACGTA-3' (SEQ ID NO. 8).

3. Selection of Immunocompatible Molecules

The types and sequences of the immunocompatible molecules used in the examples of the present invention are shown in Table 2 and Table 3.

Table 2 Types and functions of immunocompatible molecules

Table 3 Sequences of immunocompatible molecules

The shRNA or shRNA-miR immune-compatible molecule sequences of each experimental group in Tables 8 to 9 below are all shRNA or shRNA-miR immune-compatible molecules constructed by using the target sequences in Table 3. Those skilled in the art can understand that shRNA or shRNA-miR immune-compatible molecules constructed with other target sequences can also achieve the technical effects of the present invention, which all fall within the protection scope of the claims of the present invention.

4. Selection of shRNA/miRNA processing complex genes and anti-interferon effector molecules

(1) Selection of shRNA/miRNA processing complex genes

The shRNA/miRNA processing complex genes used in the present invention include shRNA and/or miRNA processing machinery that can inducibly shut down expression. The inducible shRNA and/or miRNA processing machines specifically include: Drosha (Accession number: NM_001100412), Ago1 (Accession number: NM_012199), Ago2 (Accession number: NM_001164623), Dicer1 (Accession number: NM_001195573), Exportin -5 (Accession number: NM_020750), TRBP (Accession number: NM_134323), PACT (Accession number: NM_003690), and DGCR8 (Accession number: NM_022720).

(2) Selection of anti-interferon effector molecules

The anti-interferon effector molecules used in the present invention include shRNA and/or shRNA-miR expression sequences for inhibiting PKR, 2-5As, IRF-3 and IRF-7 genes that can induce shut down expression to reduce dsRNA-induced interferon reaction to avoid cytotoxicity.

The target sequences of the anti-interferon effector molecules are shown in Table 4.

Table 4 Target sequences of anti-interferon effector molecules

The anti-interferon effector molecule sequences of each experimental group in Tables 8 to 9 below are all shRNAs or shRNA-miRs constructed by using the target sequences in Table 4. Those skilled in the art can understand that shRNA or shRNA-miR anti-interferon effector molecules constructed with other target sequences can also achieve the technical effects of the present invention, which all fall within the protection scope of the claims of the present invention.

5. Selection of synthetic genes for exosome processing

The exosome processing synthesis gene is selected from STEAP3 (NM_182915), Syndecan-4 (NM_002999), L-aspartate oxidase fragment (SEQ ID NO.68), CD63-L7Ae (SEQ ID NO.69) and Cx43S368A at least one of. Among them, Cx43S368A is obtained by mutating S (serine) at position 368 of Cx43 (NM_000165) to A (alanine).

6. Selection of Stem Cell Carriers

The stem cell carrier in the embodiment of the present invention is pluripotent stem cells, and 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 cell.

Wherein, the preparation method of described pluripotent stem cells is as follows:

ESCs: HN4 cells can be used, purchased from Shanghai Chinese Academy of Sciences.

iPSCs: Using episomal-iPSCs induction system (6F/BM1-4C), pE3.1-OG--KS and pE3.1-L-Myc--hmiR302cluster were electroporated into somatic cells, cultured in RM1 medium for 2 days, BioCISO-BM1 medium containing 2uM Parnate for 2 days, BioCISO-BM1 medium containing 2uM Parnate, 0.25mM sodium butyrate, 3uM CHIR99021 and 0.5uM PD03254901 for 2 days, then continued with BioCISO medium without other substances After culturing for about 17 days, the iPSCs cloned cells were picked, and the picked iPSCs cloned cells were purified, digested and passaged to obtain stable iPSCs. For specific construction methods, see: Stem Cell Res Ther. 2017Nov2; 8(1):245.

hPSCs-MSCs: iPSCs were cultured in BioCISO medium containing 10uM TGFβ inhibitor SB431542 for 25 days, digested and passaged (2mg/mL Dispase) until 80-90 cell confluency, and passaged 1:3 to Matrigel-coated culture plates medium, followed by ESC-MSC medium (knockout DMEM medium, containing 10% KSR, NEAA, double antibody, glutamine, β-mercaptoethanol, 10 ng/mL bFGF and SB-431542) for culture, and the medium was changed every day for continuous The cells were cultured for 20 days and passaged until the confluence of 80-90 cells (1:3 passage). For the specific construction method, please refer to: Proc Natl Acad Sci US A. 2015; 112(2):530-535.

NSCs: iPSCs were cultured with induction medium (knockout DMEM medium, containing 10% KSR, containing TGF-β inhibitor, BMP4 inhibitor) for 14 days, and rosette-shaped neurons were picked into low-adherence culture plates to cultivate. Medium in low-adherence plates included DMEM/F12 (with 1% N2, Invitrogen) and Neurobasal medium (with 2% B27, Invitrogen) in a ratio of 1:1, and 20ng/ml bFGF and 20ng/ml EGF . Digestion passages were performed using Accutase. For the specific construction method, please refer to: FASEB J. 2014; 28(11): 4642-4656.

EBs cells: iPSCs with a cell confluency of 95% were digested with BioC-PDE1 for 6 min, and the cells were scraped into clumps by mechanical scraping method to settle the cell clumps. The settled cell clumps were transferred to low-adherence plates and incubated with BioCISO-EB1 for 7 days, with medium changes every other day. After 7 days, the cells were transferred to Matrigel-coated culture plates and cultured in BioCISO medium for 7 days to obtain embryoid bodies (EBs) with inner, middle and outer germ layers. For the specific construction method, please refer to: StemCell Res Ther. 2017Nov 2;8(1):245.

The pluripotent stem cell derivatives also include adult stem cells differentiated from pluripotent stem cells, cells of each germ layer, tissues, and organs; and the adult stem cells include mesenchymal stem cells or neural stem cells.

Construct the plasmid vector:

The plasmid vectors used in the examples of the present invention include: Cas9 (D10A) plasmid, sgRNA plasmid, and Donor fragment.

The gene editing in the above plasmids all use the CRISPR-Cas9 gene editing system, the Cas 9 protein used is Cas 9 (D10A), Cas9 (D10A) is combined with sgRNA, and the sgRNA is responsible for the specific recognition of the target sequence (genomic DNA of the cell vector), and then Cas 9 (D10A) single-stranded cleavage of the target sequence. Genomic DNA double-strand break (DNA DoubleStrand Break, DSB), there must be two sets of Cas9 (D10A)/sgRNA to cut the two strands of the genomic DNA of the cell vector, and the cutting distance should not be too far. Compared with the Cas9/sgRNA scheme, the Cas9(D10A)/sgRNA scheme has the advantages of higher specificity and lower probability of off-target.

Wherein, the specific sequence of the sgRNA is:

sgRNA-AAVS1-1: 5'-TATAAGGTGGTCCCAGCTCG-3' (SEQ ID NO. 70);

sgRNA-AAVS1-2: 5'-AGGGCCGGTTAATGTGGCTC-3' (SEQ ID NO. 71);

sgRNA-B2M-1: 5'-CTCCTGTTATATTCTAGAAC-3' (SEQ ID NO. 72);

sgRNA-B2M-2: 5'-TTTCAGCATCAATGTACCCT-3' (SEQ ID NO. 73);

sgRNA-B2M-3: 5'-CGCGAGCACAGCTAAGGCCA-3' (SEQ ID NO. 74);

sgRNA-B2M-4: 5'-ACTCTCTCTTTCTGGCCTGG-3' (SEQ ID NO. 75);

sgRNA-CIITA-1: 5'-GGCACTCAGAAGACACTGAT-3' (SEQ ID NO. 76);

sgRNA-CIITA-2: 5'-AAGGTGTCTGGTCGGAGAGC-3' (SEQ ID NO. 77);

sgRNA-CIITA-3: 5'-ACCCAGCAGGGGCGTGGAGCC-3' (SEQ ID NO. 78);

sgRNA-CIITA-4: 5'-GTCAGAGCCCCAAGGTAAAA-3' (SEQ ID NO. 79).

The specific method of constructing the above-mentioned plasmid vector is:

(1) Cas9 (D10A) plasmid: directly ordered from Addgene (Plasmid 41816, Addgene).

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

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

(3) Donor fragment (KI plasmid):

a) Design PCR primers, take pUC18 plasmid (Takara, Code No.3218) as template, use high-fidelity enzyme (Nanjing Novozymes, P505-d1) to amplify the Amp(R)-pUC origin fragment by PCR method ;

b) extracting the genomic DNA of human cells and designing corresponding primers, then using the human genomic DNA as a template, using a high-fidelity enzyme to amplify the recombinant arm by PCR;

c) Design the PCR amplification primers of the KI (Knock-in) plasmid element, and then use the plasmid containing the KI plasmid element as a template to amplify the KI plasmid element (subcloning) using a high-fidelity enzyme;

d) Use multi-fragment recombinase (Nanjing Novozan, C113-02) or overlap PCR to connect the amplified Amp(R)-pUC origin fragment, recombination arm, and KI plasmid element to form a complete circular plasmid .

Wherein, the recombination arm includes B2M recombination arm, CIITA recombination arm, and AAVS1 recombination arm.

The sequences of the B2M recombination arms are shown in B2M-HR-L (SEQ ID NO. 80) and B2M-HR-R (SEQ ID NO. 81).

The sequences of the CIITA recombination arms are shown in CIITA-HR-L (SEQ ID NO. 82) and CIITA-HR-R (SEQ ID NO. 83).

The sequences of the AAVS1 recombination arms are shown in AAVS1-HR-L (SEQ ID NO. 84) and AAVS1-HR-R (SEQ ID NO. 85).

The plasmids prepared in the embodiments of the present invention can be classified into constitutive plasmids and inducible plasmids according to the type of expression framework in the plasmid.

The expression frameworks in the above-mentioned constitutive plasmids include shRNA constitutive expression frameworks and shRNAmiR constitutive expression frameworks. The Donor fragment obtained by enzyme cleavage of the above constitutive plasmid, after knocking into the stem cell vector genomic DNA, the expression function of the fragment cannot be regulated.

The expression frameworks in the above-mentioned inducible plasmids include shRNA-inducible expression frameworks and shRNAmiR-inducible expression frameworks. After digesting the Donor fragment obtained from the above inducible plasmid and knocking in the genomic DNA of the stem cell vector, the expression function of the fragment can be regulated by adding an inducer, which is equivalent to adding an on or off switch to the expression function.

The specific sequence requirements and structural composition of the above expression frameworks are shown below.

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

5'-GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACgctagcgccacc(SEQ ID NO.86)N ₁ ...N ₂₁ TTCAAGAGAN ₂₂ ...N ₄₂ TTTTTT-3'；

in,

_a ) N1... _N21 is the shRNA target sequence of the above target sequence, _N22 ... _N42 is the reverse complement of the shRNA target sequence of the above target sequence;

b) when the plasmid constructed using the above-mentioned shRNA constitutive expression framework needs to express the shRNA of multiple genes, then each gene corresponds to a shRNA expression framework, and then seamlessly connects;

c) When the above-mentioned shRNA constitutive expression framework needs to carry different resistance genes, only the resistance gene sequences in the shRNA constitutive expression framework are different, and other sequences remain the same;

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

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

5'-GAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTAAAGAAGGTATATTGCTGTTGACAGTGAGCGM ₁ N ₁ ...N ₂₁ TAGTGAAGCCACAGATGTAN ₂₂ ...N ₄₂ M ₂ TGCCTACTGCCTCGGACTTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAAT-3'；

in,

g) _N1 ... _N21 are the _shRNAmiR target sequences of the above target sequences, and N22...N42 are the reverse complementary sequences of the _shRNAmiR target sequences of the above target sequences;

H) when the plasmid constructed using the above-mentioned shRNAmiR constitutive expression framework needs to express the shRNAmiR of multiple genes, then each gene corresponds to a shRNAmiR expression framework, and then seamlessly connected;

i) When the above-mentioned shRNAmiR constitutive expression framework needs to carry different resistance genes, only the resistance gene sequences in the shRNAmiR constitutive expression framework are different, and other sequences remain the same;

j) N represents A or T or G or C base, and M base represents A or C base;

k) if N ₁ is a G base, then M ₁ is an A base; otherwise, M ₁ is a C base;

₁ ) The M1 base is complementary to the _M2 base.

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

5'-(SEQ ID NO. 87) N ₁ ...N ₂₁ TTCAAGAGAN ₂₂ ... N ₄₂ TTTTTT-3';

in,

e) N1... _N21 are the _shRNA target sequences corresponding to the above target sequences, and _N22 ... _N42 are the shRNA target sequences corresponding to the above target sequences and/or the reverse complementary sequences of the small nucleic acid molecules;

f) when the plasmid constructed using the above-mentioned shRNA constitutive expression framework needs to express the shRNA of multiple genes, then each gene corresponds to an shRNA expression framework, which is then seamlessly connected;

g) When the above-mentioned shRNA constitutive expression framework needs to carry different resistance genes, only the resistance gene sequences in the shRNA constitutive expression framework are different, and other sequences remain the same;

h) 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 Cas9 (D10A) plasmid map constructed by the above method is shown in Figure 1, the sgRNA plasmid map of the AAVS1 safety site is shown in Figure 2-3, the sgRNA plasmid map of the B2M gene is shown in Figure 4-7, and the sgRNA of the CIITA gene is shown in Figure 4-7. The plasmid map is shown in Figure 8-11, the constructed AAVS1KI plasmid map (constitutive and inducible) is shown in Figure 12-15, the B2M KI plasmid map is shown in Figure 16, and the CIITA KI plasmid map is shown in Figure 17.

Construction of stem cell vectors:

In the technical solution of the present invention, the safety sites knocked into in the genome of the stem cell vector include the AAVS1 safety site, the eGSH safety site, and the H11 safety site.

The single-cell cloning operation for AAVS1 gene knock-in includes the following steps:

a) Electric transfer procedure:

Donor cell preparation: human pluripotent stem cells;

Kit 1；Kit: Human Stem Cell

Kit 1;

Instrument: electroporator;

Medium: BioCISO;

Induction plasmid: the corresponding plasmid constructed in the above example.

b) The electrotransformed human pluripotent stem cells were screened in double antibiotic medium containing G418 and puro.

c) Screening and culturing single-cell clones to obtain single-cell clones.

d) Culturing the obtained single-cell clone.

Among them, the single-cell clone culture reagents include:

The media were: BioCISO medium, 300 μg/ml G418 and 0.5 μg/ml puro. The medium should be placed at room temperature in advance, and placed in the dark for 30 to 60 minutes until it returns to room temperature, but it should not be preheated at 37°C to avoid the reduction of biomolecular activity.

Matrigel: hESC grade Matrigel. Before passaging or resuscitating cells, add the Matrigel working solution to the cell culture flask and shake well to ensure that the Matrigel completely covers the bottom of the culture flask and that the Matrigel cannot be dried anywhere before use. In order to ensure better adherence and survival of cells, Matrigel should be placed in a 37°C incubator for coating time: 1:100X Matrigel should not be less than 0.5 hours; 1:200X Matrigel should not be less than 2 hours.

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

Freezing medium: 60% BioCISO, 30% ESCs-grade FBS and 10% DMSO.

The single-cell clone culture step adopts the routine maintenance subculture process in the art.

Among them, the best time for passage is when the overall confluence of cells reaches 80% to 90%.

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

The concrete passage steps in the present invention are as follows:

a) Discard the Matrigel in the coated cell culture flask, add an appropriate amount of the above-mentioned medium and incubate in a 37°C, 5% CO ₂ incubator;

b) When the cells meet the above passage requirements, discard the medium supernatant, and add an appropriate amount of 0.5mM EDTA digestion solution to the cell flask dish;

c) Put the cells into a 37°C, 5% CO ₂ incubator and incubate for 5-10 minutes (digested until most of the cells are shrunk and rounded but have not floated under the microscope), and the cells are detached from the wall by pipetting , suck the cell suspension into a centrifuge tube and centrifuge at 200g for 5 minutes;

d) After centrifugation, discard the supernatant, resuspend the cells with medium, pipetting the cells repeatedly to mix, then transfer the cells to a Matrigel-coated bottle, shake well, and shake well after observing no abnormality under the microscope Culture in a 37°C, 5% _CO2 incubator;

e) Observe the cell adherent survival state the next day, aspirate the medium, and change the medium on time every day.

Gene knock-in detection method:

1. Single-cell clone AAVS1 gene knock-in detection.

(1) Description of AAVS1 gene knock-in detection.

Detection principle:

Knock-in treated cells are tested for homozygosity by PCR. Since the two Donor fragments only have differences in the sequence of the resistance gene, to determine whether the cell is homozygous (two chromosomes knock-in the Donor fragments of different resistance genes respectively), it is necessary to detect whether the genome of the cell contains two genes. The Donor fragment of the resistance gene, only double knock-in cells may be correct homozygous.

Detection method:

One primer was designed inside the Donor plasmid (KI plasmid) (non-recombination arm part), and then another primer was designed in the genome of the cell (non-recombination arm part). If the Donor fragment can be inserted correctly in the genome, there will be a target band, otherwise there will be no target band.

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

Table 5 Specific sequences and amplification conditions of primers for AAVS1 gene knock-in detection

(2) Description of B2M and CIITA gene knock-in detection.

The detection method of the second nucleic acid molecule knock-in at the 3'UTR of B2M and CIITA genes is the same as that of AAVS1, and the PCR detection conditions used are shown in Tables 6 and 7.

Table 6 Specific sequences and amplification conditions of primers for B2M gene knock-in detection

Table 7 Specific sequences and amplification conditions of primers for CIITA gene knock-in detection

The selected plasmid is knocked into the stem cell vector genome using conventional techniques in the art:

According to the groupings in Tables 8 and 9 below, the sequences of LAG-3 inhibitors, immune-compatible molecules, shRNA/miRNA processing complex genes, anti-interferon effector molecules, and exosomes were processed, respectively. The synthetic gene is knocked into the safe site of the above-mentioned stem cell vector using conventional techniques in the art, and different types of constitutive stem cell expression vectors and inducible stem cell expression vectors are obtained to test the feasibility of their expression.

Table 8 Grouping of constitutive knock-in expression experiments

Among them, the "+" sign indicates the knock-in of a gene or nucleic acid sequence, and the "-" sign indicates the gene knock-out.

General principle: The shRNA (or shRNA-miR) targeting the inhibitor is placed in the shRNA (or shRNA-miR) expression framework 2 of the corresponding plasmid, and the shRNA (or shRNA-miR) of the remaining inhibitor is placed in the shRNA (or shRNA-miR) of the corresponding plasmid. or shRNA-miR) expression framework 1, the gene sequence is placed in the MCS.

B2M-3'UTR-miRNA-locus or CIITA-3'UTR-miRNA-locus, the second nucleic acid molecule (SEQ ID NO. 8), was knocked into the 3'UTR region of the B2M and CIITA genes, respectively.

B2M/CIITA-3'UTR-shRNA is the shRNA expression framework of small nucleic acid molecules, that is, the first nucleic acid molecule, which specifically targets the transcription product of the second nucleic acid molecule in the 3'UTR region of the B2M gene and CIITA gene, and the knock-in site AAVS1 is the genomic safety site.

B2M/CIITA-3'UTR-shRNA-miR is the shRNA-miR expression framework of small nucleic acid molecules, that is, the first nucleic acid molecule, targeting the transcription product of the second nucleic acid molecule in the 3'UTR region of the B2M gene and CIITA gene, knock-in The site is the genomic safety site AAVS1.

CD47 represents the CD47 expression sequence, and its knock-in site is the genomic safety site AAVS1.

If the expression cassette or MCS needs to insert multiple fragments, EMCV IRESwt (SEQ ID NO. 100) can be used to connect and then insert.

The sgRNA plasmids for gene knock-in are: sgRNA clone B2M-1, sgRNA clone B2M-2, sgRNAclone CIITA-1 and sgRNA clone CIITA-2.

The sgRNA plasmids used for gene knockout are: sgRNA clone B2M-3, sgRNA clone B2M-4, sgRNAclone CIITA-3, sgRNA clone CIITA-4.

(1) Aa1 grouping:

The shRNA expression framework 2 of the AAVS1KI Vector (shRNA, constitutive) plasmid was placed into the shRNA sequence targeting LAG-3.

(2) Aa2 grouping:

The shRNA expression frame 2 of the AAVS1KI Vector (shRNA, constitutive) plasmid is placed into the shRNA sequence targeting LAG-3, and the shRNA expression frame 1 is placed into the remaining shRNA sequences (including the target sequence of B2M/CIITA-3'UTR-shRNA) , MCS into the gene sequence.

The B2M KI Vector was placed into the B2M-3'UTR-miRNA-locus.

CIITA KI Vector was put into CIITA-3'UTR-miRNA-locus.

(3) Aa3 grouping:

The shRNA expression framework 2 of the AAVS1KI Vector (shRNA, constitutive) plasmid was put into the shRNA sequence targeting LAG-3, the shRNA expression framework 1 was put into the remaining shRNA target sequences, B2M and CIITA were knocked out, and MCS was put into the gene sequence.

(4) Aa4 grouping:

The shRNA expression frame 2 of the AAVS1KI Vector (shRNA, constitutive) plasmid is placed into the shRNA sequence targeting LAG-3, the shRNA expression frame 1 is placed into the remaining shRNA target sequences, and the MCS is placed into the gene sequence.

(5) Ab1 grouping:

The shRNA-miR expression framework 2 of the AAVS1KI Vector (shRNA-miR, constitutive) plasmid was placed into the shRNA-miR sequence targeting LAG-3.

(6) Ab2 grouping:

The shRNA-miR expression frame 2 of the AAVS1KI Vector (shRNA-miR, constitutive) plasmid is placed into the shRNA-miR sequence targeting LAG-3, and the shRNA-miR expression frame 1 is placed into the remaining shRNA-miR target sequences (including B2M/ Target sequence of CIITA-3'UTR-shRNA-miR), MCS was put into the gene sequence.

The B2M KI Vector was placed into the B2M-3'UTR-miRNA-locus.

CIITA KI Vector was put into CIITA-3'UTR-miRNA-locus.

(7) Ab3 grouping:

The shRNA-miR expression frame 2 of the AAVS1KI Vector (shRNA-miR, constitutive) plasmid was placed into the shRNA-miR sequence targeting LAG-3, and the shRNA-miR expression frame 1 was placed into the remaining shRNA-miR target sequences, knocking out B2M And CIITA, MCS into the gene sequence.

(8) Ab4 grouping:

The shRNA-miR expression frame 2 of the AAVS1KI Vector (shRNA-miR, constitutive) plasmid was placed into the shRNA-miR sequence targeting LAG-3, the shRNA-miR expression frame 1 was placed into the remaining shRNA-miR target sequences, and the MCS was placed in gene sequence.

Table 9 Groups of inducible knock-in expression experiments

Among them, the "+" sign indicates the knock-in of a gene or nucleic acid sequence, and the "-" sign indicates the gene knock-out.

The general principle is shown in the above grouping of constitutive knock-in expression experiments.

(1) Group B1:

The shRNA expression frame 2 of the AAVS1KI Vector (shRNA, inducible) plasmid is placed into the shRNA sequence targeting LAG-3, and the shRNA expression frame 1 is placed into the rest of the shRNA target sequence (including the target sequence of B2M/CIITA-3'UTR-shRNA). ), the MCS is placed into the gene sequence. Add the Tet-Off system to induce the system.

The B2M KI Vector was placed into the B2M-3'UTR-miRNA-locus.

CIITA KI Vector was put into CIITA-3'UTR-miRNA-locus.

(2) Group B2:

The shRNA-miR expression frame 2 of the AAVS1KI Vector (shRNA-miR, inducible) plasmid is placed into the shRNA-miR sequence targeting LAG-3, and the shRNA-miR expression frame 1 is placed into the remaining shRNA-miR target sequences (including B2M/ Target sequence of CIITA-3'UTR-shRNA-miR), MCS was put into the gene sequence. Add the Tet-Off system to induce the system.

The B2M KI Vector was placed into the B2M-3'UTR-miRNA-locus.

CIITA KI Vector was put into CIITA-3'UTR-miRNA-locus.

(3) Group B3:

The shRNA expression frame 2 of the AAVS1KI Vector (shRNA, inducible) plasmid is placed into the shRNA sequence targeting LAG-3, and the shRNA expression frame 1 is placed into the rest of the shRNA target sequence (including the target sequence of B2M/CIITA-3'UTR-shRNA). ), the MCS is placed into the gene sequence. Add the Tet-Off system to induce the system.

(4) Group B4:

The shRNA-miR expression frame 2 of the AAVS1KI Vector (shRNA-miR, inducible) plasmid is placed into the shRNA-miR sequence targeting LAG-3, and the shRNA-miR expression frame 1 is placed into the remaining shRNA-miR target sequences (including B2M/ Target sequence of CIITA-3'UTR-shRNA-miR), MCS was put into the gene sequence. Add the Tet-Off system to induce the system.

Among them, the above-mentioned constitutive and inducible knock-in can be transformed into hPSCs first, and then differentiated into derivatives of pluripotent stem cells for use when undergoing immune-compatible transformation; Derivatives of stem cells are then immunocompatibly engineered.

The Tet-Off system in the present invention is specifically:

In the absence of tetracycline, the tTA protein continues to act on the tet promoter, allowing the gene to continue to be expressed. This system is very useful where transgenes need to be maintained in a state of continuous expression. When tetracycline is added, tetracycline can change the structure of the tTA protein so that it cannot bind to the promoter, thereby reducing the level of gene expression it drives. To keep this system "off", tetracycline must be added continuously.

The Tet-Off system and the sequences of one or more immune-compatible molecules are knocked into the genome safe sites of pluripotent stem cells, and the expression of immune-compatible molecules can be accurately turned on or off by the addition of tetracycline, thereby reversibly regulating pluripotent stem cells or Expression of major histocompatibility complex-related genes in its derivatives.

Detection of the inhibitory effect of pluripotent stem cells or their derivatives expressing targeted LAG-3 inhibitory factors on LAG-3:

The protocols of each experimental group in Table 8 and Table 9 were knocked into the genome safety site of MSCs cells, cultured at 37°C in a 0.5% CO ₂ incubator, and the supernatant of the medium was collected, and the cells containing the knock-in exosome processing synthetic gene sequence were used Exosome extraction kit (BestBio, lot#BB-3901) was used to extract exosomes in the culture supernatant. The culture supernatant was centrifuged at 3000g for 15min at 37°C. After collecting the supernatant, centrifuged at 10,000g for 20min at 4°C. The supernatant was collected, and extract A was added at a ratio of 4:1, inverting for 1min, and overnight at 37°C. Centrifuge at 10,000g for 60min at 37°C, and collect the precipitate. Then, the LAG-3 inhibitory factor or exosomes containing LAG-3 inhibitory factor were added to the culture medium of CHO cells expressing LAG-3 for 72 hours, and then qPCR was performed to detect the expression level of LAG-3.

The N (control) group refers to LAG-3-expressing CHO cells cultured without the addition of LAG-3 inhibitory factors. Specifically: untreated CHO cells expressing LAG-3 were incubated at 37°C for 30 minutes, the cells were washed twice with PBS, and the FITC-labeled LAG-3 fusion protein was added to the test tube, and incubated at 37°C for 30 minutes.

The test results of each experimental group are shown in Table 10.

Table 10 qPCR detection results of inhibition of LAG-3 by pluripotent stem cells or their derivatives expressing LAG-3 inhibitory factors

group relative expression Deviation (±) Independent sample t-test (^*p<0.01) N (control) 1.014 0.025 - Aa1 0.339 0.014 * Aa2 0.338 0.019 * Aa3 0.315 0.038 * Aa4 0.322 0.004 * Ab1 0.322 0.029 * Ab2 0.299 0.024 * Ab3 0.297 0.024 * Ab4 0.320 0.042 * B1 0.329 0.047 * B2 0.331 0.031 * B3 0.313 0.022 * B4 0.309 0.033 *

It can be seen from the above table that the LAG-3 inhibitory factor encapsulated by exosomes expressed by the pluripotent stem cells or derivatives thereof of the present invention can effectively inhibit the expression of LAG-3 in target cells. Moreover, the degree of inhibition was relatively constant in each group, so the LAG-3 inhibitory factor expressed by pluripotent stem cell derivatives was not affected by cell differentiation morphology and other exogenous genes (immunocompatible transformation).

Effects of pluripotent stem cells or their derivatives expressing LAG-3 inhibitory factors on T cell killing of tumor cells:

The protocols of each experimental group in Table 8 and Table 9 were knocked into the genomic safety site of MSCs cells, and the ⁵¹ Cr release method was used to detect the effect of pluripotent stem cells expressing CD38 inhibitor on T cells killing tumor cells. The specific steps include:

Preparation of effector cells:

T cell isolation: use Ficoll-hypaque density gradient centrifugation to isolate human peripheral blood mononuclear cells (PBMC), and then use Dynabeads ^TM CD3 (Invitrogen ^TM , Cat. No. 11151D) kit to isolate T cells. Cells were resuspended in RPMI1640 medium containing 10% FBS, counted by trypan blue staining, and concentrated to 1 x ¹⁰⁷ cells/mL.

Preparation of target cells:

Tumor (HCC liver cancer) cells were digested and resuspended, cells were counted by trypan blue staining, and a cell suspension of 1×10 ⁷ cells/mL was prepared.

⁵¹ Cr release test:

Principle: Tumor cells are first incubated with the medium supernatant of pluripotent stem cells expressing CD38 inhibitory factors, and then when they come into contact with T cells, T will attack tumor cells and cause cell lysis and death. However, if the supernatant of pluripotent stem cells that secrete CD38 inhibitory factor is not incubated, tumor cells will not be recognized by T cells, and immune escape will occur. Therefore, by detecting the amount of ⁵¹ Cr in the medium, the ability of T cells to kill tumors can be reflected. The smaller the amount of ⁵¹ Cr released into the culture medium, the more likely the tumor cells to escape immune.

To quantitatively detect cell-mediated cytotoxicity, target cells were labeled with radioisotope ⁵¹ Cr, incubated with effector molecules or cells, and the cytotoxic activity was determined according to the number of ⁵¹ Cr radiation pulses (cpm) released by target cell lysis.

The specific steps are:

a. Label the target cells with 100 μCi (Ci, radioactivity unit) Na ⁵¹ CrO ₄ at 37°C for 120 min, shake once every 15 minutes, and then use the washing solution to centrifuge and wash 5 times after labeling, and finally resuspend in the culture solution medium, make up to 1×10 ⁶ cells/mL for later use;

b. Add target cells and T cells to a 96-well culture plate, add 100 μL of target cells (2.5×10 ³ ) and 100 μL of effector cells (E/T=1:2, 1:5, 1:10, E/T=1:2, 1:5, 1:10) to each well /T is the ratio of target cells to effector cells T), and set up natural release control wells (100μL target cells + 100uL medium) and maximum release wells (100μL target cells + 100uL 2% SDS); place at 37°C, 5% CO ₂ culture and incubate for 4h. After taking out, the supernatant of each well was sucked out with a pipette, centrifuged to take 100 μL of the supernatant, and the cpm value was measured with a gamma counter;

c. Result calculation: Calculate the natural release rate of ⁵¹ Cr and the activity of T cells according to the formula:

Among them: the natural release rate of ⁵¹ Cr is generally required to be less than 10%.

The test results of each experimental group are shown in Table 11.

The N (control) group refers to tumor cells that were not treated with the supernatant of the medium of CD38 inhibitor-expressing pluripotent stem cells.

Table 11 Effects of pluripotent stem cells or their derivatives in each experimental group on T cells killing tumor cells

Independent sample t-test ( ^* p<0.01).

Through the above experiments, it can be proved that the LAG-3 inhibitory factor-expressing stem cells or their derivatives prepared by the present invention can effectively block activated T cells and play an anti-tumor effect.

Application of pluripotent stem cells expressing targeted LAG-3 inhibitory factors in various tumor treatments:

Select B2M and CIITA gene knockout protocol groups (Aa3, Ab3) immune-compatible cells (MSCs) for testing.

In the humanized NSG mouse tumor model, each group of experimental cells (hPSCs and hPSCs-derived derivatives (hPSCs-MSCs) capable of secreting LAG-3 inhibitory factor) were injected to observe the effect of RCC kidney cancer, MC colon cancer, NIC The effect of treatment of lung cancer. In order to avoid the problem of immune compatibility, the used immune cells and hPSCs and hPSCs-derived derivatives are all derived from the same human, and the immune compatibility protocol of B2M and CIITA gene knockout is adopted.

Specific steps include:

In humanized NSG mice (The Jackson Laboratory (JAX)), 5×10 ⁶ tumor (RCC kidney cancer, MC colon cancer, NIC lung cancer) cells ^were subcutaneously injected into the right axilla, and the tumors grew to 60 mm in size. 200uL PBS (containing human immune cells and 1×10 ⁶ LAG-3 inhibitor-secreting pluripotent stem cell derivatives) was injected into the tail vein for tumor treatment, of which only the group containing human immune cells was injected as a control group . After 20 days, the mice were sacrificed, and then the tumor size between the groups was compared and the statistical analysis of differences was performed.

The N (control) group refers to the NSG mouse tumor model that was not injected with the experimental cells.

The results are shown in Table 12.

Table 12 Anti-tumor effects of pluripotent stem cells or their derivatives in each experimental group

Independent sample t-test ( ^* p<0.01).

Through the above experiments, it can be proved that the LAG-3 inhibitory factor-expressing stem cells or their derivatives prepared by the present invention can effectively block LAG-3 and play an anti-tumor effect.

Immunocompatibility testing of pluripotent stem cells expressing LAG-3 inhibitors:

Taking advantage of the low immunogenicity of MSCs, the humanized NSG mouse tumor model was injected with hPSCs-derived immunocompatible MSCs expressing LAG-3 inhibitory factors, and the effect of tumor (MM myeloma) treatment was observed. . Among them, the used immune cells and hPSCs-derived MSCs are not from the same person.

The control group was the NSG mouse tumor model without MSCs injection;

The Dox-added group was as follows: starting from the injection of cells expressing LAG-3 inhibitory factor, 0.5 mg/mL of Dox was continuously added to the mouse diet to feed the mice until the end of the experiment. The knock-in sequence (immunocompatible molecule) can be rendered inactive by using the inducer Dox.

The reversible expression test results of the immune-compatible molecule-inducible expression group are shown in Table 13.

Table 13 The reversible expression test results of the immune-compatible molecule-inducible expression group

Independent sample t-test ( ^* p<0.01).

The above experiments show that: MSCs (group 2) that only express inhibitory factors have low immunogenicity and can exist in the allogene for a certain period of time, so they can exert a certain tumor therapeutic effect, while the immune-compatible transformation (group 3- 7, including constitutive and reversible inducible immune compatibility), its immune compatibility effect is better, and it can exist in the body for a longer time (or can achieve long-term coexistence) than MSCs without immune compatibility modification, and it plays a better role in tumor treatment. , while group 4 is the B2M and CIITA gene knockout group, which completely eliminates the effects of HLA-I and HLA-II molecules, so its tumor treatment effect is the best. However, due to its constitutive immune-compatible modification (gene knock-in/knock-out), it cannot be cleared when the graft is mutated or not needed, so there is a group 6-9 protocol setting. In groups 8-9, when the inhibitor-expressing cells were injected into the mice, the Dox inducer (always used) was administered to the mice, and the immune-compatibility effect of the mice injected with the inhibitor-expressing cells was eliminated, which existed in vivo. The time is comparable to that of MSCs without immunocompatibility modification, and the tumor treatment effect is also comparable to that of MSCs without immunocompatibility modification.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Future Homo sapiens Regenerative Medicine Research Institute (Guangzhou) Co., Ltd.

Wang Linli

<120> A pluripotent stem cell expressing LAG-3 targeting inhibitor and its derivative and application

<130>

<160> 101

<170> PatentIn version 3.5

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ggtctttcct cactgccaag t 21

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ttgtactaca caaaagtact g 21

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ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccactccc tatcagtgat 60

agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa gtgaaagtcg 120

agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagctcggt acccgggtcg 180

aggtaggcgt gtacggtggg aggcctatat aagcagagct cgtttagtga accgtcagat 240

cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag 300

cctgctagcg ccacc 315

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ttcaagaga 9

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gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta 60

cttcttcagg ttaacccaac agaaggctaa agaaggtata ttgctgttga cagtgagcg 119

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tagtgaagcc acagatgta 19

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tgcctactgc ctcggacttc aaggggctac tttaggagca attatcttgt ttactaaaac 60

tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat ggtataaat 119

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attctagata cagtactttt gtgtagtaca acgtacagta cttttgtgta gtacaacgta 60

cagtactttt gtgtagtaca acgtacagta cttttgtgta gtacaacgta cagtactttt 120

gtgtagtaca acgtacagta cttttgtgta gtacaacgta cagtactttt gtgtagtaca 180

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gagcagagaa ttctcttatc c 21

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gctacctgga gcttcttaac a 21

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ggagcttctt aacagcgatg c 21

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gggtctccag tatattcatc t 21

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gctcccactc catgaggtat t 21

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ggtatttctt cacatccgtg t 21

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aggagacacg gaatgtgaag g 21

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gctcccactc catgaggtat t 21

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aagttaagaa cctgaatata a 21

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aacctgaata taaatttgtg t 21

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gggtctggtg ggcatcatta t 21

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ggtctggtgg gcatcattat t 21

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gcatcattat tgggaccatc t 21

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gatgaccaca ttcaaggaag a 21

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gaccacattc aaggaagaac t 21

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gctttcctgc ttggcagtta t 21

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gcgtaagtct gagtgtcatt t 21

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gacaatttaa ggaagaatct t 21

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ggccatagtt ctccctgatt g 21

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gccatagttc tccctgattg a 21

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gcagatgacc acattcaagg a 21

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gcaggataag tatgagtgtc a 21

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ggttcctgca cagagacatc t 21

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ggatgtggaa cccacagata c 21

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gatgtggaac ccacagatac a 21

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gcctgatagg acccatattc c 21

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gcatccaata gacgtcattt g 21

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gcgtcactgg cacagatata a 21

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ggatggattt gattatgatc c 21

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gcagttctgt tgccactctc t 21

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gctgtaagaa ggatgctttc a 21

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gctgcaggca ggattgtttc a 21

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gcctcgagtt tgagagcta 19

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ggacactggt tcaacacctg t 21

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acctgtgact tcatgtgtgc g 21

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atgaatactc tccctgaaca ttcatgtgac gtgttgatta tcggtagcgg cgcagccgga 60

ctttcactgg cgctacgcct ggctgaccag catcaggtca tcgttctaag taaggccggt 120

aacgaggttc aacattttat gcccagggcg gtattgccgc cgtgtttgat aaactgacag 180

cattgactcg catgtggaag acacattgat tgccggggct ggtatttgcg atcgccatgc 240

agttgaattt gtcgccagca atgcacgatc ctgtgtgcaa tggctaatcg accagggggt 300

gttgtttgat acccacattc aaccgaatgg cgaagaaagt taccatctga cccgtgaagg 360

tggacatagt caccgtcgta ttcttcatgc cgccgacgcc accggtagag aagtagaaac 420

cacgctggtg agcaaggcgc tgaaccatcc gaatattcgc gtgctggagc gcagcaacgc 480

ggttgatctg attgtttctg acaaaattgg cctgccgggc acgcgacggg ttgttggcgc 540

gtgggtatgg aaccgtaata aagaaacggt ggaaacctgc cacgcaaaag cggtggtgct 600

ggcaaccggc ggtgcgtcga aggtttatca gtacaccacc aatccggata tttcttctgg 660

cgatggcatt gctatggcgt ggcgcgcagg ctgccggttg ccaatctcga tttaatcagt 720

tccaccctac cgcgctatat cacccacagg cacgcaattt cctgttaaca gaagcactgc 780

gcggcgaggc gcttatctca agcgcccgga tggtacgcgt ttatccgatt ttgatgagcg 840

cggcgaactg ccccgcgcga tattgtcgcc cgcgccattg accatgaaat gaaacgcctc 900

ggcgcagatt gtatgttcct tgatatcagc cataagcccg ccgattttat tcgccagcat 960

ttcccgatga tttatgaaaa gctgctcggg ctgggattga tctcacacaa gaaccggtac 1020

cgattgtgcc tgctgcacat tatacctgcg gtggtgtaat ggttgatgat catgggcgta 1080

cggacgtcga gggcttgtat gccattggcg aggtgagtta taccggctta cacggcgcta 1140

accgcatggc ctcgaattca ttgctggagt gtctggtcta tggctggtcg gcggcggaag 1200

atatcaccag acgtatgcct tatgcccacg acatcagtac gttaccgccg tgggatgaaa 1260

gccgcgttga gaaccctgac gaacggtagt aattcagcat aactggcacg agctacgtct 1320

gtttatgtgg gattacgttg gcattgtgcg cacaacgaag cgcctggaac gcgccctgcg 1380

gcggataacc atgctccaac aagaaataga cgaatattac gcccatttcc gcgtctcaaa 1440

taatttgctg gagctgcgta atctggtaca ggttgccgag ttgattgttc gctgtgcaat 1500

gatgcgtaaa gagagtcggg gttgcatttc acgctggatt atccggaact gctcacccat 1560

tccggtccgt cgatccttcc cccggcaatc attacataaa cagataa 1607

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ggaagtggtg ccggcaccgg cggcatgtac gtgcgcttcg aggtgcccga ggacatgcag 60

aacgaggccc tgagcctgct ggaaaaagtg cgcgagagcg gcaaagtgaa gaagggcacc 120

aacgaaacca ccaaggccgt ggaacggggc ctggccaagc tggtgtatat cgccgaggac 180

gtggaccccc ccgagattgt ggcccatctg cccctgctgt gcgaagagaa gaacgtgccc 240

tacatctacg tgaagtccaa gaacgacctg ggcagagccg tgggcatcga ggtgccatgt 300

gcctctgccg ccatcatcaa cgagggcgag ctgcggaaag aactgggcag cctggtggaa 360

aagatcaagg gcctgcagaa gggttccggt ggatccggtt ccggacgggc t 411

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tataaggtgg tcccagctcg 20

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agggccggtt aatgtggctc 20

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<400> 72

ctcctgttat attctagaac 20

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tttcagcatc aatgtaccct 20

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cgcgagcaca gctaaggcca 20

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actctctctt tctggcctgg 20

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ggcactcaga agacactgat 20

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aaggtgtctg gtcggagagc 20

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<400> 78

acccagcagg gcgtggagcc 20

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gtcagagccc caaggtaaaa 20

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cctggacttc tccagtactt tctggctgga ttggtatctg aggctagtag gaagggcttg 60

ttcctgctgg gtagctctaa acaatgtatt catgggtagg aacagcagcc tattctgcca 120

gccttatttc taaccatttt agacatttgt tagtacatgg tattttaaaa gtaaaactta 180

atgtcttcct ttttttttctc cactgtcttt ttcatagatc gagacatgta agcagcatca 240

tggaggtaag tttttgacct tgagaaaatg ttttttgtttc actgtcctga ggactattta 300

tagacagctc taacatgata accctcacta tgtggagaac attgacagag taacatttta 360

gcagggaaag aagaatccta cagggtcatg ttcccttctc ctgtggagtg gcatgaagaa 420

ggtgtatggc cccaggtatg gccatattac tgaccctcta cagagagggc aaaggaactg 480

ccagtatggt attgcaggat aaaggcaggt ggttacccac attacctgca aggctttgat 540

ctttcttctg ccatttccac attggacatc tctgctgagg agagaaaatg aaccactctt 600

ttcctttgta taatgttgtt ttattcttca gacagaagag aggagttata cagctctgca 660

gacatcccat tcctgtatgg ggactgtgtt tgcctcttag aggttcccag gccactagag 720

gagataaagg gaaacagatt gttataactt gatataatga tactataata gatgtaacta 780

caaggagctc cagaagcaag agagagggag gaacttggac ttctctgcat ctttagttgg 840

agtccaaagg cttttcaatg aaattctact gcccagggta cattgatgct gaaaccccat 900

<210> 81

<211> 900

<212> DNA

<213> Artificial sequences

<400> 81

tcaaatctcc tgttatattc tagaacaggg aattgatttg ggagagcatc aggaaggtgg 60

atgatctgcc cagtcacact gttagtaaat tgtagagcca ggacctgaac tctaatatag 120

tcatgtgtta cttaatgacg gggacatgtt ctgagaaatg cttacacaaa cctaggtgtt 180

gtagcctact acacgcatag gctacatggt atagcctatt gctcctagac tacaaacctg 240

tacagcctgt tactgtactg aatactgtgg gcagttgtaa cacaatggta agtatttgtg 300

tatctaaaca tagaagttgc agtaaaaata tgctatttta atcttatgag accactgtca 360

tatatacagt ccatcattga ccaaaacatc atatcagcat ttttttcttct aagattttgg 420

gagcaccaaa gggatacact aacaggatat actctttata atgggtttgg agaactgtct 480

gcagctactt cttttaaaaa ggtgatctac acagtagaaa ttagacaagt ttggtaatga 540

gatctgcaat ccaaataaaa taaattcatt gctaaccttt ttcttttctt ttcaggtttg 600

aagatgccgc atttggattg gatgaattcc aaattctgct tgcttgcttt ttaatattga 660

tatgcttata cacttacact ttatgcacaa aatgtagggt tataataatg ttaacatgga 720

catgatcttc tttataattc tactttgagt gctgtctcca tgtttgatgt atctgagcag 780

gttgctccac aggtagctct aggagggctg gcaacttaga ggtggggagc agagaattct 840

cttatccaac atcaacatct tggtcagatt tgaactcttc aatctcttgc actcaaagct 900

<210> 82

<211> 900

<212> DNA

<213> Artificial sequences

<400> 82

cttgacaagt ctcctgctcc tcactatgaa gatcactgtc ccccagccct gtgctccccg 60

cactgtgctg cacgtccacc tccattccac tgcccctccc atccccccat cttgatagca 120

cccttcccag gtgtcaagct gcccctccta gagtgtcctg cctaaacccc ctctcctggc 180

tcctcccgct acagcatgtt ctctgaggac actaaccacg ctggaccttg aactgggtac 240

ttgtggacac agctcttctc caggctgtat cccatgagcc tcagcatcct ggcacccggc 300

ccctgctggt tcagggttgg cccctgcccg gctgcggaat gaaccacatc ttgctctgct 360

gacagacaca ggcccggctc caggctcctt tagcgcccag ttgggtggat gcctggtggc 420

agctgcggtc cacccaggag ccccgaggcc ttctctgaag gacattgcgg acagccacgg 480

ccaggccaga gggagtgaca gaggcagccc cattctgcct gcccaggccc ctgccaccct 540

ggggagaaag tacttctttt tttttatttt tagacagagt ctcactgttg cccaggctgg 600

cgtgcagtgg tgcgatctgg gttcactgca acctccgcct cttgggttca agcgattctt 660

ctgcttcagc ctcccgagta gctgggacta caggcaccca ccatcatgtc tggctaattt 720

ttcattttta gtagagacag ggttttgcca tgttggccag gctggtctca aactcttgac 780

ctcaggtgat ccacccacct cagcctccca aagtgctggg attacaagcg tgagccactg 840

caccgggcca cagagaaagt acttctccac cctgctctcc gaccagacac cttgacaggg 900

<210> 83

<211> 900

<212> DNA

<213> Artificial sequences

<400> 83

cacaccgggc actcagaaga cactgatggg caacccccag cctgctaatt ccccagattg 60

caacaggctg ggcttcagtg gcagctgctt ttgtctatgg gactcaatgc actgacattg 120

ttggccaaag ccaaagctag gcctggccag atgcaccagc ccttagcagg gaaacagcta 180

atgggacact aatggggcgg tgagagggga acagactgga agcacagctt catttcctgt 240

gtctttttttc actacattat aaatgtctct ttaatgtcac aggcaggtcc agggtttgag 300

ttcataccct gttaccattt tggggtaccc actgctctgg ttatctaata tgtaacaagc 360

caccccaaat catagtggct taaaacaaca ctcacattta ttctgctcac atatctgtca 420

tttgagcagg gctcagcggg gacagctcct tctgtcctac tctgtgtcag gtggggcagc 480

ttgagggttg ggctggtgtc acctgaagac tcattcttct gtacgtctga caggcaatgc 540

tggctgttgg ctgggggcct cagtgccact acggaatagt tggctaggac ccctccatgt 600

gggctagttg ggcttcctca tagtatggtg gctgggttgg agggtgtccc aaaaagaaag 660

gaggggatag agagagacca cttttcataa cctagcctta gaagtcacac agtattactt 720

ctgctacata tatatgtttt aagaggcagg gtctcactct gtcgcccagt ctggaatgca 780

gtggtatgat cacggctcac tgcagcctca acctcctggg ctaagtgatc ctcccacctc 840

agcctcccga atagctggga ctacaggtgt gagtcaccaa gcccagttaa tctttagttt 900

<210> 84

<211> 804

<212> DNA

<213> Artificial sequences

<400> 84

tgctttctct gacctgcatt ctctcccctg ggcctgtgcc gctttctgtc tgcagcttgt 60

ggcctgggtc acctctacgg ctggcccaga tccttccctg ccgcctcctt caggttccgt 120

cttcctccac tccctcttcc ccttgctctc tgctgtgttg ctgcccaagg atgctctttc 180

cggagcactt ccttctcggc gctgcaccac gtgatgtcct ctgagcggat cctccccgtg 240

tctgggtcct ctccgggcat ctctcctccc tcacccaacc ccatgccgtc ttcactcgct 300

gggttccctt ttccttctcc ttctggggcc tgtgccatct ctcgtttctt aggatggcct 360

tctccgacgg atgtctccct tgcgtcccgc ctccccttct tgtaggcctg catcatcacc 420

gtttttctgg acaaccccaa agtaccccgt ctccctggct ttagccacct ctccatcctc 480

ttgctttctt tgcctggaca ccccgttctc ctgtggattc gggtcacctc tcactccttt 540

catttgggca gctcccctac cccccttacc tctctagtct gtgctagctc ttccagcccc 600

ctgtcatggc atcttccagg ggtccgagag ctcagctagt cttcttcctc caacccgggc 660

ccctatgtcc acttcaggac agcatgtttg ctgcctccag ggatcctgtg tccccgagct 720

gggaccacct tatattccca gggccggtta atgtggctct ggttctgggt acttttatct 780

gtcccctcca ccccacagtg gggc 804

<210> 85

<211> 837

<212> DNA

<213> Artificial sequences

<400> 85

actagggaca ggattggtga cagaaaagcc ccatccttag gcctcctcct tcctagtctc 60

ctgatattgg gtctaacccc cacctcctgt taggcagatt ccttatctgg tgacacaccc 120

ccatttcctg gagccatctc tctccttgcc agaacctcta aggtttgctt acgatggagc 180

cagagaggat cctgggaggg agagcttggc agggggtggg agggaagggg gggatgcgtg 240

acctgcccgg ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg agctgctctg 300

acgcggccgt ctggtgcgtt tcactgatcc tggtgctgca gcttccttac acttcccaag 360

aggagaagca gtttggaaaa acaaaatcag aataagttgg tcctgagttc taactttggc 420

tcttcacctt tctagtcccc aatttattatt gttcctccgt gcgtcagttt tacctgtgag 480

ataaggccag tagccagccc cgtcctggca gggctgtggt gaggaggggg gtgtccgtgt 540

ggaaaactcc ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt ggccgcctct 600

actccctttc tctttctcca tccttctttc cttaaagagt ccccagtgct atctgggaca 660

tattcctccg cccagagcag ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt 720

tgagtccttg gcaagcccag gagaggcgct caggcttccc tgtccccctt cctcgtccac 780

catctcatgc ccctggctct cctgcccctt ccctacaggg gttcctggct ctgctct 837

<210> 86

<211> 253

<212> DNA

<213> Artificial sequences

<400> 86

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> 87

<211> 686

<212> DNA

<213> Artificial sequences

<400> 87

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> 88

<211> 22

<212> DNA

<213> Artificial sequences

<400> 88

ccatagctca gtctggtcta tc 22

<210> 89

<211> 22

<212> DNA

<213> Artificial sequences

<400> 89

ctcttcgtcc agatcatcct ga 22

<210> 90

<211> 22

<212> DNA

<213> Artificial sequences

<400> 90

ccatagctca gtctggtcta tc 22

<210> 91

<211> 20

<212> DNA

<213> Artificial sequences

<400> 91

cacaccttgc cgatgtcgag 20

<210> 92

<211> 24

<212> DNA

<213> Artificial sequences

<400> 92

gcactgaacg aacatctcaa gaag 24

<210> 93

<211> 22

<212> DNA

<213> Artificial sequences

<400> 93

ctcttcgtcc agatcatcct ga 22

<210> 94

<211> 24

<212> DNA

<213> Artificial sequences

<400> 94

gcactgaacg aacatctcaa gaag 24

<210> 95

<211> 20

<212> DNA

<213> Artificial sequences

<400> 95

cacaccttgc cgatgtcgag 20

<210> 96

<211> 22

<212> DNA

<213> Artificial sequences

<400> 96

tgctccgggt ttgtctcaga tg 22

<210> 97

<211> 22

<212> DNA

<213> Artificial sequences

<400> 97

ctcttcgtcc agatcatcct ga 22

<210> 98

<211> 22

<212> DNA

<213> Artificial sequences

<400> 98

tgctccgggt ttgtctcaga tg 22

<210> 99

<211> 20

<212> DNA

<213> Artificial sequences

<400> 99

cacaccttgc cgatgtcgag 20

<210> 100

<211> 590

<212> DNA

<213> Artificial sequences

<400> 100

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> 101

<211> 24

<212> DNA

<213> Caenorhabditis elegans

<400> 101

tcacaacctc ctagaaagag taga 24

Claims (22)

1. A pluripotent stem cell or a derivative thereof comprising an expression sequence of a LAG-3 inhibitory factor, The LAG-3 inhibitory factor is at least one of shRNA and shRNA-miR targeting LAG-3; The expression sequence of the LAG-3 inhibitory factor is preferably inserted into the genome of the pluripotent stem cell or derivative thereof. 2. A pluripotent stem cell or a derivative thereof comprising an expression sequence of a LAG-3 inhibitory factor, The LAG-3 inhibitory factor is at least one of shRNA and shRNA-miR targeting LAG-3; The expression sequence of the LAG-3 inhibitory factor is preferably inserted into the genome of the pluripotent stem cell or its derivative; The B2M gene and/or the CIITA gene of the genome of the pluripotent stem cell or its derivative is knocked out. 3. A pluripotent stem cell or a derivative thereof comprising an expression sequence of a LAG-3 inhibitory factor, The LAG-3 inhibitory factor is at least one of shRNA and shRNA-miR targeting LAG-3; The expression sequence of the LAG-3 inhibitory factor is preferably inserted into the genome of the pluripotent stem cell or its derivative; A first nucleic acid molecule is inserted into the genome of the pluripotent stem cell or its derivative; And, a second nucleic acid molecule is inserted into the 3'UTR region of the immune response-related gene in the pluripotent stem cell or its derivative; The first nucleic acid molecule encodes a small nucleic acid molecule that mediates RNA interference, the small nucleic acid molecule specifically targets the transcription product of the second nucleic acid molecule, and the small nucleic acid molecule does not target the pluripotent stem cell or its Derivatives of any other mRNA or lncRNA. 4. The pluripotent stem cell or its derivative according to claim 3, wherein the pluripotent stem cell or its derivative is derived from human; the sequence of the small nucleic acid molecule is one that does not target any human mRNA or lncRNA. Random sequences of non-human species; The small nucleic acid molecule preferably includes short interfering nucleic acid, short interfering RNA, and double-stranded RNA, preferably at least one of miRNA, shRNA, and shRNA-miR. 5. The pluripotent stem cell or derivative thereof according to claim 4, wherein the second nucleic acid molecule comprises at least 3 repeats of the reverse complement of the sequence of the small nucleic acid molecule, preferably 6 to 10 repeats The reverse complement of the small nucleic acid molecule sequence. 6. A pluripotent stem cell or a derivative thereof comprising an expression sequence of a LAG-3 inhibitory factor, The LAG-3 inhibitory factor is at least one of shRNA and shRNA-miR targeting LAG-3; The expression sequence of the LAG-3 inhibitory factor is preferably inserted into the genome of the pluripotent stem cell or its derivative; The pluripotent stem cell or its derivative further comprises an immune-compatible molecule expression sequence, and the immune-compatible molecule is used to regulate the expression of genes related to immune response in the pluripotent stem cell or its derivative; The immunocompatible molecule expression sequence is preferably inserted into the genome of the pluripotent stem cell or a derivative thereof. 7. The pluripotent stem cell or derivative thereof according to claim 6, wherein the immune compatible molecule comprises one or more of the following: (I) immune tolerance-related genes, including CD47 or HLA-G; (II) 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; (III) shRNA and/or shRNA-miR targeting the gene related to immune response. 8. The pluripotent stem cell or derivative thereof according to any one of claims 3 to 7, wherein the immune response-related gene comprises: (I) 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; (II) Major histocompatibility complex-related genes, including at least one of B2M and CIITA. 9. The pluripotent stem cell or its derivative according to claim 8, wherein The target sequence of shRNA and/or shRNA-miR targeting B2M is selected from one of SEQ ID NO.3 to SEQ ID NO.5; The target sequence of the shRNA and/or shRNA-miR targeting CIITA is selected from one of SEQ ID NO.6 to SEQ ID NO.15; The target sequence of shRNA and/or shRNA-miR targeting HLA-A is selected from one of SEQ ID NO.16 to SEQ ID NO.18; The target sequence of shRNA and/or shRNA-miR targeting HLA-B is selected from one of SEQ ID NO.19 to SEQ ID NO.24; The target sequence of shRNA and/or shRNA-miR targeting HLA-C is selected from one of SEQ ID NO.25-SEQ ID NO.30; The target sequence of shRNA and/or shRNA-miR targeting HLA-DRA is selected from one of SEQ ID NO.31 to SEQ ID NO.40; The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB1 is selected from one of SEQ ID NO.41 to SEQ ID NO.45; The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB3 is selected from one of SEQ ID NO.46 to SEQ ID NO.47; The target sequence of the shRNA and/or shRNA-miR targeting HLA-DRB4 is selected from one of SEQ ID NO.48 to SEQ ID NO.57; The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB5 is selected from one of SEQ ID NO.58-SEQ ID NO.66; The target sequence of shRNA and/or shRNA-miR targeting HLA-DQA1 is selected from one of SEQ ID NO.67-SEQ ID NO.73; The target sequence of shRNA and/or shRNA-miR targeting HLA-DQB1 is selected from one of SEQ ID NO.74-SEQ ID NO.83; The target sequence of shRNA and/or shRNA-miR targeting 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 targeting HLA-DPB1 is selected from one of SEQ ID NO. 94 to SEQ ID NO. 103. 10. The pluripotent stem cell or its derivative according to any one of claims 1 to 9, wherein the pluripotent stem cell or its derivative genome is also inserted with shRNA processing complex-related genes, miRNA processing at least one of complex-related genes and anti-interferon effector molecules; Wherein, the shRNA processing complex-related genes and miRNA processing complex-related genes include at least one of Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2), PACT (PRKRA), and DGCR8; The interferon effector molecule is preferably a shRNA and/or shRNA-miR targeting at least one of PKR, 2-5As, IRF-3 and IRF-7. 11. The pluripotent stem cell or its derivative according to claim 10, wherein, The target sequence of shRNA targeting PKR and/or shRNA-miR is selected from one of SEQ ID NO.104-SEQ ID NO.113; The target sequence of 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 shRNA and/or shRNA-miR targeting IRF-3 is selected from one of SEQ ID NO.144-SEQ ID NO.153; The target sequence of shRNA and/or shRNA-miR targeting IRF-7 is selected from one of SEQ ID NO.154-SEQ ID NO.163. 12. The pluripotent stem cell or its derivative according to any one of claims 1 to 11, characterized in that, The expression framework of the shRNA: from 5' to 3', it includes the shRNA target sequence, the stem-loop sequence, the reverse complement of the shRNA target sequence, and Poly T in turn; 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 the transcription terminator of RNA polymerase III; shRNA-miR expression framework: obtained by replacing the target sequence in microRNA-30 or microRNA-155 with the shRNA-miR target sequence. 13. The pluripotent stem cell or its derivative according to any one of claims 3, 6 or 10, wherein the pluripotent stem cell or its derivative further comprises an inducible gene expression system; A gene expression system for regulating the expression of a first nucleic acid molecule and/or an immune-compatible molecule and/or a shRNA and/or miRNA processing complex-related gene and/or an anti-interferon effector molecule; The inducible gene expression system is preferably inserted into the genome of the pluripotent stem cell or a derivative thereof. 14. The pluripotent stem cell or its derivative according to claim 13, wherein the inducible gene expression system comprises at least one of a Tet-Off system and a dimer inducible expression system. 15. The pluripotent stem cell or its derivative according to any one of claims 1 to 14, wherein the pluripotent stem cell or its derivative further comprises an exosome processing and synthesis gene; the exosome The processed synthetic gene is preferably inserted into the genome of the pluripotent stem cell or derivative thereof. 16. The pluripotent stem cell or derivative thereof according to claim 15, wherein the exosome processing synthesis gene comprises STEAP3, Syndevan-4, L-aspartate oxidase fragment, CD63-L7Ae and At least one of the Cx43 S368A. 17. The pluripotent stem cell or derivative thereof according to any one of claims 1-16, wherein the expression sequence of the LAG-3 inhibitory factor, the expression sequence of the first nucleic acid molecule, the expression sequence of the immune compatible molecule, the shRNA and/or miRNA processing complex-related genes, anti-interferon effector molecules, inducible gene expression systems, and exosome processing synthetic genes are inserted into the pluripotent stem cells by means of viral vector interference, non-viral vector transfection or gene editing methods In the genome of a derivative thereof, the gene editing is preferably a gene knock-in. 18. The pluripotent stem cell or derivative thereof according to any one of claims 1-17, wherein the expression sequence of the LAG-3 inhibitory factor, the expression sequence of the first nucleic acid molecule, the expression sequence of the immune compatible molecule, the shRNA and/or miRNA processing complex-related genes, anti-interferon effector molecules, inducible gene expression systems, exosome processing synthetic genes are inserted into the genomic safety site of the pluripotent stem cell or its derivative, the safety site It is preferably one or more of the AAVS1 security site, the eGSH security site and the H11 security site. 19. The pluripotent stem cell or its derivative according to any one of claims 1 to 18, wherein the sequence of the shRNA and/or shRNA-miR targeting LAG-3 is as shown in SEQ ID NO.1 Show. 20. The pluripotent stem cell or derivative thereof according to any one of claims 1 to 19, 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 differentiated from pluripotent stem cells, cells of each germ layer, tissues, and organs; and the adult stem cells include mesenchymal stem cells or neural stem cells. 21. Use of the pluripotent stem cells or their derivatives according to any one of claims 1 to 20 in the preparation of a tumor therapeutic drug. 22. An exosome secreted from the pluripotent stem cell or a derivative thereof according to any one of claims 1 to 20.

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