CN114457033A - A pluripotent stem cell derivative expressing IL-6 blocker and its application - Google Patents

Abstract

The invention discloses an IL-6 blocker-expressing pluripotent stem cell or a derivative thereof and application thereof. The pluripotent stem cell or its derivative includes a non-immune compatible pluripotent stem cell expressing an IL-6 blocker or At least one of a derivative thereof, an IL-6 blocker-expressing immune-compatible pluripotent stem cell or a derivative thereof, an IL-6 blocker-expressing immune-compatible reversible pluripotent stem cell or a derivative thereof. The pluripotent stem cells or derivatives thereof expressing IL-6 blocker provided by the present invention can be used for autologous cells to induce iPSCs or differentiate into low-immunogenic cells such as MSCs, and they can continuously express IL-6 blocker in vivo. It is used to treat related diseases with high expression of IL-6 or to treat rheumatoid arthritis, depression and anxiety.

Description

A pluripotent stem cell derivative expressing IL-6 blocker and its application

technical field

The invention belongs to the technical field of genetic engineering, and more particularly, relates to a pluripotent stem cell derivative expressing an IL-6 blocker and its application.

Background technique

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

IL-6 is a cytokine produced by a variety of tissue cells with complex physiological functions, and IL-6 has a wide range of biological effects. It can induce B cell differentiation and produce immunoglobulin; promote T cell proliferation and growth; promote bone marrow hematopoietic stem cell proliferation; enhance blood cell differentiation; anti-tumor effect, etc. IL-6 is widely associated with many clinical diseases, and IL-6 is elevated in patients with rheumatoid arthritis, depression, and anxiety.

Therefore, it is of great significance to develop a pluripotent stem cell or its derivative that can express an IL-6 blocker in humans.

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

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

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

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 first aspect of the present invention is to provide a pluripotent stem cell or a derivative thereof.

The purpose of the second aspect of the present invention is to provide the application of the above-mentioned pluripotent stem cells or derivatives thereof in the preparation of medicines for treating diseases.

The object of the third aspect of the present invention is to provide a preparation comprising the above-mentioned pluripotent stem cells or derivatives thereof.

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, comprising an expression sequence of a -6 blocker, the IL-6 blocker being an IL-6 antibody and/or an IL-6 receptor Antibody;

Preferably, the expression sequence of the IL-6 blocker is inserted into the genome of the pluripotent stem cell or derivative thereof.

More preferably, the expression sequence of the IL-6 blocker is inserted at a safe site in the genome of the pluripotent stem cell or derivative thereof.

Further preferably, the genome security site includes one or more of the AAVS1 security site, the eGSH security site, and the H11 security site.

The second aspect of the present invention provides a pluripotent stem cell or a derivative thereof, comprising an expression sequence of an IL-6 blocker, wherein the IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody; the B2M gene and/or the CIITA gene in the genome of the pluripotent stem cell or its derivative is knocked out.

Preferably, the expression sequence of the IL-6 blocker is inserted into the genome of the pluripotent stem cell or derivative thereof.

More preferably, the expression sequence of the IL-6 blocker is inserted at a safe site in the genome of the pluripotent stem cell or derivative thereof.

Further preferably, the genome security site includes one or more of the AAVS1 security site, the eGSH security site, and the H11 security site.

The third aspect of the present invention provides a pluripotent stem cell or a derivative thereof, comprising an expression sequence of an IL-6 blocker, wherein the IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody;

The pluripotent stem cells or derivatives thereof further comprise an immunocompatibility molecule expression sequence, and the immunocompatibility molecules are used to regulate the expression of genes related to immune response in the pluripotent stem cells or derivatives thereof.

Preferably, the expression sequence of the IL-6 blocker and the expression sequence of the immunocompatible molecule are inserted into the genome of the pluripotent stem cell or its derivative.

More preferably, the expression sequence of the IL-6 blocker and the expression sequence of the immunocompatible molecule are inserted into a safe site in the genome of the pluripotent stem cell or its derivative.

Further preferably, the genome security site includes one or more of the AAVS1 security site, the eGSH security site, and the H11 security site.

The fourth aspect of the present invention provides a pluripotent stem cell or a derivative thereof, and an expression sequence of an IL-6 blocker, wherein the IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor Antibody;

The pluripotent stem cell or its derivative further comprises an immune-compatible molecule expression sequence, and the immune-compatible molecule is used to regulate the expression of genes related to immune response in the pluripotent stem cell or its derivative;

The pluripotent stem cells or derivatives thereof also include an inducible gene expression system.

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

Preferably, the expression sequence of the IL-6 blocker, the expression sequence of the immunocompatible molecule and the inducible gene expression system are inserted into the genome of the pluripotent stem cell or derivative thereof.

More preferably, the expression sequence of the IL-6 blocker, the expression sequence of an immunocompatible molecule and the inducible gene expression system are inserted into a safe site in the genome of the pluripotent stem cell or its derivative.

Further preferably, the genome security site includes one or more of the AAVS1 security site, the eGSH security site, and the H11 security site.

According to the pluripotent stem cells or derivatives thereof according to the third aspect or the fourth aspect of the present invention, further, the immunocompatible molecule includes one or more of the following:

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

(II) HLA-C class molecules, including more than 90% of HLA-C multiple alleles in the population, or fusion protein genes composed of more than 90% of HLA-C multiple alleles and B2M;

(III) shRNA and/or shRNA-miR targeting the gene related to immune response.

According to the pluripotent stem cells or derivatives thereof according to the third or fourth aspect of the present invention, further, the genes related to immune response include:

(I) Major histocompatibility complex genes, including HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA - at least one of DQB1, HLA-DPAl and HLA-DPB1;

(II) Major histocompatibility complex-related genes, including at least one of B2M and CIITA.

The target sequence of shRNA and/or shRNA-miR targeting B2M is selected from one of SEQ ID NO.6 to SEQ ID NO.8;

The target sequence of shRNA and/or shRNA-miR targeting CIITA is selected from one of SEQ ID NO.9 to SEQ ID NO.18;

The target sequence of shRNA and/or shRNA-miR targeting HLA-A is selected from one of SEQ ID NO.19 to SEQ ID NO.21;

The target sequence of shRNA and/or shRNA-miR targeting HLA-B is selected from one of SEQ ID NO.22-SEQ ID NO.27;

The target sequence of shRNA and/or shRNA-miR targeting HLA-C is selected from one of SEQ ID NO.28 to SEQ ID NO.33;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DRA is selected from one of SEQ ID NO.34 to SEQ ID NO.43;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB1 is selected from one of SEQ ID NO.44-SEQ ID NO.48;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB3 is selected from one of SEQ ID NO.49 to SEQ ID NO.50;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB4 is selected from one of SEQ ID NO.51 to SEQ ID NO.60;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB5 is selected from one of SEQ ID NO.61 to SEQ ID NO.69;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DQA1 is selected from one of SEQ ID NO.70 to SEQ ID NO.76;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DQB1 is selected from one of SEQ ID NO.77-SEQ ID NO.86;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DPA1 is selected from one of SEQ ID NO.87-SEQ ID NO.96;

The target sequence of shRNA and/or shRNA-miR targeting HLA-DPB1 is selected from one of SEQ ID NO.97-SEQ ID NO.106.

According to the pluripotent stem cell or the derivative thereof according to the third aspect or the fourth aspect of the present invention, further, the genome of the pluripotent stem cell or the derivative thereof is further introduced into shRNA processing complex-related genes, miRNA processing complex At least one of a body-related gene and an anti-interferon effector molecule.

Preferably, the shRNA processing complex-related genes and miRNA processing complex-related genes include at least one of Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2), PACT (PRKRA), and DGCR8;

The anti-interferon effector molecule is preferably shRNA and/or shRNA-miR targeting at least one of PKR, 2-5As, IRF-3 and IRF-7.

The target sequence of shRNA targeting PKR and/or shRNA-miR is selected from one of SEQ ID NO.107～SEQ ID NO.116;

The target sequence of shRNA and/or shRNA-miR targeting 2-5As is selected from one of SEQ ID NO.117～SEQ ID NO.146;

The target sequence of shRNA and/or shRNA-miR targeting IRF-3 is selected from one of SEQ ID NO.147-SEQ ID NO.156;

The target sequence of shRNA targeting IRF-7 and/or shRNA-miR is selected from one of SEQ ID NO.157-SEQ ID NO.166.

According to the pluripotent stem cell or the derivative thereof according to the third aspect or the fourth aspect of the present invention, further, the shRNA expression framework: from 5' to 3', sequentially includes shRNA target sequence, stem-loop sequence, shRNA The reverse complement of the target sequence, Poly T;

Wherein, the shRNA target sequence, the stem-loop sequence and the reverse complementary sequence of the shRNA target sequence form a hairpin structure;

Poly T is the transcription terminator of RNA polymerase III;

shRNA-miR expression framework: obtained by replacing the target sequence in microRNA-30 or microRNA-155 with the shRNA-miR target sequence.

More preferably, the length of the stem-loop sequence in the shRNA expression framework is 3-9 bases; the length of the poly T is 5-6 bases.

According to the pluripotent stem cells or derivatives thereof according to 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 cells or derivatives thereof according to the first to fourth aspects of the present invention, further, the pluripotent stem cell derivatives include adult stem cells, germ layer cells or tissues differentiated from pluripotent stem cells;

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

According to the pluripotent stem cells or their derivatives according to the first to fourth aspects of the present invention, further, the heavy chain sequence of the IL-6 antibody is shown in SEQ ID NO. 1, and the light chain sequence is shown in SEQ ID NO.1 ID NO.2.

According to the pluripotent stem cells or their derivatives according to the first to fourth aspects of the present invention, further, the heavy chain sequence of the IL-6 receptor antibody is shown in SEQ ID NO. 3, and the light chain sequence is shown in SEQ ID NO. As shown in SEQ ID NO.4.

Those skilled in the art can understand that other IL-6 antibody or IL-6 receptor antibody expression sequences can also be used to achieve the purpose of the present invention.

The fifth aspect of the present invention provides the use of the pluripotent stem cells or their derivatives according to any one of the first to fourth aspects of the present invention in the preparation of a medicine for treating a disease, wherein the disease is rheumatoid arthritis, At least one of depression and anxiety.

A sixth aspect of the present invention provides a preparation comprising the pluripotent stem cells or derivatives thereof according to any one of the first to fourth aspects.

The beneficial effects of the present invention are:

The present invention provides an IL-6 blocker-expressing pluripotent stem cell or a derivative thereof, which can be used for autologous cells to induce iPSCs or differentiate into low-immunogenic cells such as MSCs, which can continuously express IL-6 in vivo. -6 blocker for the treatment of related diseases with high IL-6 expression or rheumatoid arthritis, depression and anxiety.

The present invention also provides an immune-compatible pluripotent stem cell or a derivative thereof that expresses an IL-6 blocker, because the B2M and CIITA genes in the pluripotent stem cell or its derivative are knocked out, or the gene has been introduced into the pluripotent stem cell or its derivative. Immunocompatible molecular expression sequences, so such pluripotent stem cells or their derivatives have low immunogenicity, and when transplanted into recipients, can overcome the problem of allogeneic immune rejection between donor cells and recipients, making The donor cells were able to express the IL-6 blocker continuously for a long time in the recipient.

The present invention also provides an inducible gene expression system and an immune-compatible molecular expression sequence introduced into the genome of an immune-compatible and reversible pluripotent stem cell or a derivative thereof expressing an IL-6 blocker. The inducible gene expression system is regulated by exogenous inducers. By adjusting the addition amount, duration and type of exogenous inducers, the inducible gene expression system can be turned on and off, thereby controlling the expression of immune-compatible molecular expression sequences. . And immune-compatible molecules can regulate the expression of immune response-related genes in 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, which can eliminate or reduce the allogeneic immune rejection response between donor cells and recipients, This enables the donor cells to continue expressing the IL-6 blocker in the recipient for a long time. 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 making the recipient cell. The body can clear the diseased cells, thereby improving the clinical safety of such universal pluripotent stem cells or their derivatives, and greatly expanding their value in clinical applications.

In addition, by adjusting the amount of exogenous inducer and the duration of action, the graft can gradually express low concentrations of HLA molecules to stimulate the recipient, so that the recipient can gradually develop tolerance to the graft, and finally achieve a stable tolerance. . At this time, even if the unmatched HLA class I molecules expressed on the surface of the transplanted cells can be compatible with the recipient immune system, so that after inducing and shutting down the expression of immune compatible molecules in the transplanted cells, the recipient immune system can, on the one hand, be able to Re-identify the cells with genetic mutations presented by HLA class I molecules in the transplant, and remove the diseased cells; Cleared by the recipient's immune system. Therefore, the immune system of the recipient only removes the harmful mutation of the graft, and retains the normal function of the graft. When the harmful graft is removed, it can be transferred to the mode of silencing of HLA class I molecules on the surface of the graft cell. The graft immune tolerance program mediated by exogenous inducers can also turn on or off the surface expression of HLA class I molecules after implantation without induction or other induction after complete tolerance of the recipient.

Description of drawings

Figure 1, AAVS1 KI Vector (shRNA, constitutive) plasmid map.

Figure 2, AAVS1 KI Vector (shRNA, inducible) plasmid map.

Figure 3, AAVS1 KI Vector (shRNA-miR, constitutive) plasmid map.

Figure 4, AAVS1 KI Vector (shRNA-miR, inducible) plasmid map.

Figure 5, sgRNA clone B2M-1 plasmid map.

Figure 6, sgRNA clone B2M-2 plasmid map.

Figure 7, sgRNA clone CIITA-1 plasmid map.

Figure 8, sgRNA clone CIITA-2 plasmid map.

Figure 9, Cas9 (D10A) plasmid map.

Figure 10, sgRNA Clone AAVS1-1 plasmid map.

Figure 11, sgRNA Clone AAVS1-2 plasmid map.

Detailed ways

The content of the present invention will be described in further detail below with reference to specific embodiments and accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

The experimental method of unreceipted specific conditions in the following examples, usually according to normal conditions, such as people such as Sambrook, molecular cloning: the conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to manufacture conditions recommended by the manufacturer. Various common chemical reagents used in the examples are all commercially available products.

1 Experimental materials and methods

1.1 IL-6 blockers

The IL-6 blocking agent can be selected from IL-6 antibody and/or IL-6 receptor antibody.

The heavy chain (HC) sequence of the IL-6 antibody is shown in SEQ ID NO.1, and the light chain (LC) sequence is shown in SEQ ID NO.2; the heavy chain (HC) sequence of the IL-6 receptor antibody is shown in SEQ ID NO.2 The light chain (LC) sequence is shown in SEQ ID NO.4 as shown in ID NO.3.

The front end of IL-6 antibody and IL-6 receptor antibody has a signal peptide sequence, and the signal peptide sequence is shown in SEQ ID NO.5.

GAGGTGCAGCTGGTGGAGAGCGGCGGCAAGCTGCTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTTCGCCATGAGCTGGTTCAGGCAGAGCCCCGAGAAGAGGCTGGAGTGGGTGGCCGAGATCAGCAGCGGCGGCAGCTACACCTACTACCCCGACACCGTGACCGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACACCCTGTACCTGGAGATGAGCAGCCTGAGGAGCGAGGACACCGCCATGTACTACTGCGCCAGGGGCCTGTGGGGCTACTACGCCCTGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCA TCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCAGGGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG(SEQ ID NO.1)。

CAGATCGTGCTGATCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGTACTGGTACCAGCAGAAGCCCGGCAGCAGCCCCAGGCTGCTGATCTACGACACCAGCAACCTGGCCAGCGGCGTGCCCGTGAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGGATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCGGCTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC(SEQ ID NO.2)。

GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCAGGAGCCTGAGGCTGAGCTGCGCCGCCAGCAGGTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCGGCATCAGCTGGAACAGCGGCAGGATCGGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCGAGAACAGCCTGTTCCTGCAGATGAACGGCCTGAGGGCCGAGGACACCGCCCTGTACTACTGCGCCAAGGGCAGGGACAGCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGA CCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGACCTACCTGCCCCCCAGCAGGGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG(SEQ ID NO.3)。

GACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCAGCTACTACTGCCAGCAGGCCAACAGCTTCCCCTACACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC(SEQ ID NO.4)。

ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG (SEQ ID NO. 5).

Those skilled in the art can understand that other IL-6 antibody or IL-6 receptor antibody expression sequences can also be used to achieve the purpose of the present invention.

1.2 Pluripotent stem cells or their derivatives

Pluripotent stem cells can be selected from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and other forms of pluripotent stem cells, such as hPSCs-MSCs, NSCs, EBs cells. in:

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

iPSCs: Using our third-generation efficient and safe episomal-iPSCs induction system (6F/BM1-4C), pE3.1-OG--KS and pE3.1-L-Myc--hmiR302cluster were electroporated into somatic cells Medium, RM1 was cultured for 2 days, BioCISO-BM1 containing 2uM Parnate was cultured for 2 days, BioCISO-BM1 containing 2uM Parnate, 0.25mM sodium butyrate, 3uMCHIR99021 and 0.5uM PD03254901 was cultured for 2 days, and the stem cell medium BioCISO was cultured for 17 days The iPSCs clones can be picked around left and right, and the picked iPSCs clones 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 stem cell medium (BioCISO, containing 10uM TGFβ inhibitor SB431542) for 25 days, digested and passaged at 80-90 confluency (2mg/mL Dispase digestion), and passaged 1:3 into Matrigel-coated cells In the culture plate, then ESC-MSC medium (knockout DMEM medium, containing 10% KSR, NEAA, double antibody, glutamine, β-mercaptoethanol, 10ng/mL bFGF and SB-431542) was cultured, and the medium was changed every day , 80-90 confluency for passage (1:3 passage), continuous culture for 20 days. For the specific construction method, please refer to: Proc Natl Acad Sci US A. 2015; 112(2):530-535.

NSCs: iPSCs were cultured in 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 Cultivated in a 1:1 ratio of DMEM/F12 (containing 1% N2, Invitrogen) and Neurobasal medium (containing 2% B27, Invitrogen), and also containing 20ng/ml bFGF and 20ng/ml EGF, for culture , Digestion using Accutase for digestion and passage. For the specific construction method, please refer to: FASEB J. 2014; 28(11): 4642-4656.

EBs cells: iPSCs with a confluency of 95% were digested with BioC-PDE1 for 6 min, and then scraped into a block using a mechanical scraping method, settled down to the cell mass, and the settled cell mass was transferred to a low-adherence culture plate for use BioCISO-EB1 was cultured for 7 days, and the medium was changed every other day. After 7 days, the cells were transferred to Matrigel-coated culture plates to continue adherent culture with BioCISO. After 7 days, embryoid bodies (EBs) with inner, middle and outer germ layers were obtained. 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.

1.3 Genome Safety Sites

As a more preferred solution, in order to ensure the stable expression of the knock-in gene/expression structure, the gene/expression structure can be knocked into the genome safety site, and the genome safety site can be selected from the AAVS1 safety site, the eGSH safety site, or Other safe spots:

(1) AAVS1 safety site

The AAVS1 locus (alias "PPP1R2C locus") is located on chromosome 19 of the human genome and is a validated "safe harbor" site that ensures the intended function of the transferred DNA fragment. This site is an open chromosomal structure, which can ensure that the transferred gene can be transcribed normally, and the insertion of exogenous target fragments at this site has no known side effects on cells.

(2) eGSH safety site

The eGSH safety site is located on chromosome 1 of the human genome and is another "safe harbor" site that has been validated by the paper and can ensure the intended function of the transferred DNA fragment.

(3) Other security sites

The H11 safety site (also called Hipp11), located on human chromosome 22, is a site between the two genes Eif4enif1 and Drg1. It was discovered and named by Simon Hippenmeyer in 2010. Since the H11 site is located in two genes Therefore, the risk of affecting the expression of the endogenous gene after the insertion of the exogenous gene is very small. The H11 locus was verified to be a safe transcriptional activation region between genes and a new "safe harbor" site besides the AAVS1 and eGSH loci.

1.4 Inducible gene expression system

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

(1) tet-Off system

In the absence of tetracycline, the tTA protein continues to act on the tet promoter, 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.

Knock the sequences of the tet-Off system and one or more immune-compatible molecules into the genomic safety site of pluripotent stem cells, and accurately turn on or off the expression of immune-compatible molecules by adding tetracycline, thereby reversibly regulating pluripotent stem cells Expression of major histocompatibility complex-related genes in or derivatives thereof.

(2) Dimer shutdown expression system

Dimer-mediated gene expression regulation system: There are many ways to chemically regulate the transcription of target genes, the most common is the use of allosteric regulators that affect the activity of transcription factors. One such approach is to use dimerization inducers or dimers to reconstitute active transcription factors on inactive fusion proteins. 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-related protein, or mTOR ( Mammalian target of rapamycin)] has high affinity and the function of binding with these two proteins, so these 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.

1.5 Immunocompatible molecules

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

The types and sequences of specific immunocompatible molecules are shown in Table 1.

Table 1 Immunocompatible molecules

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

Table 2 Target sequences of shRNA or shRNA-miR

In the protocol of knock-in of immune-compatible molecules in subsequent experiments, the shRNA or shRNA-miR sequences of each experimental group were the shRNA or shRNA-miR immune-compatible molecules constructed using target sequence 1 in Table 2. 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.

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

The primary miRNA (pri-miRNA) in the nucleus is micro-processed by the complex Drosha-DGCR8, and the pri-miRNA is cleaved into the precursor miRNA (pre-miRNA), which will form a hairpin structure. Next, the pre-miRNA is transported out of the nucleus via the Exportin-5-Ran-GTP complex. The RNaseDicer enzyme, which binds to the double-stranded RNA-binding protein TRBP (TARBP2) in the cytoplasm, cleaves pre-miRNAs to their mature lengths, while the miRNAs are still double-stranded. Finally, it is transported into AGO2 to form RISC (RNA-induced silencing complex). One strand of the final miRNA duplex remains in the RISC complex, while the other is excreted and rapidly degraded. As the main binding protein of Drosha, DGCR8 can bind to pri-miRNA through its two double-stranded RNA binding regions at the C-terminus, recruit and guide Drosha to cleave at the correct position of pri-miRNA to produce pre-miRNA, pre-miRNA It is further processed and cleaved by Dicer and TRBP/PACT to form mature miRNA. The deletion or abnormal expression of DGCR8 can affect the splicing activity of Drosha, which in turn affects the activity of miRNAs, leading to the occurrence of diseases. TRBP can recruit Dicer complex miRNAs to form RISC Ago2.

Using gene knock-in technology, when knocking in shRNA-miR expression sequences targeting HLA class I molecules and HLA class II molecules that can inducibly shut down expression at a safe site in the genome, it is preferable to simultaneously knock in shRNA and/or shRNA that can inducibly shut down expression. miRNA processing machines 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), so that cells do not occupy the processing of other miRNAs and affect cell function.

In addition, in the process of IFN induction, double-stranded RNA-dependent Protein Kinase (PKR), which is a key factor in the entire cell signal transduction pathway, also has 2',5 'Oligoadenylate synthase (2,5-Oligoadenylate Synthetase, 2-5As), these two enzymes are closely related to dsRNA-induced IFN. PKR can inhibit protein synthesis by phosphorylating eukaryotic transcription factors, make cells stagnate in G0/G1 and G2/M phases, and induce apoptosis, while dsRNA can promote 2-5As synthesis, resulting in non-specific RNase, RNaseL. Heterogeneous activation, degrades all mRNA in cells, and causes cell death. The induction specificity of type I interferon is achieved by members of the IRF transcription factor family. In the absence of the expression of IRF-3 and IRF-7 in cells, type I interferon cannot be induced and secreted in many viral infections. In the absence of an IFN response, co-expression of the two proteins is required to restore it.

Using gene knock-in technology, when knocking in the expression sequence of the immune-compatible molecule shRNA-miR at a safe site in the genome, it is preferable to simultaneously knock in the expression sequences that can inducibly shut down the expression of PKR, 2-5As, IRF-3 and IRF-7 genes. shRNA and/or shRNA-miR expression sequences that reduce dsRNA-induced interferon responses, thereby avoiding cytotoxicity.

There is no restriction on the insertion positions of shRNA/miRNA processing complex-related genes, anti-interferon effector molecules, and immune-compatible molecules in the safe sites of the genome, and they can be arranged in any order without interfering with each other or affecting other genes in the genome. structure and function.

The target sequences of specific anti-interferon effector molecules are shown in Table 3.

Table 3 Target sequences of anti-interferon effector molecules

In the follow-up experiments including the anti-interferon effector molecule knock-in scheme, the target sequences of the anti-interferon effector molecules in each experimental group were the anti-interferon effector molecules constructed by using the target sequence 1 in Table 3. Those skilled in the art can understand:

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.

1.7 General framework of immune-compatible molecules, shRNAs against interferon effector molecules or shRNA-miRs The general framework sequences of immune-compatible molecules, shRNAs against interferon effector molecules or shRNA-miRs are shown below:

(1) The shRNA constitutive expression framework is:

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGCTAGCGCCACC(SEQ ID NO.167)N ₁ ...N ₂₁ TTCAAGAGA(SEQ IDNO.168)N ₂₂ ...N ₄₂ TTTTTT；

in:

_a . N1... _N21 is the shRNA target sequence of the corresponding gene, and _N22 ... _N42 is the reverse complementary sequence of the shRNA target sequence of the corresponding gene;

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

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

d, N represent A, T, G, C bases;

e, SEQ ID NO.167 is the U6 promoter sequence;

f. SEQ ID NO. 168 is the stem-loop sequence.

(2) The shRNA-inducible expression framework is:

(SEQ ID NO. 169) N ₁ ... N ₂₁ TTCAAGAGA (SEQ ID NO. 168) N ₂₂ ... N ₄₂ TTTTTT;

in:

_a . N1... _N21 is the shRNA target sequence of the corresponding gene, and _N22 ... _N42 is the reverse complementary sequence of the shRNA target sequence of the corresponding gene;

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

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

d, N represent A, T, G, C bases;

e, SEQ ID NO.166 is the H1TO promoter sequence;

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

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

Obtained by replacing the target sequence in microRNA-30 with the shRNA-miR target sequence, the specific sequence is as follows:

GAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTAAAGAAGGTATATTGCTGTTGACAGTGAGCG(SEQ ID NO.170)M ₁ N ₁ ...N ₂₁ TAGTGAAGCCACAGATGTA(SEQ ID NO.171)N ₂₂ ...N ₄₂ M ₂ TGCCTACTGCCTCGGACTTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAAT(SEQ ID NO.172)；

in:

_a . N1... _N21 is the shRNA-miR target sequence of the corresponding gene, and _N22 ... _N42 is the reverse complementary sequence of the shRNA-miR target sequence of the corresponding gene;

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

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

d, M base represents A or C base, N represents A, T, G, 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 . M1 base is complementary to _M2 base.

1.8 Gene editing system, gene editing method and testing method

1.8.1 Gene editing system

The gene editing technology uses the CRISPR-Cas9 gene editing system. The Cas 9 protein used by the inventors is Cas 9 (D10A). Cas 9 (D10A) binds to sgRNA, and the sgRNA is responsible for specific recognition of the target sequence (genomic DNA), and then Cas 9 (D10A) performs single-stranded cleavage of the target sequence. Genomic DNA double-strand break (DNA Double Strand Break, DSB), there must be two sets of Cas 9 (D10A)/sgRNA to cut the two strands of genomic DNA, 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. The plasmids or Donor fragments used in this gene editing system are: Cas9 (D10A) plasmid, sgRNA clone plasmid, and Donor fragment, respectively.

(1) Cas9 (D10A) plasmid: a plasmid expressing Cas 9 (D10A) protein, which specifically cuts genomic DNA under the guidance of sgRNA.

(2) sgRNA plasmid: a plasmid expressing sgRNA, sgRNA (small guide RNA) is a guide RNA (guideRNA, gRNA), which is responsible for guiding the targeted cleavage of the expressed Cas 9 (D10A) protein in gene editing.

(3) Donor fragment: the two ends contain recombination arms, which are located on the left and right sides of the genomic DNA break position, and the middle contains the gene, fragment or expression element to be inserted. In the presence of Donor fragments, cells undergo a homologous recombination (HR) reaction at the site of genome breakage. If the Donor fragment is not added, the non-homologous end joining (NHEJ) reaction occurs at the genomic break site of the cell. The fragment was recovered by KI (Knock-in, the same below) vector plasmid digestion.

1.8.2 Constitutive and inducible plasmids

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

Inducible plasmid: After the Donor fragment obtained from the inducible plasmid is knocked into the genomic DNA, 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.

1.8.3 Plasmid construction method

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

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

(3) KI (Knock-in, the same below) Vector plasmid:

a. Acquisition of Amp(R)-pUC origin fragment: Design PCR primers, use the pUC18 (Takara, CodeNo.3218) plasmid as a template and use a high-fidelity enzyme (Nanjing Novozyme, P505-d1) to pass the PCR method. The fragment is amplified and recovered;

b. Acquisition of AAVS1 or eGSH recombination arm: extract the genomic DNA of human cells and design corresponding primers, and then use the human genomic DNA as a template to use a high-fidelity enzyme (Nanjing Novozymes, P505-d1) to pass the PCR method, Amplify and recover such fragments;

c. Acquisition of each plasmid element: Design the PCR amplification primers of each element, and then use the plasmid containing the element as a template and use a high-fidelity enzyme (Nanjing Novozyme, P505-d1) to pass the PCR method to separate each plasmid. Elements are amplified and recovered;

d. Assemble into a complete plasmid: Use multi-fragment recombinase (Nanjing Novozymes, C113-02) to connect the fragments obtained in the previous steps to form a complete plasmid.

1.8.4 Gene editing process

1. Single-cell cloning operation steps of AAVS1 gene knock-in

(1) Electric transfer procedure:

Donor cell preparation: Human pluripotent stem cells.

Kit 1。Kit: Human Stem Cell

Kit 1.

Instrument: electroporator.

Medium: BioCISO.

Induction plasmids: Cas9D10A, sgRNA clone AAVS1-1, sgRNA clone AAVS1-2, AAVS1neoVectoⅠ, AAVS1neo VectorⅡ.

Note: The induction plasmids used for eGSH gene knock-in: Cas9D10A, sgRNA clone eGSH-1, sgRNA clone eGSH-2, eGSH-neo/eGSH-puro(donor) The donor plasmid here is compared with AAVS1, only the left and right recombination arms are different. All other components are the same. Since the gene editing process of eGSH is the same as that of AAVS1, it will not be repeated later.

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

(3) Screening and culturing single-cell clones to obtain single-cell clones.

2. Culture reagents for single-cell clones with AAVS1 gene knock-in

(1) Culture medium: BioCISO + 300μg/mL G418 + 0.5μg/mL puro (should be placed at room temperature in advance and placed in the dark for 30 to 60 minutes until it returns to room temperature. Note: BioCISO should not be placed at 37°C for Preheat to avoid reduced biomolecular activity.).

(2) Matrigel: hESC-grade Matrigel (before passage or resuscitation of 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 the Matrigel cannot be placed anywhere before use. Dry. In order to ensure better adherence and survival of cells, Matrigel should be placed in a 37°C incubator for coating time: 1:100X Matrigel should not be less than 0.5 hours; 1:200X Matrigel should not be less than 2 hours.).

(3) Digestion solution: Use DPBS to dissolve EDTA to a final concentration of 0.5mM, pH 7.4 (Note: EDTA cannot be diluted with water, otherwise cells will die due to reduced osmotic pressure.).

(4) Cryopreservation solution: 60% BioCISO+30% ESCs grade FBS+10% DMSO (the cryopreservation solution is best prepared and used immediately).

3. Routine maintenance subculture process

(1) The best time for passage and passage ratio

a. The best time for passage: the overall confluence of cells reaches 80% to 90%;

b. The optimal ratio of passage: 1:4～1:7 passage, and the best confluence the next day should be maintained at 20%～30%.

(2) Passaging process

a. Aspirate and discard the Matrigel in the coated cell culture flask in advance, add an appropriate amount of medium (BioCISO+300μg/mL G418+0.5μg/mL puro), and put it into 37 ℃, 5% CO ₂ culture incubation in the box;

b. When the cells meet the requirements of passage, aspirate 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 5-10 minutes (digest until most cells shrink and become rounded but not floating under the microscope, gently pipette the cells to detach from the wall , 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, gently pipette the cells several times to mix well, and then transfer the cells to a bottle prepared to be coated with Matrigel;

e. After the cells are transferred to the cell flask dish, shake them horizontally from front to back and left and right. After no abnormality is observed under the microscope, shake well and place them in a 37°C, 5% CO ₂ incubator for culture;

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

4. Cell cryopreservation

(1) According to the routine passaging procedure, digest the cells with 0.5mM EDTA until most of the cells shrink and become round but have not floated, gently pipet the cells, collect the cell suspension, centrifuge at 200g for 5 minutes, discard the supernatant, and add an appropriate amount of freezing solution Resuspend the cells and transfer the cells to a cryopreservation tube (it is recommended to freeze one tube at a confluence of 80% of the six-well plate, and the volume of the freezing solution is 0.5mL/tube);

(2) Place the cryovials in a programmed cooling box, and immediately put them at -80°C overnight (the temperature of the cryopreservation tube must be lowered by 1°C per minute);

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

5. Cell recovery

(1) Prepare the Matrigel-coated cell flask dish in advance. Before resuscitating the cells, aspirate the Matrigel, add an appropriate amount of BioCISO to the cell flask dish, and incubate in a 37°C, 5% CO ₂ incubator;

(2) Quickly take out the cryopreservation tube from the liquid nitrogen, and immediately put it into a 37°C water bath and shake it quickly to thaw the cells quickly. After careful observation, stop shaking when the ice crystals completely disappear, and transfer the cells to a biological safety cabinet;

(3) Add 10 mL of DMEM/F12 (1:1) basal medium to a 15 mL centrifuge tube in advance, and equilibrate to room temperature. Use a Pasteur pipette to pipette 1 mL of DMEM/F12 (1:1) into the cryopreservation tube slowly, and mix gently. Homogenize, transfer the cell suspension to a prepared 15mL centrifuge tube containing DMEM/F12 (1:1), and centrifuge at 200g for 5 minutes;

(4) Carefully discard the supernatant, add an appropriate amount of BioCISO, mix the cells gently, and plant them in the prepared cell flask dish. , 5% CO ₂ incubator;

(5) Observe the cell adherent survival state the next day, and change the medium regularly every day. If the adherence is good, replace BioCISO with BioCISO+300μg/mL G418+0.5μg/mL puro.

1.8.5 AAVS1 gene knock-in detection method

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

(1) Description of AAVS1 gene knock-in detection

a. Purpose of the test: PCR detects the cells that have undergone gene knock-in treatment to test whether the cells are homozygous; since the two Donor fragments only have differences in the sequence of the resistance gene, it is necessary to determine whether the cells are homozygous (two Donor fragments). Chromosome knock-in Donor fragments of different resistance genes respectively), it is necessary to detect whether the genome of the cell contains Donor fragments of two resistance genes, only double-knock-in cells may be correct homozygotes;

b. Test method: first design a primer inside the Donor plasmid (non-recombination arm part), and then design another primer in the genome PPP1R12C (non-recombination arm part); if the Donor fragment can be inserted correctly in the genome, there will be a target band appears, otherwise no purposeful bands appear);

c. Test protocol The primer sequences and PCR protocol are shown in Table 4.

Table 4 Test protocol primer sequences and PCR protocol

2. The detection method of eGSH gene knock-in is the same as that of AAVS1 gene knock-in detection.

1.8.6 Test methods for knock-in gene methods at safe genomic sites

(1) Test purpose: PCR detection of gene knock-in-treated cells to test whether the cells are homozygous. 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.

(2) Test method: firstly design a primer inside the Donor plasmid (non-recombination arm part), and then design another primer in the genome (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.

1.9 Determination method of pluripotent stem cells expressing IL-6 blocker

IL-6 antibody detection: ELISA (double antigen sandwich method) was used to detect the anti-IL-6 antibody expressed by pluripotent stem cells and their derivatives. Collect the culture supernatant of pluripotent stem cells expressing anti-IL-6 antibodies and their derivatives, and load them on an enzyme-labeled plate that has been coated with human IL-6 antigen. Add 10ul of the sample to be tested, and add the culture supernatant of pluripotent stem cells and their derivatives that do not express anti-IL-6 antibody to the control group, and mix gently. After sealing, the plate was incubated at 37 °C for 30 min, washed 5 times, and then added 50 ul of enzyme-labeled IL-6 antigen reagent. After sealing, the plate was incubated at 37 °C for 30 min. Stop solution 50ul, read and measure the absorbance value at 450nm, the expression of IL-6 antibody is positively correlated with the color depth.

IL-6 receptor antibody detection: ELISA (competitive method) was used to detect the IL-6 receptor antibody expressed by pluripotent stem cells and their derivatives. Collect the culture supernatant of pluripotent stem cells and their derivatives expressing IL-6 receptor antibody, add sample diluent to dilute 5 times, and mix with enzyme-labeled IL-6 antigen (1:1), and then coat with IL-6. The samples were loaded on the enzyme-labeled plate with -6 receptor antigen, and the culture supernatant of pluripotent stem cells and their derivatives that did not express IL-6 receptor antibody was added to the control group, and mixed gently. After sealing, the plates were incubated at 37°C for 30 min, washed 5 times, and then added with a chromogenic solution for 15 min. Add 50 ul of stop solution, read and measure the absorbance at 450 nm. The expression of IL-6 receptor antibody is negatively correlated with the color depth.

1.10 Detecting the therapeutic effect of IL-6 blocker in mouse rheumatoid arthritis 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, collagen was used to induce the establishment of an arthritis model in mice. After emulsification with bovine type II collagen and complete adjuvant, the mice were injected at 4-6 points on the back of the mice, 200ul/mice. After the complete adjuvant and bovine type II collagen were mixed and emulsified, the mice were injected at 3-5 points at the base of the tail, 100ul/mice. After the mice developed arthritis symptoms, mice with comparable symptoms were selected for grouping and then the experiment was carried out. A tail vein injection of 200 uL PBS (containing 106 of IL-6 blocker-expressing pluripotent stem cell derivatives, which is derived from the same donor as human immune cells) was performed for rheumatoid arthritis treatment. Then the treatment of rheumatoid arthritis was scored and the difference was statistically analyzed.

1.11 Detecting the therapeutic effect of IL-6 blocker in mouse depression 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. Mice were modeled by chronic unpredictable mild stress, stress factors such as: tail clipping for 1 min, foot click for 10 s, swimming in ice water at 4°C for 5 min, day and night reversal, fasting for 24 h, water deprivation for 24 h, and wet cage for 24 h , Tilt 45° rearing for 24h, etc. Choose 1-2 stress methods every day and try to avoid repetition for 6 weeks. Use the sugar water preference test to judge whether the modeling is successful. After successful modeling, 200uL PBS (containing 10 ⁶ IL-6 blocker-expressing pluripotent stem cell derivatives, which is derived from the same donor as human immune cells) was injected into the tail vein for depression treatment. The effect of treating depressive symptoms was then tested using the sugar water preference test.

Anhedonia is a typical symptom in patients with depression, and the symptoms of depressed mice are marked by a marked decrease in preference for sugar water. We used the sugar water preference test to examine the changes in the preference degree of sugar water in the depressed mice, so as to judge whether the mice developed depressive symptoms.

Sugar water preference test: firstly train mice for 48 hours to make the mice adapt to drinking sugar water. All mice were housed in equal cages, with 2 vials in each cage, one with 1% aqueous sugar solution and the other with pure water. Change the bottle position every 12h. After the training, the animals were deprived of water and food for 12 hours, and then each caged mice were given 2 bottles of water that had been weighed in advance: 1% aqueous sugar solution and pure water, with free access to water for 1 hour. . After the test, the weight of the drinking water bottle was weighed to calculate the consumption of sugar water and pure water.

Then calculate the preference percentage of sugar water in mice according to the formula: animal preference percentage for sugar water (%) = sugar water consumption (g) / total liquid consumption (sugar water consumption (g) + pure water consumption (g)) × 100 %.

1.12 Detecting the therapeutic effect of IL-6 blocker in mouse anxiety disorder 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. Two weeks later, 200 uL of PBS (containing 10 ⁶ IL-6 blocker-expressing pluripotent stem cell derivatives, which was derived from the same donor as human immune cells) was injected into the tail vein for anxiety treatment. The mice were then tested for anxiety using the elevated plus maze test.

The elevated plus maze has a pair of open arms and a pair of closed arms. Rodents tend to move in the closed arms due to their dark addiction, but they move in the open arms out of curiosity and exploration. When stimulated, animals have the impulse to explore and fear at the same time, which leads to the conflicting behavior of exploration and avoidance, resulting in anxiety. Anti-anxiety drugs can significantly increase the number and time of entering the open arm. The plus maze is higher from the ground, which is equivalent to standing on a cliff, causing the subjects to feel fear and anxiety. The number of times the mice entered the open arm and the stay time were negatively correlated with the anxiety of the rats.

2. Experimental protocol

First, the gene expressing the IL-6 blocker was knocked into a safe harbor in the genome to enable pluripotent stem cell derivatives to express the IL-6 blocker. Thus, pluripotent stem cell derivatives can be used in disease treatment. Then, it can also be modified by gene editing technology (pluripotent stem cell derivatives expressing IL-6 blocker) into constitutive immune-compatible universal pluripotent stem cell derivatives and immune-compatible and reversible universal multipotent stem cell derivatives. Stem cell derivatives, which in turn can be used for disease treatment in allogeneic.

Specifically, the following genetic manipulations were performed in pluripotent stem cell derivatives expressing IL-6 blockers (IL-6 receptor antibody, IL-6 antibody) to achieve allogeneic hPSCs and hPSCs-derived derivatives (hPSCs- MSCs, NSCs, EBs) immunocompatibilities.

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

Construct the following vectors according to the plasmid construction method in 1.8.3:

Table 5 Experimental protocol for constitutive expression

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

IL-6 antibody is a closed type antibody. Antibody structure: signal peptide + light chain (including stop codon) + IRESwt (SEQ ID NO. 181) + signal peptide + heavy chain (including stop codon), the stop codon is generally TGA .

IL-6 receptor antibody is a closed antibody, antibody structure: signal peptide + light chain (including stop codon) + IRESwt (SEQ ID NO. 181) + signal peptide + heavy chain (including stop codon), stop codon TGA is generally used.

The signal peptide sequence was selected as ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG (SEQ ID NO.5).

General principle: IL-6 receptor antibody sequence and anti-IL-6 antibody sequence are placed in the position of MCS2 of the corresponding plasmid, shRNA is placed in the shRNA expression frame of the corresponding plasmid, shRNAmiR is placed in the shRNAmiR expression frame of the corresponding plasmid, and other genes Put the position of MCS1 corresponding to the plasmid. The maps of each plasmid are shown in Figures 1 to 11.

Among them, the sgRNA clone B2M plasmids include sgRNA clone B2M-1 and sgRNA clone B2M-2 plasmids. The sgRNA clone CIITA plasmid contains the sgRNA clone CIITA-1 and sgRNA clone CIITA-2 plasmids.

(1) Aa1 group (IL-6 receptor antibody)

The MCS2 of the AAVS1 KI Vector (shRNA, constitutive) plasmid was placed into the IL-6 receptor antibody sequence (the LC light chain and HC heavy chain sequences of the antibody were connected by EMCV IRESwt, and the sorting order was as follows: LC light chain, EMCV IRESwt, HC heavy chain, the same below).

(2) Aa2 grouping (IL-6 receptor antibody, shRNA, gene)

The MCS2 of the AAVS1 KI Vector (shRNA, constitutive) plasmid was placed into the IL-6 receptor antibody sequence. The shRNA expression framework is placed into the shRNA target sequence (seamlessly linked if multiple shRNAs are present). MCS1 was placed into the gene sequence (connected using EMCV IRESwt if multiple genes were present).

(3) Aa3 grouping (IL-6 receptor antibody, shRNA-miR, gene)

The MCS2 of the AAVS1 KI Vector (shRNA-miR, constitutive) plasmid was put into the IL-6 receptor antibody sequence. The shRNA-miR expression framework is placed into the shRNA target sequence (seamlessly linked if multiple shRNA-miRs are present). MCS1 was placed into the gene sequence (connected using EMCV IRESwt if multiple genes were present).

(4) Aa4 grouping (IL-6 receptor antibody, B2M and CIITA double knockout, gene)

The MCS2 of the AAVS1 KI Vector (shRNA, constitutive) plasmid was placed into the IL-6 receptor antibody sequence. MCS1 was placed into the gene sequence (connected using EMCV IRESwt if multiple genes were present).

The target sequence of the sgRNA clone B2M plasmid was put into the sgRNA target sequence of B2M (SEQ ID NO.182 and SEQ ID NO.183), and the target sequence of the sgRNA clone CIITA plasmid was put into the sgRNA target sequence of CIITA (SEQ ID NO.184 and SEQ ID NO.184). NO.185).

(5) Aa5 grouping (IL-6 receptor antibody, shRNA, gene)

The method of grouping with Aa2.

(6) Aa6 grouping (IL-6 receptor antibody, shRNA-miR, gene)

The method of grouping with Aa3.

(7) Ab1 grouping (IL-6 antibody)

The MCS2 of the AAVS1 KI Vector (shRNA, constitutive) plasmid is put into the LC light chain and HC heavy chain sequences of the antibody, and the EMCV IRESwt is used in the middle to connect, and the sorting order is as follows: LC light chain, EMCV IRESwt, HC heavy chain.

(8) Ab2 grouping (IL-6 antibody, shRNA, gene)

The MCS2 of the AAVS1 KI Vector (shRNA, constitutive) plasmid is put into the LC light chain and HC heavy chain sequences of the antibody, and the EMCV IRESwt is used in the middle to connect, and the sorting order is as follows: LC light chain, EMCV IRESwt, HC heavy chain. The shRNA expression framework is placed into the shRNA target sequence (seamlessly linked if multiple shRNAs are present). MCS1 was placed into the gene sequence (connected using EMCV IRESwt if multiple genes were present).

(9) Ab3 grouping (IL-6 antibody, shRNA-miR, gene)

The MCS2 of the AAVS1 KI Vector (shRNA-miR, constitutive) plasmid was put into the LC light chain and HC heavy chain sequences of the antibody, and EMCV IRESwt was used to connect them in the middle. The sorting order was as follows: LC light chain, EMCV IRESwt, HC heavy chain. The shRNA-miR expression framework is placed into the shRNA target sequence (seamlessly linked if multiple shRNA-miRs are present). MCS1 was placed into the gene sequence (connected using EMCV IRESwt if multiple genes were present).

(10) Ab4 grouping (IL-6 antibody, B2M and CIITA double knockout, gene)

The MCS2 of the AAVS1 KI Vector (shRNA, constitutive) plasmid is put into the LC light chain and HC heavy chain sequences of the antibody, and the EMCV IRESwt is used in the middle to connect, and the sorting order is as follows: LC light chain, EMCV IRESwt, HC heavy chain. MCS1 was placed into the gene sequence (connected using EMCV IRESwt if multiple genes were present).

The target sequence of the sgRNA clone B2M plasmid was put into the sgRNA target sequence of B2M (SEQ ID NO.182 and SEQ ID NO.183), and the target sequence of the sgRNA clone CIITA plasmid was put into the sgRNA target sequence of CIITA (SEQ ID NO.184 and SEQ ID NO.184). NO.185).

(11) Ab5 grouping (IL-6 antibody, shRNA, gene)

Method of grouping with Ab2.

(12) Ab6 grouping (IL-6 antibody, shRNA-miR, gene)

Method of grouping with Ab3.

Table 6 Experimental protocol for inducible expression

(1) Ba1 group (IL-6 receptor antibody, shRNA, gene)

The MCS2 of the AAVS1 KI Vector (shRNA, inducible) plasmid was put into the IL-6 receptor antibody sequence. The shRNA expression framework is placed into the shRNA target sequence (seamlessly linked if multiple shRNAs are present). MCS1 was placed into the gene sequence (connected using EMCV IRESwt if multiple genes were present).

(2) Ba2 group (IL-6 receptor antibody, shRNA-miR, gene)

The MCS2 of the AAVS1 KI Vector (shRNA-miR, inducible) plasmid was put into the IL-6 receptor antibody sequence. The shRNA-miR expression framework is placed into the shRNA target sequence (seamlessly linked if multiple shRNA-miRs are present). MCS1 was placed into the gene sequence (connected using EMCV IRESwt if multiple genes were present).

(3) Ba3 group (IL-6 receptor antibody, shRNA, gene)

The method of grouping with Ba1.

(4) Ba4 group (IL-6 receptor antibody, shRNA-miR, gene)

The method of grouping with Ba2.

(5) Bb1 grouping (IL-6 antibody, shRNA, gene)

The MCS2 of the AAVS1 KI Vector (shRNA, inducible) plasmid was placed into the anti-IL-6 antibody sequence. The shRNA expression framework is placed into the shRNA target sequence (seamlessly linked if multiple shRNAs are present). MCS1 was placed into the gene sequence (connected using EMCV IRESwt if multiple genes were present).

(6) Bb2 grouping (IL-6 antibody, shRNA-miR, gene)

MCS2 of AAVS1 KI Vector (shRNA-miR, inducible) plasmid was put into anti-IL-6 antibody sequence. The shRNA-miR expression framework is placed into the shRNA target sequence (seamlessly linked if multiple shRNA-miRs are present). MCS1 was placed into the gene sequence (connected using EMCV IRESwt if multiple genes were present).

(7) Bb3 grouping (IL-6 antibody, shRNA, gene)

The method of grouping with Bb1.

(8) Bb4 grouping (IL-6 antibody, shRNA-miR, gene)

The method of grouping with Bb2.

Example 1 Detection of IL-6 receptor antibody expressed by pluripotent stem cell derivatives

The protocols of each experimental group in Tables 5 and 6 were knocked into the genomic safety sites of iPSCs, EBs, MSCs, and NSCs cells, cultured at 37°C in a 0.5% CO ₂ incubator, and pluripotent stem cells expressing IL-6 receptor antibody and The culture supernatant of its derivatives is diluted 5 times with sample diluent, mixed with enzyme-labeled IL-6 antigen (1:1), and then loaded on the enzyme-labeled plate that has been loaded with IL-6 receptor antigen. , the control group added the culture supernatant of pluripotent stem cells and their derivatives that did not express IL-6 receptor antibody, and mixed gently. After sealing, the plates were incubated at 37°C for 30 min, washed 5 times, and then added with color developing solution for 15 min, adding 50 ul of stop solution, and reading and measuring the absorbance at 450 nm. The results are shown in Table 7.

Table 7 ELISA detection of IL-6 receptor antibody expressed in each experimental group

It can be seen from the above table that the pluripotent stem cells or derivatives thereof prepared by the present invention can effectively express IL-6 receptor antibody. And its expression level is relatively constant in each group, so the IL-6 receptor antibody expressed by pluripotent stem cell derivatives is not affected by cell differentiation morphology and other exogenous genes (immunocompatible transformation).

Example 2 Detection of IL-6 antibody expressed by pluripotent stem cell derivatives

The protocols of each experimental group in Table 5 and Table 6 were knocked into the genomic safety sites of iPSCs, EBs, MSCs, and NSCs cells, and cultured at 37°C in a 0.5% CO ₂ incubator to collect IL-6 antibody-expressing pluripotent stem cells and their derivatives The culture supernatant of the sample was loaded on an enzyme-labeled plate that had been loaded with human IL-6 antigen, and 40ul of sample diluent was added to the well of the sample to be tested, and then 10ul of the sample to be tested was added. 6. The culture supernatant of pluripotent stem cells and their derivatives with antibody, mix gently. After the plate was sealed, it was incubated at 37°C for 30min. After washing 5 times, 50ul of enzyme-labeled IL-6 antigen reagent was added. After sealing, the plate was incubated at 37°C for 30min. Stop solution 50ul, read and measure the absorbance value at 450nm, the results are shown in Table 8.

Table 8 ELISA detection of IL-6 antibody expressed by each experimental group

It can be seen from the above table that the pluripotent stem cells or derivatives thereof prepared by the present invention can effectively express IL-6 antibody. And its expression level is relatively constant in each group, so the IL-6 antibody expressed by pluripotent stem cell derivatives is not affected by cell differentiation morphology and other exogenous genes (immunocompatible transformation).

Example 3 The effect of IL-6 blocker in the treatment of rheumatoid arthritis

The inventors selected cells (MSCs) expressing only the blocker protocol group (Aal, Ab1) for testing. In the humanized NSG mouse rheumatoid arthritis model, mice were injected with hPSCs and hPSCs-derived derivatives (hPSCs- MSCs, hPSCs-NSCs, hPSCs-EBs) to observe the effect of treating rheumatoid arthritis, the results are shown in Table 9.

Note: To avoid immunocompatibility issues, the immune cells used are from the same human as hPSCs and hPSCs-derived derivatives.

Table 9 Effect of each experimental group expressing IL-6 blocker in the treatment of rheumatoid arthritis

Through the above experiments, it can be proved that the IL-6 blocker-expressing stem cells or their derivatives prepared by the present invention can effectively treat rheumatoid arthritis.

Example 4 The effect of IL-6 blocker in the treatment of depression

The inventors selected cells (MSCs) expressing only the blocker protocol group (Aal, Ab1) for testing. In the humanized NSG mouse model of depression, the mice were injected with hPSCs and hPSCs-derived derivatives (hPSCs-MSCs, hPSCs-MSCs, hPSCs-NSCs, hPSCs-EBs) to observe the effect of treating depression.

Note: To avoid immunocompatibility issues, the immune cells used are from the same human as hPSCs and hPSCs-derived derivatives.

Table 10 Each experimental group expresses the effect of IL-6 blocker on depression

Through the above experiments, it can be proved that the IL-6 blocker-expressing stem cells or derivatives thereof prepared by the present invention can effectively treat depression.

Example 5 The effect of IL-6 blocker in the treatment of anxiety disorders

The inventors selected cells (MSCs) expressing only the blocker protocol group (Aal, Ab1) for testing. In the humanized NSG mouse model of anxiety, mice were injected with hPSCs and hPSCs-derived derivatives (hPSCs-MSCs, hPSCs-MSCs, hPSCs-NSCs, hPSCs-EBs) to observe the effect of treating depression, the results are shown in Table 11.

Note: To avoid immunocompatibility issues, the immune cells used are from the same human as hPSCs and hPSCs-derived derivatives.

Table 11 Effect of each experimental group expressing IL-6 blocker on anxiety disorder

Note: *p<0.01 compared to control.

Through the above experiments, it can be proved that the IL-6 blocker-expressing stem cells or derivatives thereof prepared by the present invention can play an anxiolytic effect and can be used for the treatment of anxiety disorders.

Example 6 The effect of immune compatible molecule inducible expression group in the treatment of rheumatoid arthritis

Through the above examples, hPSCs and hPSCs-derived derivatives expressing IL-6 blocker can effectively play a role in the treatment of rheumatoid arthritis, depression and anxiety.

In addition, the immunocompatibility of hPSCs and hPSCs-derived derivatives must also be considered. Therefore, the inventors selected a suitable combination to test the immunocompatibility. Taking advantage of the low immunogenicity of MSCs, in the humanized NSG mouse disease model, the mice were injected with hPSCs-derived immune-compatible MSCs capable of expressing IL-6 blocker (IL-6 antibody), and their treatment was observed. The effect of rheumatoid arthritis, the results are shown in Table 12.

Note: The immune cells and hPSCs-derived MSCs used are not from the same person.

The control group refers to the NSG mouse disease model without MSCs injection.

The treatment of the Dox-added group was as follows: 0.5 mg/mL Dox was added to the mouse diet, and the mice were fed, starting from the injection of cells expressing the blocker, and used until the end of the experiment.

Table 12 The reversible expression test results of the immune-compatible molecule-inducible expression group

The above experiments show that: in the treatment of diseases. MSCs expressing only the blocker (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, while the immunocompatibility transformation (group 3-11, including composition Type and reversible inducible immune compatibility), its immune compatibility effect is better, than the MSCs without immune compatibility modification in the body for a longer time (or can achieve long-term coexistence), its therapeutic effect is better, and group 5 is The B2M and CIITA gene knockout groups completely eliminated the effects of HLA-I and HLA-II molecules, so their therapeutic effects were 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 8-15 protocol setting. In Groups 12-15, the Dox inducer (always used) was administered to the mice at the same time as the injection of the blocker-expressing cells into the mice. The existence time in vivo is comparable to that of MSCs without immune-compatibility modification, and its therapeutic effect is also comparable to that of MSCs without immune-compatibility modification.

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

SEQUENCE LISTING

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

Wang Linli

<120> A pluripotent stem cell derivative expressing IL-6 blocker and its application

<130>

<160> 185

<170> PatentIn version 3.5

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gacatccaga tgacccagag ccccagcagc gtgagcgcca gcgtgggcga cagggtgacc 60

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ggtctggtgg gcatcattat t 21

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gcacatggag gtgatggtgt t 21

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ggaggtgatg gtgtttctta g 21

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ggctttacaa agctggcaat a 21

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gctttacaaa gctggcaata t 21

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gctccgtact ctaacatcta g 21

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gatgaccaca ttcaaggaag a 21

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gaccacattc aaggaagaac t 21

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gctttcctgc ttggcagtta t 21

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ggcagttatt cttccacaag a 21

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gcagttattc ttccacaaga g 21

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<213> human

<400> 49

gcgtaagtct gagtgtcatt t 21

<210> 50

<211> 21

<212> DNA

<213> human

<400> 50

gacaatttaa ggaagaatct t 21

<210> 51

<211> 21

<212> DNA

<213> human

<400> 51

ggccatagtt ctccctgatt g 21

<210> 52

<211> 21

<212> DNA

<213> human

<400> 52

gccatagttc tccctgattg a 21

<210> 53

<211> 21

<212> DNA

<213> human

<400> 53

gcagatgacc acattcaagg a 21

<210> 54

<211> 21

<212> DNA

<213> human

<400> 54

gatgaccaca ttcaaggaag a 21

<210> 55

<211> 21

<212> DNA

<213> human

<400> 55

gaccacattc aaggaagaac c 21

<210> 56

<211> 21

<212> DNA

<213> human

<400> 56

gctttgtcag gaccaggttg t 21

<210> 57

<211> 21

<212> DNA

<213> human

<400> 57

gaccaggttg ttactggttc a 21

<210> 58

<211> 21

<212> DNA

<213> human

<400> 58

gaagcctcac agctttgatg g 21

<210> 59

<211> 21

<212> DNA

<213> human

<400> 59

gatggcagtg cctcatcttc a 21

<210> 60

<211> 21

<212> DNA

<213> human

<400> 60

ggcagtgcct catcttcaac t 21

<210> 61

<211> 21

<212> DNA

<213> human

<400> 61

gcagcaggat aagtatgagt g 21

<210> 62

<211> 21

<212> DNA

<213> human

<400> 62

gcaggataag tatgagtgtc a 21

<210> 63

<211> 21

<212> DNA

<213> human

<400> 63

ggttcctgca cagagacatc t 21

<210> 64

<211> 21

<212> DNA

<213> human

<400> 64

gcacaagagac atctataacc a 21

<210> 65

<211> 21

<212> DNA

<213> human

<400> 65

gagacatcta taaccaagag g 21

<210> 66

<211> 21

<212> DNA

<213> human

<400> 66

gagtactgga acagccagaa g 21

<210> 67

<211> 21

<212> DNA

<213> human

<400> 67

gctttcctgc ttggctctta t 21

<210> 68

<211> 21

<212> DNA

<213> human

<400> 68

ggctcttatt cttccacaag a 21

<210> 69

<211> 21

<212> DNA

<213> human

<400> 69

gctcttattc ttccacaaga g 21

<210> 70

<211> 21

<212> DNA

<213> human

<400> 70

ggatgtggaa cccacagata c 21

<210> 71

<211> 21

<212> DNA

<213> human

<400> 71

gatgtggaac ccacagatac a 21

<210> 72

<211> 21

<212> DNA

<213> human

<400> 72

gtggaaccca cagatacaga g 21

<210> 73

<211> 21

<212> DNA

<213> human

<400> 73

ggaacccaca gatacagaga g 21

<210> 74

<211> 21

<212> DNA

<213> human

<400> 74

gagccaactg tattgcctat t 21

<210> 75

<211> 21

<212> DNA

<213> human

<400> 75

agccaactgt attgcctatt t 21

<210> 76

<211> 21

<212> DNA

<213> human

<400> 76

gccaactgta ttgcctattt g 21

<210> 77

<211> 21

<212> DNA

<213> human

<400> 77

gggtagcaac tgtcaccttg a 21

<210> 78

<211> 21

<212> DNA

<213> human

<400> 78

ggatttcgtg ttccagttta a 21

<210> 79

<211> 21

<212> DNA

<213> human

<400> 79

gcatgtgcta cttcaccaac g 21

<210> 80

<211> 21

<212> DNA

<213> human

<400> 80

gcgtcttgtg accagataca t 21

<210> 81

<211> 21

<212> DNA

<213> human

<400> 81

gcttatgcct gcccagaatt c 21

<210> 82

<211> 21

<212> DNA

<213> human

<400> 82

gcaggaaatc actgcagaat g 21

<210> 83

<211> 21

<212> DNA

<213> human

<400> 83

gctcagtgca ttggccttag a 21

<210> 84

<211> 21

<212> DNA

<213> human

<400> 84

ggtgagtgct gtgtaaataa g 21

<210> 85

<211> 21

<212> DNA

<213> human

<400> 85

gacatatata gtgatccttg g 21

<210> 86

<211> 21

<212> DNA

<213> human

<400> 86

ggaaagtcac atcgatcaag a 21

<210> 87

<211> 21

<212> DNA

<213> human

<400> 87

gctcacagtc atcaattata g 21

<210> 88

<211> 21

<212> DNA

<213> human

<400> 88

gccctgaaga cagaatgttc c 21

<210> 89

<211> 21

<212> DNA

<213> human

<400> 89

gcggaccatg tgtcaactta t 21

<210> 90

<211> 21

<212> DNA

<213> human

<400> 90

ggaccatgtg tcaacttatg c 21

<210> 91

<211> 21

<212> DNA

<213> human

<400> 91

gcgtttgtac agacgcatag a 21

<210> 92

<211> 21

<212> DNA

<213> human

<400> 92

ggctggctaa cattgctata t 21

<210> 93

<211> 21

<212> DNA

<213> human

<400> 93

gctggctaac attgctatat t 21

<210> 94

<211> 21

<212> DNA

<213> human

<400> 94

ggaccaggtc acatgtgaat a 21

<210> 95

<211> 21

<212> DNA

<213> human

<400> 95

ggaaaggtct gaggatattg a 21

<210> 96

<211> 21

<212> DNA

<213> human

<400> 96

ggcagattag gattccattc a 21

<210> 97

<211> 21

<212> DNA

<213> human

<400> 97

gcctgatagg acccatattc c 21

<210> 98

<211> 21

<212> DNA

<213> human

<400> 98

gcatccaata gacgtcattt g 21

<210> 99

<211> 21

<212> DNA

<213> human

<400> 99

gcgtcactgg cacagatata a 21

<210> 100

<211> 21

<212> DNA

<213> human

<400> 100

gctgtcacat aataagctaa g 21

<210> 101

<211> 21

<212> DNA

<213> human

<400> 101

gctaaggaag acagtatata g 21

<210> 102

<211> 21

<212> DNA

<213> human

<400> 102

gggatttcta aggaaggatg c 21

<210> 103

<211> 21

<212> DNA

<213> human

<400> 103

ggagttgaag agcagagatt c 21

<210> 104

<211> 21

<212> DNA

<213> human

<400> 104

gccagtgaac acttaccata g 21

<210> 105

<211> 21

<212> DNA

<213> human

<400> 105

gcttctctga agtctcattg a 21

<210> 106

<211> 21

<212> DNA

<213> human

<400> 106

ggctgcaact aacttcaaat a 21

<210> 107

<211> 21

<212> DNA

<213> human

<400> 107

ggatggattt gattatgatc c 21

<210> 108

<211> 21

<212> DNA

<213> human

<400> 108

ggaccttgga acaatggatt g 21

<210> 109

<211> 21

<212> DNA

<213> human

<400> 109

gctaattctt gctgaacttc t 21

<210> 110

<211> 21

<212> DNA

<213> human

<400> 110

gctgaacttc ttcatgtatg t 21

<210> 111

<211> 21

<212> DNA

<213> human

<400> 111

gcctcatctc tttgttctaa a 21

<210> 112

<211> 21

<212> DNA

<213> human

<400> 112

gctctggaga agatatattt g 21

<210> 113

<211> 21

<212> DNA

<213> human

<400> 113

gctcttgagg gaactaatag a 21

<210> 114

<211> 21

<212> DNA

<213> human

<400> 114

gggacggcat taatgtattc a 21

<210> 115

<211> 21

<212> DNA

<213> human

<400> 115

ggacaaacat gcaaactata g 21

<210> 116

<211> 21

<212> DNA

<213> human

<400> 116

gcagcaacca gctaccattc t 21

<210> 117

<211> 21

<212> DNA

<213> human

<400> 117

gcagttctgt tgccactctc t 21

<210> 118

<211> 21

<212> DNA

<213> human

<400> 118

gggagagttc atccaggaaa t 21

<210> 119

<211> 21

<212> DNA

<213> human

<400> 119

ggagagttca tccaggaaat t 21

<210> 120

<211> 21

<212> DNA

<213> human

<400> 120

gagagttcat ccaggaaatt a 21

<210> 121

<211> 21

<212> DNA

<213> human

<400> 121

gcctgtcaaa gagagagagc a 21

<210> 122

<211> 21

<212> DNA

<213> human

<400> 122

gctcagcttc gtactgagtt c 21

<210> 123

<211> 21

<212> DNA

<213> human

<400> 123

gcttcacaga actacagaga g 21

<210> 124

<211> 21

<212> DNA

<213> human

<400> 124

gcatctactg gacaaagtat t 21

<210> 125

<211> 21

<212> DNA

<213> human

<400> 125

ggctgaatta cccatgcttt a 21

<210> 126

<211> 21

<212> DNA

<213> human

<400> 126

gctgaattac ccatgcttta a 21

<210> 127

<211> 21

<212> DNA

<213> human

<400> 127

gggttggttt atccaggaat a 21

<210> 128

<211> 21

<212> DNA

<213> human

<400> 128

ggatcagaag agaagccaac g 21

<210> 129

<211> 21

<212> DNA

<213> human

<400> 129

ggttcaccat ccaggtgttc a 21

<210> 130

<211> 21

<212> DNA

<213> human

<400> 130

gctctcttct ctggaactaa c 21

<210> 131

<211> 21

<212> DNA

<213> human

<400> 131

gctagagtga ctccatctta a 21

<210> 132

<211> 21

<212> DNA

<213> human

<400> 132

gctgaccacc aattataatt g 21

<210> 133

<211> 21

<212> DNA

<213> human

<400> 133

gcagaatatt taaggccata c 21

<210> 134

<211> 21

<212> DNA

<213> human

<400> 134

gcccacttaa aggcagcatt a 21

<210> 135

<211> 21

<212> DNA

<213> human

<400> 135

ggtcatcaat accactgtta a 21

<210> 136

<211> 21

<212> DNA

<213> human

<400> 136

gcattcctcc ttctcctttc t 21

<210> 137

<211> 21

<212> DNA

<213> human

<400> 137

ggaggaactt tgtgaacatt c 21

<210> 138

<211> 21

<212> DNA

<213> human

<400> 138

gctgtaagaa ggatgctttc a 21

<210> 139

<211> 21

<212> DNA

<213> human

<400> 139

gctgcaggca ggattgtttc a 21

<210> 140

<211> 21

<212> DNA

<213> human

<400> 140

gcagttcgag gtcaagtttg a 21

<210> 141

<211> 21

<212> DNA

<213> human

<400> 141

gccaattagc tgagaagaat t 21

<210> 142

<211> 21

<212> DNA

<213> human

<400> 142

gcaggtttac agtgtatatg t 21

<210> 143

<211> 21

<212> DNA

<213> human

<400> 143

gcctacagag actagagtag g 21

<210> 144

<211> 21

<212> DNA

<213> human

<400> 144

gcagttgggt accttccatt c 21

<210> 145

<211> 21

<212> DNA

<213> human

<400> 145

gcaactcagg tgcatgatac a 21

<210> 146

<211> 21

<212> DNA

<213> human

<400> 146

gcatggcgct ggtacgtaaa t 21

<210> 147

<211> 19

<212> DNA

<213> human

<400> 147

gcctcgagtt tgagagcta 19

<210> 148

<211> 19

<212> DNA

<213> human

<400> 148

agacattctg gatgagtta 19

<210> 149

<211> 19

<212> DNA

<213> human

<400> 149

gggtctgtta cccaaagaa 19

<210> 150

<211> 19

<212> DNA

<213> human

<400> 150

ggtctgttac ccaaagaat 19

<210> 151

<211> 19

<212> DNA

<213> human

<400> 151

ggaaggaagc ggacgctca 19

<210> 152

<211> 19

<212> DNA

<213> human

<400> 152

ggaggcagta cttctgata 19

<210> 153

<211> 19

<212> DNA

<213> human

<400> 153

cgctctagag ctcagctga 19

<210> 154

<211> 19

<212> DNA

<213> human

<400> 154

ccaccacctc aaccaataa 19

<210> 155

<211> 19

<212> DNA

<213> human

<400> 155

atttcaagaa gtcgatcaa 19

<210> 156

<211> 19

<212> DNA

<213> human

<400> 156

gaagatctga ttaccttca 19

<210> 157

<211> 21

<212> DNA

<213> human

<400> 157

ggacactggt tcaacacctg t 21

<210> 158

<211> 21

<212> DNA

<213> human

<400> 158

ggttcaacac ctgtgacttc a 21

<210> 159

<211> 21

<212> DNA

<213> human

<400> 159

acctgtgact tcatgtgtgc g 21

<210> 160

<211> 21

<212> DNA

<213> human

<400> 160

gctggacgtg accatcatgt a 21

<210> 161

<211> 21

<212> DNA

<213> human

<400> 161

ggacgtgacc atcatgtaca a 21

<210> 162

<211> 21

<212> DNA

<213> human

<400> 162

gacgtgacca tcatgtacaa g 21

<210> 163

<211> 21

<212> DNA

<213> human

<400> 163

acgtgaccat catgtacaag g 21

<210> 164

<211> 21

<212> DNA

<213> human

<400> 164

acgctatacc atctacctgg g 21

<210> 165

<211> 21

<212> DNA

<213> human

<400> 165

gcctctatga cgacatcgag t 21

<210> 166

<211> 21

<212> DNA

<213> human

<400> 166

gacatcgagt gcttccttat g 21

<210> 167

<211> 253

<212> DNA

<213> Artificial sequences

<400> 167

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> 168

<211> 9

<212> DNA

<213> Artificial sequences

<400> 168

ttcaagaga 9

<210> 169

<211> 686

<212> DNA

<213> Artificial sequences

<400> 169

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> 170

<211> 119

<212> DNA

<213> Artificial sequences

<400> 170

gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta 60

cttcttcagg ttaacccaac agaaggctaa agaaggtata ttgctgttga cagtgagcg 119

<210> 171

<211> 19

<212> DNA

<213> Artificial sequences

<400> 171

tagtgaagcc acagatgta 19

<210> 172

<211> 119

<212> DNA

<213> Artificial sequences

<400> 172

tgcctactgc ctcggacttc aaggggctac tttaggagca attatcttgt ttactaaaac 60

tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat ggtataaat 119

<210> 173

<211> 22

<212> DNA

<213> Artificial sequences

<400> 173

ccatagctca gtctggtcta tc 22

<210> 174

<211> 22

<212> DNA

<213> Artificial sequences

<400> 174

tcaggatgat ctggacgaag ag 22

<210> 175

<211> 20

<212> DNA

<213> Artificial sequences

<400> 175

ccggtcctgg actttgtctc 20

<210> 176

<211> 20

<212> DNA

<213> Artificial sequences

<400> 176

ctcgacatcg gcaaggtgtg 20

<210> 177

<211> 20

<212> DNA

<213> Artificial sequences

<400> 177

cgcattggag tcgctttaac 20

<210> 178

<211> 24

<212> DNA

<213> Artificial sequences

<400> 178

cgagctgcaa gaactcttcc tcac 24

<210> 179

<211> 23

<212> DNA

<213> Artificial sequences

<400> 179

cacggcactt acctgtgttc tgg 23

<210> 180

<211> 23

<212> DNA

<213> Artificial sequences

<400> 180

cagtacaggc atccctgtga aag 23

<210> 181

<211> 590

<212> DNA

<213> Artificial sequences

<400> 181

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> 182

<211> 23

<212> DNA

<213> Artificial sequences

<400> 182

cgcgagcaca gctaaggcca cgg 23

<210> 183

<211> 23

<212> DNA

<213> Artificial sequences

<400> 183

actctctctt tctggcctgg agg 23

<210> 184

<211> 23

<212> DNA

<213> Artificial sequences

<400> 184

acccagcagg gcgtggagcc agg 23

<210> 185

<211> 23

<212> DNA

<213> Artificial sequences

<400> 185

gtcagagccc caaggtaaaa agg 23

Claims (21)

1. A pluripotent stem cell or a derivative thereof comprising the expression sequence of an IL-6 blocker, The IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody; The expression sequence of the IL-6 blocker is preferably inserted into the genome of the pluripotent stem cell or derivative thereof. 2. A pluripotent stem cell or derivative thereof comprising the expression sequence of an IL-6 blocker, The IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody; The expression sequence of the IL-6 blocker is preferably inserted into the genome of the pluripotent stem cell or derivative thereof; The B2M gene and/or the CIITA gene of the genome of the pluripotent stem cell or its derivative is knocked out. 3. A pluripotent stem cell or derivative thereof comprising the expression sequence of an IL-6 blocker, The IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody; The expression sequence of the IL-6 blocker is preferably inserted into the genome of the pluripotent stem cell or derivative thereof; The pluripotent stem cell or its derivative further comprises an immune-compatible molecule expression sequence, and the immune-compatible molecule is used to regulate the expression of genes related to immune response in the pluripotent stem cell or its derivative; The immunocompatible molecule expression sequence is preferably inserted into the genome of the pluripotent stem cell or a derivative thereof. 4. A pluripotent stem cell or a derivative thereof comprising the expression sequence of an IL-6 blocker, The IL-6 blocker is an IL-6 antibody and/or an IL-6 receptor antibody; The expression sequence of the IL-6 blocker is preferably inserted into the genome of the pluripotent stem cell or derivative thereof; The pluripotent stem cell or its derivative further comprises an immune-compatible molecule expression sequence, and the immune-compatible molecule is used to regulate the expression of genes related to immune response in the pluripotent stem cell or its derivative; The immunocompatible molecule expression sequence is preferably inserted into the genome of the pluripotent stem cell or its derivative; The pluripotent stem cells or derivatives thereof further comprise an inducible gene expression system; The inducible gene expression system is preferably inserted into the genome of the pluripotent stem cell or a derivative thereof. 5 . The pluripotent stem cell or its derivative according to claim 4 , wherein the inducible gene expression system is at least one of a Tet-Off system and a dimer inducible expression system. 6 . 6. The pluripotent stem cell or derivative thereof according to claim 3 or 4, wherein the immune compatible molecule comprises one or more of the following: (I) immune tolerance-related genes, including CD47 or HLA-G; (II) HLA-C class molecules, including HLA-C multiple alleles with a proportion of more than 90% in the population, or fusion protein genes composed of more than 90% of HLA-C multiple alleles and B2M; (III) shRNA and/or shRNA-miR targeting the gene related to immune response. 7. The pluripotent stem cell or derivative thereof according to claim 3 or 4, wherein the gene related to immune response comprises: (I) Major histocompatibility complex genes, including HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA - at least one of DQB1, HLA-DPAl and HLA-DPB1; (II) Major histocompatibility complex-related genes, including at least one of B2M and CIITA. 8. The pluripotent stem cell or its derivative according to claim 6, wherein, The target sequence of shRNA and/or shRNA-miR targeting B2M is selected from one of SEQ ID NO.6 to SEQ ID NO.8; The target sequence of shRNA and/or shRNA-miR targeting CIITA is selected from one of SEQ ID NO.9 to SEQ ID NO.18; The target sequence of shRNA and/or shRNA-miR targeting HLA-A is selected from one of SEQ ID NO.19 to SEQ ID NO.21; The target sequence of shRNA and/or shRNA-miR targeting HLA-B is selected from one of SEQ ID NO.22-SEQ ID NO.27; The target sequence of shRNA and/or shRNA-miR targeting HLA-C is selected from one of SEQ ID NO.28 to SEQ ID NO.33; The target sequence of shRNA and/or shRNA-miR targeting HLA-DRA is selected from one of SEQ ID NO.34-SEQ ID NO.43; The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB1 is selected from one of SEQ ID NO.44-SEQ ID NO.48; The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB3 is selected from one of SEQ ID NO.49-SEQ ID NO.50; The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB4 is selected from one of SEQ ID NO.51 to SEQ ID NO.60; The target sequence of shRNA and/or shRNA-miR targeting HLA-DRB5 is selected from one of SEQ ID NO.61 to SEQ ID NO.69; The target sequence of shRNA and/or shRNA-miR targeting HLA-DQA1 is selected from one of SEQ ID NO.70-SEQ ID NO.76; The target sequence of shRNA and/or shRNA-miR targeting HLA-DQB1 is selected from one of SEQ ID NO.77-SEQ ID NO.86; The target sequence of shRNA and/or shRNA-miR targeting HLA-DPA1 is selected from one of SEQ ID NO.87-SEQ ID NO.96; The target sequence of the shRNA and/or shRNA-miR targeting HLA-DPB1 is selected from one of SEQ ID NO.97-SEQ ID NO.106. 9. The pluripotent stem cell or its derivative according to claim 3 or 4, wherein the shRNA processing complex-related gene and the miRNA processing complex-related gene are also introduced into the genome of the pluripotent stem cell or its derivative and at least one of anti-interferon effector molecules. 10. The pluripotent stem cell or derivative thereof according to claim 9, wherein the shRNA processing complex-related genes and miRNA processing complex-related genes comprise Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP At least one of (TARBP2), PACT (PRKRA), DGCR8; the anti-interferon effector molecule is preferably shRNA and/or shRNA targeting at least one of PKR, 2-5As, IRF-3 and IRF-7 -miRs. 11. The pluripotent stem cell or its derivative according to claim 10, wherein, The target sequence of shRNA targeting PKR and/or shRNA-miR is selected from one of SEQ ID NO.107～SEQ ID NO.116; The target sequence of shRNA and/or shRNA-miR targeting 2-5As is selected from one of SEQ ID NO.117-SEQ ID NO.146; The target sequence of shRNA and/or shRNA-miR targeting IRF-3 is selected from one of SEQ ID NO.147-SEQ ID NO.156; The target sequence of shRNA targeting IRF-7 and/or shRNA-miR is selected from one of SEQ ID NO.157-SEQ ID NO.166. 12. The pluripotent stem cell or its derivative according to claim 6 or 9, wherein, Described shRNA expression frame: from 5 ' to 3 ' sequentially including shRNA target sequence, stem-loop sequence, the reverse complement sequence of shRNA target sequence, Poly T; Wherein, the shRNA target sequence, the stem-loop sequence and the reverse complementary sequence of the shRNA target sequence form a hairpin structure; Poly T is the transcription terminator of RNA polymerase III; shRNA-miR expression framework: obtained by replacing the target sequence in microRNA-30 or microRNA-155 with the shRNA-miR target sequence. 13. The pluripotent stem cell or its derivative according to claim 12, wherein the length of the stem-loop sequence in the shRNA expression framework is 3-9 bases; the length of the Poly T is 5-6 bases base. 14. The pluripotent stem cell or derivative thereof according to any one of claims 1 to 4, wherein the expression sequence of the IL-6 blocker, the expression sequence of the immune compatible molecule or the inducible gene The expression system is inserted at a safe site in the genome of the pluripotent stem cell or derivative thereof. 15. The pluripotent stem cell or derivative thereof according to claim 14, wherein the genome security site comprises one or more of the AAVS1 security site, the eGSH security site, and the H11 security site . 16. The pluripotent stem cell or derivative thereof according to any one of claims 1 to 4, wherein the pluripotent stem cell comprises embryonic stem cells, embryonic germ cells, embryonic cancer cells, or induced pluripotent stem cells. 17. The pluripotent stem cell or its derivative according to any one of claims 1 to 4, wherein the pluripotent stem cell derivative comprises an adult stem cell, each germ layer cell or tissue differentiated by the pluripotent stem cell; The adult stem cells include mesenchymal stem cells or neural stem cells. 18. The pluripotent stem cell or its derivative according to any one of claims 1 to 4, wherein the heavy chain sequence of the IL-6 antibody is shown in SEQ ID NO.1, and the light chain sequence is shown in SEQ ID NO. 1 NO.2 is shown. 19. The pluripotent stem cell or its derivative according to any one of claims 1 to 4, wherein the heavy chain sequence of the IL-6 receptor antibody is shown in SEQ ID NO. 3, and the light chain sequence is shown in SEQ ID NO. 3. shown in SEQ ID NO.4. 20. Use of the pluripotent stem cells or their derivatives according to any one of claims 1 to 19 in the preparation of a medicament for treating a disease, wherein the disease is at least one of rheumatoid arthritis, depression and anxiety. 21. A preparation comprising the pluripotent stem cells or derivatives thereof according to any one of claims 1 to 19.

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