CN114657130A - Pluripotent stem cell expressing VEGF-A targeted inhibitory factor, derivative and application thereof - Google Patents

Abstract

The invention discloses a pluripotent stem cell expressing a VEGF-A targeting inhibitor and its derivative and application. An expression sequence of a VEGF-A inhibitor is introduced into the genome of the pluripotent stem cell or its derivative, and the VEGF-A inhibitor is at least one of shRNA and/or shRNA-miR targeting VEGF-A. After the VEGF-A inhibitory factor expressed by VEGF-A pluripotent stem cells or their derivatives is encapsulated by exosomes, the exosomes carry the VEGF-A inhibitory factor and bind to the target cells, releasing the VEGF-A inhibitory factor contained in the exosomes, thereby preventing the VEGF-A inhibitory factor. Blocking the VEGF-A pathway, releasing immunosuppression and activating the immune system can effectively remove tumor cells and treat macular degeneration.

Description

A pluripotent stem cell expressing VEGF-A 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 VEGF-A targeting inhibitor, a derivative thereof and an application thereof.

Background technique

VEGF-A (vascular endothelial growth factor A) is a member of the PDGF/VEGF family of growth factors. VEGF-A encodes a heparin-binding protein that exists as a disulfide-linked homodimer. According to the prior art, VEGF-A has a certain role in angiogenesis, angiogenesis and endothelial cell growth, and can induce endothelial cell proliferation, promote cell migration, inhibit apoptosis and induce vascular permeability. Angiogenesis is required. The VEGF-A gene is upregulated in many known tumors, and its expression correlates with tumor stage and progression.

On the other hand, in the field of cell therapy, the issue of allogeneic immunocompatibility is still a major problem. 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. 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 HLA-C antigens of HLA-C antigens vary greatly by ethnicity. Through our verification and calculation, some regions can only account for 70% of the proportion, and many countries with large populations in the world 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.

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

The 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 applications of the pluripotent stem cells or derivatives thereof and the exosomes secreted by them in the preparation of macular degeneration treatment 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 pluripotent stem cell or a derivative thereof comprising a VEGF-A targeting inhibitor, the VEGF-A targeting inhibitor comprising at least one of VEGF-A-expressing shRNA and shRNA-miR A; the sequences of the VEGF-A-expressing shRNA and shRNA-miR are preferably inserted into the genome of the pluripotent stem cell or a derivative thereof.

The inventors found that after the expression sequence of VEGF-A inhibitory factor was introduced into pluripotent stem cells or derivatives thereof, the VEGF-A inhibitory factors targeting VEGF-A secreted in pluripotent stem cells or their derivatives were similar to Binding to target cells can effectively remove tumor cells and inhibit macular degeneration.

Further, the sequences of the VEGF-A-targeting shRNA and shRNA-miR are shown in SEQ ID NO.1 to SEQ ID NO.2.

A second aspect of the present invention provides:

A pluripotent stem cell or a derivative thereof, the pluripotent stem cell or a derivative thereof comprising a VEGF-A targeting inhibitor, the VEGF-A targeting inhibitor comprising at least one of VEGF-A-expressing shRNA and shRNA-miR One; the sequences of the shRNA and shRNA-miR expressing VEGF-A are preferably inserted into the genome of the pluripotent stem cell or a derivative thereof;

The B2M gene and/or the CIITA gene of 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 their tumor and macular degeneration treatments were the best.

A third aspect of the present invention provides:

A pluripotent stem cell or a derivative thereof, the pluripotent stem cell or a derivative thereof comprising a VEGF-A targeting inhibitor, the VEGF-A targeting inhibitor comprising at least one of VEGF-A-expressing shRNA and shRNA-miR One; the sequences of the shRNA and shRNA-miR expressing VEGF-A are preferably inserted into the genome of the pluripotent stem cell or a derivative thereof;

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. 101);

5'-TCACAACCTCCTAGAAAGAGTAGA-3' (SEQ ID NO. 102).

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 pluripotent stem cell or a derivative thereof comprising a VEGF-A targeting inhibitor, the VEGF-A targeting inhibitor comprising at least one of VEGF-A-expressing shRNA and shRNA-miR A; the sequences of the VEGF-A-expressing shRNA and shRNA-miR are preferably inserted into the genome of the pluripotent stem cell or a derivative thereof.

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 VEGF-A in the recipient. After these inhibitory factors are encapsulated by exosomes, the exosomes carry them to bind to the target cells, and then release them, thereby preventing the inhibition of VEGF-A. Blocking the VEGF-A pathway, relieves immunosuppression, activates the immune system, can effectively remove tumor cells and improve macular degeneration.

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 Cx43 S368A.

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 VEGF-A 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 expression sequence of the VEGF-A inhibitor, the first nucleic acid molecule, 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 VEGF-A inhibitor, the expression sequence of the immune compatible molecule, the shRNA and/or miRNA processing complex related genes, the anti-interferon effect The introduction site of molecules, inducible gene expression systems, and exosome processing synthetic genes is a genome safety site, preferably one or more of the AAVS1 safety site, eGSH safety site, and H11 safety site.

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:

Use of the above-mentioned pluripotent stem cells or derivatives thereof and exosomes secreted by them in the preparation of macular degeneration 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. After introducing the expression sequence of shRNA/shRNA-miR targeting VEGF-A inhibitory factor of VEGF-A into the genome of iPSCs induced by autologous cells, iPSCs can express shRNA/shRNA-miR targeting VEGF-A in large quantities, and Encapsulated by exosomes secreted by cells. The exosomes carry these inhibitory factors to bind to target cells and release them, thereby blocking the VEGF-A pathway, relieving immunosuppression, activating the immune system, and restoring T cell activity, enabling it 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 VEGF-A in the recipient. After these inhibitory factors are encapsulated by exosomes, the exosomes carry them to bind to the target cells, and then release them, thereby preventing the inhibition of VEGF-A. Blocking the VEGF-A pathway, relieves immunosuppression, activates the immune system, can effectively remove tumor cells and improve macular degeneration.

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. An inducible gene expression system is also introduced into the genome of the pluripotent stem cell or its derivative, which is used to regulate the expression of the first nucleic acid molecule, so as to realize the immunocompatibility and reversibility of the pluripotent stem cell or its derivative.

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/shRNA-miR targeting VEGF-A in the recipient. After these inhibitory factors are encapsulated by exosomes, the exosomes carry them to bind to the target cells, and then release them, thereby preventing the inhibition of VEGF-A. Blocking the VEGF-A pathway, relieves immunosuppression, activates the immune system, can effectively remove tumor cells and improve macular degeneration.

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 remove 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 AAVS1 KI Vector (shRNA, constitutive).

Figure 13 is a plasmid map of AAVS1 KI Vector (shRNA, inducible).

Figure 14 is a plasmid map of AAVS1 KI Vector (shRNA-miR, constitutive).

Figure 15 is a plasmid map of AAVS1 KI 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 VEGF-A Targeting Inhibitors

VEGF-A targeting inhibitor: shRNA or shRNA-miR targeting VEGF-A.

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

Table 1 Target sequences of shRNA or shRNA-miR sequences targeting VEGF-A

2. Construction of small nucleic acid molecules

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

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

5'-TTGTACTACACAAAAGTACTG-3' (SEQ ID NO. 101);

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 Target 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 Cx43 S368A at least one of them. Among them, Cx43 S368A 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--hmiR302 cluster 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 BioCISO medium without other substances Continue to culture for about 17 days, pick iPSCs cloned cells, and the picked iPSCs cloned cells are purified, digested, and passaged to obtain stable iPSCs. For the specific construction method, please refer to: Stem Cell Res Ther. 2017Nov 2;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 adopts the CRISPR-Cas9 gene editing system, and the Cas 9 protein used is Cas 9 (D10A), and Cas 9 (D10A) is combined with sgRNA, and the sgRNA is responsible for the specific recognition of the target sequence (genomic DNA of the cell vector), Cas 9 (D10A) then single-stranded cleavage of the target sequence. Genomic DNA double-strand break (DNA DoubleStrand Break, DSB), there must be two sets of Cas 9 (D10A)/sgRNA to cut the two strands of the genomic DNA of the cell vector respectively, and the cutting distance should not be too far. The advantages of Cas 9(D10A)/sgRNA protocol compared with Cas 9/sgRNA protocol are 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 AAVS1 KI 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 wrapping 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) Incubate the cells in a 37°C, 5% CO ₂ incubator for 5 to 10 minutes (digested until most of the cells are contracted 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 no target band will appear.

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 groups in Tables 8 and 9 below, the sequences of VEGF-A targeting inhibitors, immune-compatible molecules, shRNA/miRNA processing complex genes, anti-interferon effector molecules, exocytosis The body-processed synthetic gene is knocked into the safe site of the 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 AAVS1 KI Vector (shRNA, constitutive) plasmid was placed into the shRNA sequence targeting VEGF-A.

(2) Aa2 grouping:

The shRNA expression frame 2 of the AAVS1 KI Vector (shRNA, constitutive) plasmid was placed into the shRNA sequence targeting VEGF-A, and the shRNA expression frame 1 was placed into the remaining shRNA sequences (including the target sequence of B2M/CIITA-3'UTR-shRNA). ), the MCS is placed 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 AAVS1 KI Vector (shRNA, constitutive) plasmid was placed into the shRNA sequence targeting VEGF-A, the shRNA expression framework 1 was placed into the remaining shRNA target sequences, B2M and CIITA were knocked out, and MCS was placed in the gene sequence.

(4) Aa4 grouping:

The shRNA expression framework 2 of the AAVS1 KI Vector (shRNA, constitutive) plasmid is placed into the shRNA sequence targeting VEGF-A, the shRNA expression framework 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 AAVS1 KI Vector (shRNA-miR, constitutive) plasmid was placed into the shRNA-miR sequence targeting VEGF-A.

(6) Ab2 grouping:

The shRNA-miR expression framework 2 of the AAVS1 KI Vector (shRNA-miR, constitutive) plasmid was placed into the shRNA-miR sequence targeting VEGF-A, and the shRNA-miR expression framework 1 was placed into the remaining shRNA-miR target sequences (including B2M /CIITA-3'UTR-shRNA-miR target sequence), 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.

(7) Ab3 grouping:

The shRNA-miR expression framework 2 of the AAVS1 KI Vector (shRNA-miR, constitutive) plasmid was placed into the shRNA-miR sequence targeting VEGF-A, and the shRNA-miR expression framework 1 was placed into the remaining shRNA-miR target sequences, knocked out B2M and CIITA, MCS into the gene sequence.

(8) Ab4 grouping:

The shRNA-miR expression frame 2 of the AAVS1 KI Vector (shRNA-miR, constitutive) plasmid is placed into the shRNA-miR sequence targeting VEGF-A, the shRNA-miR expression frame 1 is placed into the remaining shRNA-miR target sequences, and the MCS is placed into the 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 AAVS1 KI Vector (shRNA, inducible) plasmid is placed into the shRNA sequence targeting VEGF-A, and the shRNA expression frame 1 is placed into the remaining shRNA target sequences (including the target of B2M/CIITA-3'UTR-shRNA). sequence), 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 AAVS1 KI Vector (shRNA-miR, inducible) plasmid is placed into the shRNA-miR sequence targeting VEGF-A, and the shRNA-miR expression frame 1 is placed into the remaining shRNA-miR target sequences (including B2M /CIITA-3'UTR-shRNA-miR target sequence), MCS 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 AAVS1 KI Vector (shRNA, inducible) plasmid is placed into the shRNA sequence targeting VEGF-A, and the shRNA expression frame 1 is placed into the remaining shRNA target sequences (including the target of B2M/CIITA-3'UTR-shRNA). sequence), 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 AAVS1 KI Vector (shRNA-miR, inducible) plasmid is placed into the shRNA-miR sequence targeting VEGF-A, and the shRNA-miR expression frame 1 is placed into the remaining shRNA-miR target sequences (including B2M /CIITA-3'UTR-shRNA-miR target sequence), MCS 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 sequence of one or more immune-compatible molecules are knocked into the genome safety site 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 VEGF-A targeting inhibitor on VEGF-A:

The protocols of each experimental group in Table 8 and Table 9 were knocked into the genomic safety sites of iPSCs, MSCs, NSCs, and EBs cells, cultured at 37°C in a 0.5% CO ₂ incubator, and the supernatant of the medium was collected, containing the knock-in exosome processing. Cells with synthetic gene sequences were used to extract exosomes from the culture supernatant using an exosome extraction kit (BestBio, lot #BB-3901). The culture supernatant was centrifuged at 3000g for 15min at 37°C, the supernatant was collected and centrifuged at 10,000g for 20min at 37°C to collect the supernatant, add extract A at a ratio of 4:1, invert upside down for 1min, and overnight at 37°C. Centrifuge at 10,000 g for 60 min at 4°C, and collect the precipitate.

The blocking effect of VEGF-A inhibitory factor expressed by pluripotent stem cells was then tested by flow cytometry. The exosomes containing VEGF-A inhibitor were added to the medium of CHO cells, and the CHO cells expressing VEGF-A were cultured for 72 h. After being digested into single cells and washed twice with PBS, the FITC-labeled VEGF-A antibody fusion protein was added to the test tube and incubated at 37°C for 30 minutes. Flow cytometric analysis was performed using a flow cytometer. According to the mean fluorescence intensity (MFI) of staining, the effect of VEGF-A inhibitor expressed by pluripotent stem cells on inhibiting the expression of VEGF-A by CHO cells can be measured.

Group N (control) refers to VEGF-A-expressing CHO cells cultured without the addition of VEGF-A inhibitor or exosomes containing VEGF-A inhibitor.

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

Table 10 VEGF-A expression results of cells expressing VEGF-A detected by flow cytometry

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

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

Detection of anti-tumor cell migration effect of pluripotent stem cells or their derivatives expressing VEGF-A targeting inhibitor:

VEGF-A targeting inhibitor-containing exosomes are extracted from the culture supernatant of the VEGF-A targeting inhibitory factor-expressing pluripotent stem cells or derivatives thereof, and added to serum-free basal medium to treat tumors. HepG2 cells were starved for 12 hours, and tumor cells hepG2 were digested and resuspended in serum-free basal medium containing VEGF-A inhibitory exosomes to adjust the cell number to 1X10 ⁶ cells/mL. 100 μL of cells were seeded into the upper chamber of an 8.0 μm Transwell chamber of a 24-well plate, and 500 μL of basal medium containing 15% FBS was added to the lower chamber, and cultured at 37° C. and 5% CO ₂ for 24 h. The control group used the culture supernatant of pluripotent stem cells and their derivatives that did not express VEGF-A antibody, and other operations were the same as the experimental group. The cells on the upper layer of the filter membrane in the experimental group and the control group were wiped with cotton swabs, fixed with methanol for 5 min, and stained with Giemsa dye for 15 min. Under the 10x objective lens, the transmembrane cells in 5 different fields of view in the upper, lower, left and right are selected for counting, and the average value is calculated.

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

Table 11 Effects of VEGF-A targeting inhibitor expressed in each group on tumor cell migration

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

Through the above experiments, it can be proved that the VEGF-A inhibitory factor encapsulated by exosomes expressed by the pluripotent stem cells or derivatives thereof prepared by the present invention can effectively inhibit the migration of tumor cells.

The application of pluripotent stem cells expressing VEGF-A targeted inhibitory factors in the treatment of various tumors:

Selected B2M and CIITA knockout protocol group (Aa3) immune-compatible cells (MSCs) were tested.

In the humanized NSG mouse tumor model, each group of experimental cells was injected to observe the therapeutic effect on RCC kidney cancer, MC colon cancer, and NIC lung cancer. To avoid immunocompatibility issues, the immune cells and hPSCs-derived derivatives used were all derived from the same human.

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 of PBS (containing human immune cells and 1×10 ⁶ pluripotent stem cell derivatives expressing exosome-encapsulated VEGF-A inhibitory factor) for tumor treatment, in which only human immune cells were injected. group as the 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 results are shown in Table 12.

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

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

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

Immunocompatibility testing of pluripotent stem cells expressing targeted VEGF-A inhibitors in tumor models:

Taking advantage of the low immunogenicity of MSCs, in a humanized NSG mouse tumor model, they were injected with hPSCs-derived immune-compatible MSCs capable of expressing VEGF-A inhibitory factor (shRNA targeting VEGF-A), and the results were observed. Effect of tumor (NIC lung cancer) therapy. 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;

Add Dox group: starting from the injection of cells expressing inhibitory factors, continue to add 0.5 mg/mL Dox to the mouse diet to feed the mice until the end of the experiment.

The results 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 expressing only inhibitory factors (group 2) 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 immunocompatibility transformation (groups 3-7) , including constitutive and reversible inducible immune compatibility), its immune compatibility effect is better, and its existence in the body is longer than that of MSCs without immune compatibility modification (or can achieve long-term coexistence), and its tumor treatment effect is better, And 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.

Test of the therapeutic effect of pluripotent stem cells expressing targeting VEGF-A inhibitor on macular degeneration:

Select B2M and CIITA gene knockout protocol group (Aa3) immunocompatible cells for testing.

Construction of a macular degeneration mouse model:

In humanized NSG mice (The Jackson Laboratory (JAX)), human immune cells from the same donor were injected to reconstitute the immune system of the mice. After 2 weeks, macular degeneration mice were established by retinal laser injury choroidal neovascularization (CNV).

The specific construction method is as follows: the mouse model was anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital, the injection volume was 45 mg per kg, and the eyes were dilated with 0.5% tropicamide + 0.5% phenylephrine hydrochloride. Under the slit lamp, use a 532nm double-ton Nd:YAG laser (spot diameter 75lrm, exposure time 100ms, energy 180mW) through the front mirror, about 1-1.5PD from the optic disc, shoot the retina around the optic disc, and shoot each eye 8-10 points, avoiding direct shots of retinal vessels. Subsequently, 200 uL of PBS (containing 10 ⁶ of VEGF-A inhibitor-expressing pluripotent stem cell derivatives, which are derived from the same donor as human immune cells) was injected into the tail vein to induce macular degeneration.

Detection method: Fundus angiography (FFA) was used, 20% sodium fluorescein was diluted with water for injection into 2% sodium fluorescein, injected intraperitoneally with an injection volume of 0.3 mL, and the angiography was recorded using a Heidelberg fundus fluorescein angiography (FFA) instrument. process. The treatment effect was judged by the CNV formation rate.

The pluripotent stem cell derivatives expressing VEGF-A inhibitory factors in this example are hPSCs and hPSCs-derived derivatives (hPSCs-MSCs, hPSCs-NSCs, hPSCs-EBs) expressing shRNA targeting VEGF-A.

The formation rate of CNV was judged according to the integral of FFA fluorescence leakage intensity.

The formula is as follows:

The intensity of fluorescein leakage is judged with reference to the Takehana grading standard, namely:

Grade 0: no fluorescent leakage;

Grade 1: mild fluorescent leakage;

Grade 2: moderate fluorescence leakage;

Grade 3: Strong fluorescent leakage.

Integral calculation of fluorescence leakage intensity:

The total score of each laser spot is set to 3 points, with 0 points for level 0; 1 point for level 1; 2 points for level 2; and 3 points for level 3.

The results are shown in Table 14.

Table 14. Therapeutic effect of VEGF-A inhibitory factor-expressing pluripotent stem cells and their derivatives on macular degeneration

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

The results show that the VEGF-A inhibitory factor-expressing stem cells or derivatives thereof prepared by the present invention can effectively block VEGF-A and play a therapeutic role in macular degeneration.

Immunocompatibility testing of pluripotent stem cells expressing targeted VEGF-A inhibitors in a macular degeneration model:

Taking advantage of the low immunogenicity of MSCs, in a humanized NSG mouse model of macular degeneration, they were injected with hPSCs-derived immune-compatible MSCs capable of expressing VEGF-A inhibitor (shRNA targeting VEGF-A). Its macular degeneration treatment effect. Among them, the used immune cells and hPSCs-derived MSCs are not from the same person.

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

Add Dox group: starting from the injection of cells expressing inhibitory factors, continue to add 0.5 mg/mL Dox to the mouse diet to feed the mice until the end of the experiment.

The results are shown in Table 15.

Table 15 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 expressing only inhibitory factors (group 2) have low immunogenicity and can exist in the allogene for a certain period of time, so they can exert a certain therapeutic effect on macular degeneration. 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 the treatment of macular degeneration. The group 4 is the B2M and CIITA gene knockout group, which completely eliminates the effects of HLA-I and HLA-II molecules, so the treatment effect of macular degeneration 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 treatment effect of macular degeneration is also comparable to that of MSCs without immunocompatibility modification.

The described embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the described embodiments, and any other changes, modifications, substitutions, Combinations and simplifications should all be equivalent replacement modes, 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 VEGF-A targeting inhibitor and its derivative and application

<130>

<160> 102

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> DNA

<213> human

<400> 1

gagtacatct tcaagccatc c 21

<210> 2

<211> 21

<212> DNA

<213> human

<400> 2

gcagattatg cggatcaaac c 21

<210> 3

<211> 315

<212> DNA

<213> Artificial sequences

<400> 3

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

<210> 4

<211> 9

<212> DNA

<213> Artificial sequences

<400> 4

ttcaagaga 9

<210> 5

<211> 119

<212> DNA

<213> Artificial sequences

<400> 5

gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta 60

cttcttcagg ttaacccaac agaaggctaa agaaggtata ttgctgttga cagtgagcg 119

<210> 6

<211> 19

<212> DNA

<213> Artificial sequences

<400> 6

tagtgaagcc acagatgta 19

<210> 7

<211> 119

<212> DNA

<213> Artificial sequences

<400> 7

tgcctactgc ctcggacttc aaggggctac tttaggagca attatcttgt ttactaaaac 60

tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat ggtataaat 119

<210> 8

<211> 210

<212> DNA

<213> Artificial sequences

<400> 8

attctagata cagtactttt gtgtagtaca acgtacagta cttttgtgta gtacaacgta 60

cagtactttt gtgtagtaca acgtacagta cttttgtgta gtacaacgta cagtactttt 120

gtgtagtaca acgtacagta cttttgtgta gtacaacgta cagtactttt gtgtagtaca 180

acgtacagta cttttgtgta gtacaacgta 210

<210> 9

<211> 21

<212> DNA

<213> human

<400> 9

gggagcagag aattctctta t 21

<210> 10

<211> 21

<212> DNA

<213> human

<400> 10

ggagcagaga attctcttat c 21

<210> 11

<211> 21

<212> DNA

<213> human

<400> 11

gagcagagaa ttctcttatc c 21

<210> 12

<211> 21

<212> DNA

<213> human

<400> 12

gctacctgga gcttcttaac a 21

<210> 13

<211> 21

<212> DNA

<213> human

<400> 13

ggagcttctt aacagcgatg c 21

<210> 14

<211> 21

<212> DNA

<213> human

<400> 14

gggtctccag tatattcatc t 21

<210> 15

<211> 21

<212> DNA

<213> human

<400> 15

gctcccactc catgaggtat t 21

<210> 16

<211> 21

<212> DNA

<213> human

<400> 16

ggtatttctt cacatccgtg t 21

<210> 17

<211> 21

<212> DNA

<213> human

<400> 17

aggagacacg gaatgtgaag g 21

<210> 18

<211> 21

<212> DNA

<213> human

<400> 18

gctcccactc catgaggtat t 21

<210> 19

<211> 21

<212> DNA

<213> human

<400> 19

ggtatttcta cacctccgtg t 21

<210> 20

<211> 21

<212> DNA

<213> human

<400> 20

ggaccggaac acacagatct a 21

<210> 21

<211> 21

<212> DNA

<213> human

<400> 21

ttcttacttc cctaatgaag t 21

<210> 22

<211> 21

<212> DNA

<213> human

<400> 22

aagttaagaa cctgaatata a 21

<210> 23

<211> 21

<212> DNA

<213> human

<400> 23

aacctgaata taaatttgtg t 21

<210> 24

<211> 21

<212> DNA

<213> human

<400> 24

gggtctggtg ggcatcatta t 21

<210> 25

<211> 21

<212> DNA

<213> human

<400> 25

ggtctggtgg gcatcattat t 21

<210> 26

<211> 21

<212> DNA

<213> human

<400> 26

gcatcattat tgggaccatc t 21

<210> 27

<211> 21

<212> DNA

<213> human

<400> 27

gatgaccaca ttcaaggaag a 21

<210> 28

<211> 21

<212> DNA

<213> human

<400> 28

gaccacattc aaggaagaac t 21

<210> 29

<211> 21

<212> DNA

<213> human

<400> 29

gctttcctgc ttggcagtta t 21

<210> 30

<211> 21

<212> DNA

<213> human

<400> 30

gcgtaagtct gagtgtcatt t 21

<210> 31

<211> 21

<212> DNA

<213> human

<400> 31

gacaatttaa ggaagaatct t 21

<210> 32

<211> 21

<212> DNA

<213> human

<400> 32

ggccatagtt ctccctgatt g 21

<210> 33

<211> 21

<212> DNA

<213> human

<400> 33

gccatagttc tccctgattg a 21

<210> 34

<211> 21

<212> DNA

<213> human

<400> 34

gcagatgacc acattcaagg a 21

<210> 35

<211> 21

<212> DNA

<213> human

<400> 35

gcagcaggat aagtatgagt g 21

<210> 36

<211> 21

<212> DNA

<213> human

<400> 36

gcaggataag tatgagtgtc a 21

<210> 37

<211> 21

<212> DNA

<213> human

<400> 37

ggttcctgca cagagacatc t 21

<210> 38

<211> 21

<212> DNA

<213> human

<400> 38

ggatgtggaa cccacagata c 21

<210> 39

<211> 21

<212> DNA

<213> human

<400> 39

gatgtggaac ccacagatac a 21

<210> 40

<211> 21

<212> DNA

<213> human

<400> 40

gtggaaccca cagatacaga g 21

<210> 41

<211> 21

<212> DNA

<213> human

<400> 41

gggtagcaac tgtcaccttg a 21

<210> 42

<211> 21

<212> DNA

<213> human

<400> 42

ggatttcgtg ttccagttta a 21

<210> 43

<211> 21

<212> DNA

<213> human

<400> 43

gcatgtgcta cttcaccaac g 21

<210> 44

<211> 21

<212> DNA

<213> human

<400> 44

gctcacagtc atcaattata g 21

<210> 45

<211> 21

<212> DNA

<213> human

<400> 45

gccctgaaga cagaatgttc c 21

<210> 46

<211> 21

<212> DNA

<213> human

<400> 46

gcggaccatg tgtcaactta t 21

<210> 47

<211> 21

<212> DNA

<213> human

<400> 47

gcctgatagg acccatattc c 21

<210> 48

<211> 21

<212> DNA

<213> human

<400> 48

gcatccaata gacgtcattt g 21

<210> 49

<211> 21

<212> DNA

<213> human

<400> 49

gcgtcactgg cacagatata a 21

<210> 50

<211> 21

<212> DNA

<213> human

<400> 50

ggatggattt gattatgatc c 21

<210> 51

<211> 21

<212> DNA

<213> human

<400> 51

ggaccttgga acaatggatt g 21

<210> 52

<211> 21

<212> DNA

<213> human

<400> 52

gctaattctt gctgaacttc t 21

<210> 53

<211> 21

<212> DNA

<213> human

<400> 53

gcagttctgt tgccactctc t 21

<210> 54

<211> 21

<212> DNA

<213> human

<400> 54

gggagagttc atccaggaaa t 21

<210> 55

<211> 21

<212> DNA

<213> human

<400> 55

ggagagttca tccaggaaat t 21

<210> 56

<211> 21

<212> DNA

<213> human

<400> 56

gggttggttt atccaggaat a 21

<210> 57

<211> 21

<212> DNA

<213> human

<400> 57

ggatcagaag agaagccaac g 21

<210> 58

<211> 21

<212> DNA

<213> human

<400> 58

ggttcaccat ccaggtgttc a 21

<210> 59

<211> 21

<212> DNA

<213> human

<400> 59

ggaggaactt tgtgaacatt c 21

<210> 60

<211> 21

<212> DNA

<213> human

<400> 60

gctgtaagaa ggatgctttc a 21

<210> 61

<211> 21

<212> DNA

<213> human

<400> 61

gctgcaggca ggattgtttc a 21

<210> 62

<211> 19

<212> DNA

<213> human

<400> 62

gcctcgagtt tgagagcta 19

<210> 63

<211> 19

<212> DNA

<213> human

<400> 63

agacattctg gatgagtta 19

<210> 64

<211> 19

<212> DNA

<213> human

<400> 64

gggtctgtta cccaaagaa 19

<210> 65

<211> 21

<212> DNA

<213> human

<400> 65

ggacactggt tcaacacctg t 21

<210> 66

<211> 21

<212> DNA

<213> human

<400> 66

ggttcaacac ctgtgacttc a 21

<210> 67

<211> 21

<212> DNA

<213> human

<400> 67

acctgtgact tcatgtgtgc g 21

<210> 68

<211> 1607

<212> DNA

<213> human

<400> 68

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

<210> 69

<211> 411

<212> DNA

<213> human

<400> 69

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

<210> 70

<211> 20

<212> DNA

<213> Artificial sequences

<400> 70

tataaggtgg tcccagctcg 20

<210> 71

<211> 20

<212> DNA

<213> Artificial sequences

<400> 71

agggccggtt aatgtggctc 20

<210> 72

<211> 20

<212> DNA

<213> Artificial sequences

<400> 72

ctcctgttat attctagaac 20

<210> 73

<211> 20

<212> DNA

<213> Artificial sequences

<400> 73

tttcagcatc aatgtaccct 20

<210> 74

<211> 20

<212> DNA

<213> Artificial sequences

<400> 74

cgcgagcaca gctaaggcca 20

<210> 75

<211> 20

<212> DNA

<213> Artificial sequences

<400> 75

actctctctt tctggcctgg 20

<210> 76

<211> 20

<212> DNA

<213> Artificial sequences

<400> 76

ggcactcaga agacactgat 20

<210> 77

<211> 20

<212> DNA

<213> Artificial sequences

<400> 77

aaggtgtctg gtcggagagc 20

<210> 78

<211> 20

<212> DNA

<213> Artificial sequences

<400> 78

acccagcagg gcgtggagcc 20

<210> 79

<211> 20

<212> DNA

<213> Artificial sequences

<400> 79

gtcagagccc caaggtaaaa 20

<210> 80

<211> 900

<212> DNA

<213> Artificial sequences

<400> 80

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> 21

<212> DNA

<213> Caenorhabditis elegans

<400> 101

ttgtactaca caaaagtact g 21

<210> 102

<211> 24

<212> DNA

<213> Caenorhabditis elegans

<400> 102

tcacaacctc ctagaaagag taga 24

Claims (22)

1. A pluripotent stem cell or a derivative thereof, comprising a VEGF-A targeted inhibitor, wherein the VEGF-A targeted inhibitor comprises at least one of shRNA and shRNA-miR expressing VEGF-A; the sequences of the shRNA and shRNA-miR expressing VEGF-A are preferably inserted into the genome of the pluripotent stem cell or the derivative thereof.

2. The pluripotent stem cell or the derivative thereof according to claim 1, wherein the sequences of the shRNA and shRNA-miR targeting VEGF-A are shown in SEQ ID No. 1-SEQ ID No. 2.

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

4. The pluripotent stem cell or the derivative thereof according to claim 1, wherein: the genome of the pluripotent stem cell or the derivative thereof is also introduced with a first nucleic acid molecule;

and, a second nucleic acid molecule is further introduced into the 3' UTR region of the immune response-associated gene in the pluripotent stem cell or a derivative thereof;

the first nucleic acid molecule encodes a small nucleic acid molecule that mediates RNA interference, which small nucleic acid molecule specifically targets the transcript of the second nucleic acid molecule, and which small nucleic acid molecule does not target any other mRNA or incrna of the pluripotent stem cell or a derivative thereof.

5. The pluripotent stem cell or the derivative thereof according to claim 4, wherein the small nucleic acid molecule comprises at least one of a short interfering nucleic acid, a short interfering RNA, a double-stranded RNA, preferably miRNA, shRNA-miR.

6. The pluripotent stem cells or the derivatives thereof according to claim 5, wherein the pluripotent stem cells or the derivatives thereof are derived from a human; the sequence of the small nucleic acid molecule is a random sequence of a non-human species that does not target any mRNA or incrna of a human.

7. The pluripotent stem cell or the derivative thereof according to claim 4, wherein the second nucleic acid molecule comprises the reverse complement of at least 3 repeats of the sequence of the small nucleic acid molecule, preferably 6 to 10 repeats of the sequence of the small nucleic acid molecule.

8. The pluripotent stem cells or the derivatives thereof according to claim 1, wherein the genome of the pluripotent stem cells or the derivatives thereof further comprises an expression sequence of at least one immune-compatible molecule for regulating the expression of genes associated with an immune response in the pluripotent stem cells or the derivatives thereof.

9. The pluripotent stem cell or the derivative thereof according to claim 4 or 8, wherein the genes associated with the immune response comprise:

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

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

10. The pluripotent stem cell or derivative thereof of claim 8, wherein the immune-compatible molecule comprises at least one of:

(1) an immune tolerance-related gene including at least one of CD47 and HLA-G;

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

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

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

11. The pluripotent stem cell or derivative thereof of claim 10, wherein:

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

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

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

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

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

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

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

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

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

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

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

the target sequence of the shRNA and/or shRNA-miR of the target HLA-DPA1 is selected from one of SEQ ID NO. 44-SEQ ID NO. 46; the target sequence of the shRNA and/or shRNA-miR of the target HLA-DPB1 is selected from one of SEQ ID NO. 47-SEQ ID NO. 49.

12. The pluripotent stem cell or the derivative thereof according to any one of claims 1 to 11, wherein a shRNA and/or miRNA processing complex-associated gene and/or an anti-interferon effector molecule is further introduced into the genome of the pluripotent stem cell or the derivative thereof, wherein: the shRNA and/or miRNA processing complex related gene comprises at least one of Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2), PACT (PRKRA) and DGCR 8; the anti-interferon effector molecule is shRNA and/or shRNA-miR of at least one of target PKR, 2-5As, IRF-3 and IRF-7.

13. The pluripotent stem cell or the derivative thereof according to claim 12, wherein the target sequence of the shRNA and/or shRNA-miR targeting the PKR is selected from one of SEQ ID No.50 to SEQ ID No. 52;

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

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

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

14. The pluripotent stem cells or derivatives thereof of claim 2, 11 or 13, wherein the expression frameworks of the VEGF-a targeting shRNA and/or shRNA-miR, major histocompatibility complex gene, major histocompatibility complex-related gene, anti-interferon effector molecule are as follows:

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

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

shRNA-miR expression framework: and replacing the shRNA target sequence in the shRNA expression frame by using the shRNA-miR target sequence.

15. The pluripotent stem cell or the derivative thereof according to claim 4, 8 or 12, wherein an inducible gene expression system is further introduced into the genome of the pluripotent stem cell or the derivative thereof for regulating the expression of the first nucleic acid molecule and/or the immune-compatible molecule and/or the shRNA and/or the miRNA processing complex-associated gene and/or the anti-interferon effector molecule.

16. The pluripotent stem cells or derivatives thereof according to claim 15, wherein the inducible gene expression system comprises at least one of a Tet-Off system, a dimer inducible expression system.

17. The pluripotent stem cell or derivative thereof according to any one of claims 1 to 16, wherein the genome of the pluripotent stem cell or derivative thereof further comprises an exosome-processing synthetic gene comprising at least one of STEAP3, Syndevan-4, an L-aspartate oxidase fragment, CD63-L7Ae and Cx 43S 368A.

18. The pluripotent stem cell or the derivative thereof according to any one of claims 1 to 17, wherein the VEGF-a inhibitory factor expression sequence, the first nucleic acid molecule, the immune-compatible molecule expression sequence, the shRNA and/or miRNA processing complex-associated gene, the anti-interferon effector molecule, the inducible gene expression system, the exosome processing synthetic gene are introduced by viral vector interference, non-viral vector transfection or gene editing, preferably by knock-in.

19. The pluripotent stem cell or the derivative thereof according to any one of claims 1 to 17, wherein the VEGF-a inhibitory factor expression sequence, the immune-compatible molecule expression sequence, the shRNA and/or miRNA processing complex-associated gene, the anti-interferon effector molecule, the inducible gene expression system, the exosome processing synthetic gene are introduced at a genome-safe site, preferably at one or more of an AAVS 1-safe site, an eGSH-safe site, and an H11-safe site.

20. The pluripotent stem cell or derivative thereof of any one of claims 1 to 19, wherein:

the pluripotent stem cells comprise embryonic stem cells, embryonic germ cells, embryonic cancer cells, or induced pluripotent stem cells;

the pluripotent stem cell derivative comprises adult stem cells differentiated by the pluripotent stem cells, cells of each germ layer or tissues and organs;

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

21. Use of the pluripotent stem cells or derivatives thereof and exosomes secreted therefrom of claim 20 for the preparation of a macular degeneration therapeutic agent or medicament.

22. An exosome secreted from the pluripotent stem cell or derivative thereof of any one of claims 1 to 20.

Images