Universal cells and their preparation method

Abstract

A method for preparing universal cells is disclosed. After major histocompatibility complex class I (MHC-I) and class II genes are inactivated in cells, fusion proteins containing functional domains of CD47 and / or CD24 are overexpressed, so that the obtained human pluripotent stem cells or human cell lines avoid T cell attack and further avoid cell killing by NK cells, the effect of which is even better than that of the reported prominent target CD47. Meanwhile, the less immunogenic pluripotent stem cells retain their stemness and differentiation ability.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS The present invention claims priority to the prior applications of Patent Application No. 202210307870.8 entitled "Universal cell and method for preparation same" filed with the China Intellectual Property Office on March 25, 2022, and Patent Application No. 202210806871.7 entitled "Universal cell and method for preparation same" filed with the China Intellectual Property Office on July 8, 2022. The contents of these two prior applications are incorporated herein by reference in their entireties.

[0002] The present invention belongs to the intersection of genetic engineering and stem cell technology, and in particular relates to universal cells and methods for their preparation. [Background technology]

[0003] Through in vitro cell culture or induced differentiation of stem cells, healthy functional cells can be regenerated in large quantities in vitro, and diseases can be treated by allogeneic functional cell transplantation. However, immune incompatibility and immune rejection of transplanted cells remain important obstacles to their clinical application. Stem cells are a kind of "seed" cell with the ability of self-renewal and differentiation into specific functional cells. According to the different degrees of stem cell properties, stem cells are mainly classified into totipotent stem cells, pluripotent stem cells (PSCs), and adult stem cells. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the potential to proliferate indefinitely, self-renew, and differentiate into various cell types, and have significant application prospects in the treatment of cancer, neurological related diseases, cardiovascular diseases, etc.

[0004] Although autologous cell transplantation can avoid the problem of immune rejection, the cost of producing autologous cells from patients is high, the preparation process cycle is long (Khera et al., 2013), and the quality and efficacy of the cell product from each source is uncertain. Data indicates that the cells in a patient's body are different from those in a normal person's body, and the efficacy of the treatment may be affected.

[0005] Immunogenicity, or the rejection of the host immune system against allogeneic transplanted cells, can be reduced by immunosuppressive drugs, HLA matching, and gene editing. Immunosuppressive drugs have severe side effects and can cause bone marrow suppression, hepatotoxicity, hair loss, and gastrointestinal adverse reactions. Currently, HLA-matched iPSC libraries have been established in the United States, Japan, and China, but the construction and maintenance of libraries is costly because HLA antigen genes are the most polymorphic genes observed in the human genome. Currently, iPSC libraries in various regions cannot provide matches for most people in each country and can only cover certain populations (Solomon et al., 2015, Turner et al., 2013). Although allogeneic cell therapy for large patient groups may have very clear advantages over matching libraries in terms of economics, as well as construction and operation costs, allogeneic cell therapy is subject to strong immune rejection. Therefore, there is an urgent need to construct universal PSCs that are allogeneic immune compatible.

[0006] The human major histocompatibility complex (MHC), or human leukocyte antigens (HLA), is the main cause of immune incompatibility. The HLA complex consists of a set of genes that can be classified into class I, class II, and class III. MHC-I genes are expressed in almost all tissue cell types, and transplanted cells expressing "non-self" MHC class I molecules stimulate the activation of CD8+ T cells and are eliminated. CD4+ helper T cells recognize the MHC-II genes of "non-self" cells, resulting in immune rejection, whereas class III molecules are not involved in immune activity. The use of gene editing methods to modify immunogenic elements to generate less immunogenic cells allows for the large-scale manufacture of immune-privileged "off-the-shelf" cell therapy products.

[0007] In recent years, it has been previously reported that it is possible to generate immune-compatible universal PSCs by knocking out genes such as B2M and CIITA, thereby deleting the expression of MHC-I and MHC-II on the cell surface or the genes themselves, thereby conferring immune tolerance to the cells or avoiding T / B cell-specific immune responses, which lays an important foundation for the more widespread application of cells, tissues, and organs derived from this universal PSC. However, HLA molecules are the major inhibitory ligands for natural killer cells (NK cells), and MHC class I-negative cells are susceptible to lysis by natural killer (NK) cells. In vitro and in vivo data have shown that host NK cells can inhibit the expression of transplanted B2M - / - It has been shown that donor cells can be eliminated (Flahou et al., 2021). Therefore, it is necessary to improve previous methods to generate universal donor cells that can avoid immune responses. It has been reported that by disrupting the expression of MHC class I and MHC class II genes, cells can express non-classical HLA class I molecules such as HLA-E / G, or express immune inhibitory checkpoint proteins such as PD-L1, CTLA4-Ig, CD47, and CD24, and thus effectively avoid cell killing by NK cells (Zhao, W. et al., 2020; Ye, Q. et al., 2020; WO2021041316A1).

[0008] These schemes have technical problems such as incomplete, unclear or non-sustained immune compatibility, narrow effective dose window, based only on the direct application of common prominent molecules known in the art, thus indicating that more innovative and better optimized strategies are needed to achieve a more optimized modification of stem cells to obtain a better immune privilege scheme. Summary of the Invention

[0009] In response to the shortcomings of existing technology, the present invention provides a novel method for modifying cells to obtain low immunogenicity, which involves adopting the strategy of novel genes with domain fusion for the first time, carrying out the strategic implementation of modifying human stem cells based on CD47 and CD24 to obtain low immunogenicity, and finally identifying a representative novel fusion gene through multiple rounds of screening and sequence combination testing. This novel fusion gene can significantly reduce or avoid the recognition and attack of the immune system, especially the attack of natural killer cells, macrophages, etc. The present invention provides a feasible strategy for realizing immune privilege of cells by developing novel genes with domain fusion.

[0010] In the present invention, a positive clone with B2M / CIITA double allele knockout (DKO cell) is successfully constructed by knocking out β-2-microglobulin (B2M) in the endoplasmic reticulum of human pluripotent stem cells and knocking out CIITA, a positive regulator of MHC-II gene transcription. Then, a lentiviral vector is used to overexpress the novel fusion gene identified by the present invention in this DKO cell, so that the obtained human pluripotent stem cells can further avoid cell killing by NK cells based on avoiding T cell attack, thus achieving an unexpectedly superior immune evasion effect compared with DKO+CD47 (hereinafter "CD47 prior art"). Meanwhile, the less immunogenic pluripotent stem cells retain the important biological functions of pluripotent stem cells, such as their stemness and differentiation ability.

[0011] To achieve the above objectives, the present invention adopts the following technical solutions:

[0012] In a first aspect, the present invention provides a universal cell which, compared to a wild-type cell, comprises: 1) reduced or absent expression of MHC-I and / or MHC-II human leukocyte antigens; and 2) expression of a fusion protein containing an immune inhibitory checkpoint; Here, the cells are able to avoid attack by T cells and killing by NK cells.

[0013] In certain embodiments, the fusion protein comprising an immune inhibitory checkpoint comprises two or more of PD-L1, CTLA4-Ig, CD47, and CD24. Preferably, a fusion protein comprising two of the immune inhibitory checkpoints is expressed.

[0014] According to the present invention, fusion proteins containing functional domains of CD47 and / or CD24 are expressed.

[0015] In certain aspects, the cells comprise reduced or no expression of MHC-I and MHC-II human leukocyte antigens.

[0016] In certain embodiments, gene editing tools (such as TALEN and / or CRISPR systems) are used in cells to target one or more genes encoding one or more transcriptional regulators of MHC-I and one or more genes encoding one or more transcriptional regulators of MHC-II to achieve reduced or no expression of MHC-I and MHC-II genes.

[0017] In certain embodiments, to achieve reduced or non-expression of MHC-I and MHC-II genes, the transcriptional regulator of MHC-I may preferably be selected from one or more of B2M, TAP1, TAP2, TAP-related glycoprotein (Tapasin) or NLRC5, and the transcriptional regulator of MHC-II may preferably be selected from one or more of CIITA, RFXANK, RFX5 and RFXAP.

[0018] The transcriptional regulator is preferably B2M or CIITA.

[0019] In certain embodiments, the cells further comprise a genetic modification that targets the CIITA gene with a rare-cutting endonuclease that selectively inactivates the CIITA gene.

[0020] In certain embodiments, the cells further comprise a genetic modification that targets the B2M gene with a rare-cutting endonuclease that selectively inactivates the B2M gene.

[0021] In certain embodiments, the genetic modification targeting the CIITA gene or B2M gene by a rare-cutting endonuclease comprises a CAS protein or a polynucleotide encoding a CAS protein and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene or B2M.

[0022] In a specific embodiment, the CRISPR / CAS9 system is used to directly knock out the exon segments of B2M and CIITA at both ends, where the target sequences of gRNA for B2M gene are SEQ ID NO:2 and SEQ ID NO:3, and the target sequences of gRNA for CIITA gene are SEQ ID NO:4 and SEQ ID NO:5.

[0023] In certain aspects, gene expression modifying molecules for one or more genes encoding one or more transcriptional regulators of MHC-I or one or more genes encoding one or more transcriptional regulators of MHC-II are introduced into the cells, thereby achieving reduced or non-expression of MHC-I and / or MHC-II genes, the gene expression modifying molecules comprising one selected from siRNA, shRNA, microRNA, antisense RNA, and another RNA-mediated inhibitory molecule.

[0024] In a particular aspect, the functional domain of CD47 in a fusion protein comprising the functional domains of CD47 and / or CD24 is the transmembrane domain of CD47, and preferably, the amino acid sequence of the transmembrane domain of CD47 is as set forth in any one of SEQ ID NOs: 6 to 10.

[0025] In a specific aspect, the functional domain of CD24 in a fusion protein comprising the functional domains of CD47 and / or CD24 is the signal peptide sequence of CD24, the mature peptide of CD24, the extracellular peptide of CD24, the membrane anchor sequence of CD24, and the extracellular mature peptide of CD24, and preferably, the amino acid sequence of the functional domain of CD24 is as set forth in any one of SEQ ID NOs: 11 to 16.

[0026] In a particular embodiment, the fusion protein constructed with functional domains of CD47 and CD24 is a fusion protein in which the SIRPα binding domain of CD47 is connected to the membrane anchor sequence of CD24.

[0027] In a specific embodiment, the fusion protein constructed with the functional domains of CD47 and CD24 is a fusion protein in which the SIRPα-binding domain of CD47 is inserted at the junction of the extracellular sequence and the membrane anchor sequence of CD24.

[0028] In a particular embodiment, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the SIRPα binding domain of CD47 is linked after the extracellular sequence of CD24.

[0029] In a particular embodiment, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the mature peptide of CD24 is linked after the SIRPα binding domain of CD47.

[0030] In a specific embodiment, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the extracellular mature peptide of CD24 is inserted at the junction of the SIRPα binding domain and the transmembrane domain of CD47.

[0031] In a particular embodiment, the fusion protein constructed with the functional domains of CD47 and CD24 is a fusion protein in which the mature peptide sequence of CD24 is connected to the transmembrane domain of CD47.

[0032] Preferably, the amino acid sequence of the fusion protein constructed with the functional domains of CD47 and CD24 has more than 70% homology, such as more than 80% homology, such as more than 90%, more than 95% or more than 98% homology, with the sequence shown in SEQ ID NO:1.

[0033] More preferably, the amino acid sequence of the fusion protein constructed by the functional domains of CD47 and CD24 is as shown in SEQ ID NO:1.

[0034] In a particular embodiment, the nucleic acid sequence encoding the fusion protein constructed by the functional domains of CD47 and CD24 can be obtained based on the amino acid sequence of the fusion protein.

[0035] In certain aspects, the cells further comprise modifications to increase expression of one or more of the following polypeptides: DUX4, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, Cl inhibitor, IL-10, IL-35, CCL21, Mfge8, and SerpinB9.

[0036] In a particular embodiment, the cell is an embryonic stem cell.

[0037] In certain aspects, the cells are pluripotent stem cells.

[0038] In certain embodiments, the cells are poorly immunogenic stem cells.

[0039] In certain embodiments, the cells are human stem cells or human somatic cells.

[0040] In a second aspect, the present invention provides a method for preparing a universal cell of the first aspect, the method comprising the steps of: 1) knocking out one or more genes for one or more transcriptional regulators of MHC-I in a cell; and / or 2) knocking out one or more genes of one or more transcriptional regulators of MHC-II in the cell; and 3) introducing into the cell a nucleic acid sequence encoding a fusion protein comprising an immunoinhibitory checkpoint.

[0041] In certain embodiments, the fusion protein comprising the immunoinhibitory checkpoints comprises more than one of PD-L1, CTLA4-Ig, CD47, and CD24. Preferably, a nucleic acid sequence encoding a fusion protein comprising two of the immunoinhibitory checkpoints is introduced.

[0042] According to the present invention, a nucleic acid sequence is introduced that encodes a fusion protein constructed by including functional domains of CD47 and / or CD24.

[0043] In certain aspects, the transcriptional regulator of MHC-I may preferably be selected from one or more of B2M, TAP1, TAP2, TAP-related glycoprotein (Tapasin), or NLRC5. The transcriptional regulator of MHC-II may preferably be selected from one or more of CIITA, RFXANK, RFX5, and RFXAP.

[0044] In certain embodiments, the transcription factor is selected from B2M and CIITA.

[0045] In a particular embodiment, the knockout in steps 1) and 2) is a genetic modification that targets the CIITA gene or the B2M gene with a rare-cutting endonuclease that selectively inactivates the CIITA gene or the B2M gene.

[0046] Preferably, the rare-cutting endonuclease is selected from a CAS protein, a TALE nuclease, a zinc finger nuclease, a large nuclease, and a homing nuclease.

[0047] More preferably, the genetic modification targeting the CIITA gene or B2M gene by a rare-cutting endonuclease comprises a CAS protein or a polynucleotide encoding a CAS protein and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene or B2M.

[0048] In a specific embodiment, the CRISPR system is used to directly knock out the exon segments of B2M and CIITA at both ends in steps 1) and 2), respectively, where the target sequences of gRNA for B2M gene are SEQ ID NO:2 and SEQ ID NO:3, and the target sequences of gRNA for CIITA gene are SEQ ID NO:4 and SEQ ID NO:5.

[0049] In a particular embodiment, the knockout in step 1) or 2) is achieved by introducing a gene expression modifying molecule for one or more genes encoding one or more transcriptional regulators of MHC-I or one or more genes encoding one or more transcriptional regulators of MHC-II, thereby achieving reduced or no expression of MHC-I and / or MHC-II genes, the gene expression modifying molecule comprising one selected from siRNA, shRNA, microRNA, antisense RNA, and another RNA-mediated inhibitory molecule.

[0050] In a particular embodiment, in step 3), an expression vector is used to introduce into the cells a nucleic acid sequence encoding a fusion protein comprising functional domains of CD47 and / or CD24.

[0051] Preferably, the expression vector used in step 3) is a viral vector.

[0052] In certain embodiments, the viral vector used in step 3) is a lentivirus.

[0053] In a particular embodiment, step 3) comprises introducing a nucleic acid sequence encoding a fusion protein comprising a functional domain of CD47 and / or CD24 into a selected site of the cell, preferably the selected site of the cell being a safe harbor gene site.

[0054] In a particular aspect, the functional domain of CD47 in a fusion protein comprising the functional domains of CD47 and / or CD24 is the transmembrane domain of CD47, and preferably, the amino acid sequence of the transmembrane domain of CD47 is as set forth in any one of SEQ ID NOs: 6 to 10.

[0055] In a specific aspect, the functional domain of CD24 in a fusion protein comprising the functional domains of CD47 and / or CD24 is the signal peptide sequence of CD24, the mature peptide of CD24, the extracellular peptide of CD24, the membrane anchor sequence of CD24, and the extracellular mature peptide of CD24, and preferably, the amino acid sequence of the functional domain of CD24 is as set forth in any one of SEQ ID NOs: 11 to 16.

[0056] In a particular embodiment, the fusion protein constructed with functional domains of CD47 and CD24 is a fusion protein in which the SIRPα binding domain of CD47 is connected to the membrane anchor sequence of CD24.

[0057] In a specific embodiment, the fusion protein constructed with the functional domains of CD47 and CD24 is a fusion protein in which the SIRPα-binding domain of CD47 is inserted at the junction of the extracellular sequence and the membrane anchor sequence of CD24.

[0058] In a particular embodiment, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the SIRPα binding domain of CD47 is linked after the extracellular sequence of CD24.

[0059] In a particular embodiment, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the mature peptide of CD24 is linked after the SIRPα binding domain of CD47.

[0060] In a specific embodiment, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the extracellular mature peptide of CD24 is inserted at the junction of the SIRPα binding domain and the transmembrane domain of CD47.

[0061] In a particular embodiment, the fusion protein constructed with the functional domains of CD47 and CD24 is a fusion protein in which the mature peptide sequence of CD24 is connected to the transmembrane domain of CD47.

[0062] Preferably, the fusion protein constructed with the functional domains of CD47 and CD24 has at least more than 70% homology, such as more than 80% homology, such as more than 90%, more than 95% or more than 98% homology, with the sequence shown in SEQ ID NO: 1. More preferably, the amino acid sequence of the fusion protein constructed with the functional domains of CD47 and CD24 is as shown in SEQ ID NO: 1.

[0063] In certain aspects, the universal cell further comprises a second expression vector comprising a polynucleotide sequence encoding one selected from CD35, CD27, DUX4, CD26, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, IDO2, TDO, CTLA4-IgG, Cl inhibitor, IL-10, CD46, CD55, CD59, CCL21, Mfge8, SerpinB9, and IL-35.

[0064] In certain embodiments, the second expression vector is an inducible expression vector, and preferably, the second expression vector is a viral vector.

[0065] In a third aspect, the present invention provides a method for preparing a differentiated universal cell, the method comprising culturing under differentiation conditions a universal cell prepared according to the method according to the second aspect, thereby preparing a less immunogenic differentiated cell.

[0066] In certain aspects, the differentiation conditions are suitable for differentiating cells into a cell type selected from cardiomyocytes, neuronal cells, glial cells, endothelial cells, T cells, NK cells, NKT cells, macrophages, hematopoietic progenitor cells, mesenchymal cells, pancreatic islet cells, chondrocytes, retinal pigment epithelial cells, kidney cells, liver cells, thyroid cells, skin cells, blood cells, and epithelial cells.

[0067] In a fourth aspect, the present invention provides a method for treating a patient in need of cell therapy comprising administering a population of less immunogenic differentiated cells prepared according to the method according to the third aspect.

[0068] In a fifth aspect, the present invention provides a composition comprising a universal cell according to the first aspect.

[0069] In certain aspects, the composition comprises a universal cell as described in the first aspect and one or more therapeutic agents, including peptides, cytokines, small molecule compounds, polymers, ADCs, antibodies, nanoparticles, biological analogs, mRNA, Chinese herbologies, proteins, vaccines, checkpoint inhibitors, mitogens, growth factors, small RNA, double-stranded RNA (dsRNA), mononuclear blood cells, feeder cells, feeder cell components or substitutes thereof, vectors comprising one or more polynucleic acids of interest, antibodies, etc.

[0070] In a sixth aspect, the present invention provides a fusion protein comprising a functional domain of CD47 and / or CD24.

[0071] In a particular aspect, the functional domain of CD47 in a fusion protein comprising the functional domains of CD47 and / or CD24 is the transmembrane domain of CD47, and preferably, the amino acid sequence of the transmembrane domain of CD47 is as set forth in any one of SEQ ID NOs: 6 to 10.

[0072] In a specific aspect, the functional domain of CD24 in a fusion protein comprising the functional domains of CD47 and / or CD24 is the signal peptide sequence of CD24, the mature peptide of CD24, the extracellular peptide of CD24, the membrane anchor sequence of CD24, and the extracellular mature peptide of CD24, and preferably, the amino acid sequence of the functional domain of CD24 is as set forth in any one of SEQ ID NOs: 11 to 16.

[0073] In a particular embodiment, the fusion protein constructed with functional domains of CD47 and CD24 is a fusion protein in which the SIRPα binding domain of CD47 is connected to the membrane anchor sequence of CD24.

[0074] In a specific embodiment, the fusion protein constructed with the functional domains of CD47 and CD24 is a fusion protein in which the SIRPα-binding domain of CD47 is inserted at the junction of the extracellular sequence and the membrane anchor sequence of CD24.

[0075] In a particular embodiment, the fusion protein constructed with the functional domains of CD47 and CD24 is a fusion protein in which CD47 is linked after the extracellular sequence of CD24.

[0076] In a particular embodiment, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the mature peptide of CD24 is linked after the SIRPα binding domain of CD47.

[0077] In a specific embodiment, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the extracellular mature peptide of CD24 is inserted at the junction of the SIRPα binding domain and the transmembrane domain of CD47.

[0078] In a particular embodiment, the fusion protein constructed with the functional domains of CD47 and CD24 is a fusion protein in which the mature peptide sequence of CD24 is connected to the transmembrane domain of CD47.

[0079] Preferably, the fusion protein constructed with the functional domains of CD47 and CD24 has at least more than 70% homology, such as more than 80% homology, such as more than 90%, more than 95% or more than 98% homology, with the sequence shown in SEQ ID NO: 1. More preferably, the amino acid sequence of the fusion protein constructed with the functional domains of CD47 and CD24 is as shown in SEQ ID NO: 1.

[0080] In a seventh aspect, the present invention provides a nucleic acid sequence encoding the fusion protein of the sixth aspect, said nucleic acid sequence being obtainable based on the amino acid sequence of said fusion protein.

[0081] In an eighth aspect, the present invention provides an expression vector or expression cassette comprising a nucleic acid sequence of the seventh aspect.

[0082] In a ninth aspect, the present invention provides a cell comprising expression of a fusion protein of the sixth aspect and reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0083] In a tenth aspect, the present invention provides a cell comprising no expression of CIITA, expression of a fusion protein of the sixth aspect, and reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0084] In an eleventh aspect, the present invention provides a cell comprising no expression of B2M, expression of a fusion protein of the sixth aspect, and reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0085] In a twelfth aspect, the present invention provides a cell comprising no expression of CIITA and B2M, expression of a fusion protein of the sixth aspect, and reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0086] In a thirteenth aspect, the present invention provides a cell comprising a fusion protein of the sixth aspect and expression of at least one polypeptide selected from DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, Cl inhibitor, CD46, CD55, CD59 and IL-35, and reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0087] In a fourteenth aspect, the present invention provides a cell comprising no expression of CIITA, expression of a fusion protein of the sixth aspect and at least one polypeptide selected from CD35, CD27, DUX4, CD26, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, IDO2, TDO, CTLA4-IgG, Cl inhibitor, IL-10, CD46, CD55, CD59, CCL21, Mfge8, SerpinB9 and IL-35, and reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0088] In a fifteenth aspect, the invention provides a cell comprising no expression of B2M, expression of a fusion protein of the sixth aspect and at least one polypeptide selected from DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, Cl inhibitor, CD46, CD55, CD59 and IL-35, and reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0089] In a sixteenth aspect, the present invention provides a cell comprising no expression of CIITA and B2M, expression of a fusion protein of the sixth aspect and at least one polypeptide selected from CD35, CD27, DUX4, CD26, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, IDO2, TDO, CTLA4-IgG, Cl inhibitor, IL-10, CD46, CD55, CD59, CCL21, Mfge8, SerpinB9 and IL-35, and reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0090] In a seventeenth aspect, the present invention provides a cell as described in any one of the ninth to sixteenth aspects above, wherein the cell is selected from a stem cell, a differentiated cell, a pluripotent stem cell, an induced pluripotent stem cell, an adult stem cell, a progenitor cell, a somatic cell, a primary T cell, and a chimeric antigen receptor T cell.

[0091] In an eighteenth aspect, the present invention provides the use of a universal cell according to the first aspect, a composition according to the fifth aspect, or an expression vector or expression cassette according to the eighth aspect in the preparation of a cell therapy product.

[0092] In a nineteenth aspect, the present invention provides the use of a universal cell according to the first aspect, a composition according to the fifth aspect, or an expression vector or expression cassette according to the eighth aspect in the preparation of a product for organ transplantation.

[0093] In a twentieth aspect, the present invention provides the use of a universal cell according to the first aspect, a composition according to the fifth aspect, or an expression vector or expression cassette according to the eighth aspect in constructing a cell library of universal PSCs.

[0094] In a twenty-first aspect, the present invention provides the use of a universal cell according to the first aspect, a composition according to the fifth aspect, or an expression vector or expression cassette according to the eighth aspect as a gene drug carrier.

[0095] Beneficial Effects of the Invention After major histocompatibility complex class I (MHC-I) and class II genes are inactivated in stem cells in the present invention, the fusion protein XSG006 constructed by the functional domains of CD47 and CD24 is overexpressed, so that the obtained human pluripotent stem cells further avoid cell killing by NK cells based on avoiding the attack of T cells, and the effect is even better than that of the reported positive targets CD47 and CD24. Meanwhile, the less immunogenic pluripotent stem cells retain their stemness and differentiation ability. Compared with neural cells differentiated from WT cells, neural cells differentiated from DKO+G6 cells overexpressing the fusion protein XSG006 can effectively avoid the attack of the immune system in vivo. [Brief description of the drawings]

[0096] [Figure 1] FIG. 1 shows the knockout strategy and results of the B2M gene in the human embryonic stem cell line H1. [Diagram 2] FIG. 1 shows the knockout strategy and results of the CIITA gene in human embryonic stem cell line H1. [Diagram 3] FIG. 1 shows the expression of B2M and CIITA at the RNA level in DKO cells detected by RT-qPCR in Example 1. [Figure 4] FIG. 1 shows the results of B2M at the protein level detected by Western blot in Example 1. [Diagram 5] FIG. 1 shows the expression of HLA-I / II in various cells detected by a flow cytometer after stimulating wild-type H1 (WT) and DKO with INF-gamma in Example 1. Here, T cells are a positive control for detecting HLA class I / II molecules. [Figure 6] FIG. 2 shows the karyotypes of B2M / CIITA double allele knockout (DKO) positive clones obtained in Example 1. [Figure 7]1 shows the expression of stemness genes POU5F1 / NANOG / SOX2 at RNA and protein levels in WT and DKO cells, as detected by immunofluorescence and RT-qPCR in Example 2. MSCs are the negative control. [Figure 8] FIG. 1 shows the expression of stemness genes SSEA-4 and Tra1-81 on the surface of WT and DKO cells detected by immunofluorescence in Example 2. [Figure 9] Immunohistochemistry has been used to show that DKO cells can form teratomas with three germ layers: endoderm, mesoderm, and ectoderm. [Figure 10] FIG. 1 shows the results of verifying the immune function of DKO cells detected by RTCA in Example 2. [Figure 11] FIG. 1 is a schematic diagram of the structure of pGC-EF1a plasmid. [Figure 12] FIG. 1 shows the expression of CD47 in the DKO+CD47 cell line constructed in Example 3, as detected by a flow cytometer. [Figure 13] FIG. 1 shows the results of NK cell killing against CD47-overexpressing DKO+CD47 cell lines, WT, and DKO cells, as detected by RTCA in Example 3. [Figure 14] FIG. 13 shows the results of NK cell killing against cell lines DKO+G1 to G20 overexpressing XSG1 to XSG20 fusion proteins, H1WT (positive control), and H1 DKO cells (negative control), detected by RTCA in Example 4. [Figure 15] 1 shows the sequence PCR identification of the genome of the DKO+G6 cell line constructed in Example 5. [Figure 16] FIG. 13 shows the results of NK cell killing against the DKO+G6 cell line, WT cells, and DKO cells overexpressing the XSG006 protein, as detected by RTCA in Example 5. [Figure 17]FIG. 1 shows the results of NK cell killing against DKO+G6 cell line overexpressing XSG006 protein, WT, DKO, and DKO+CD24 / CD47 cells overexpressing CD24 or CD47 protein, detected by RTCA in Example 6. FIG. 1 shows the results of cell killing. FIG. 1 shows a summary of multiple results. [Figure 18] FIG. 1 shows the results of NK cell killing against DKO+G6 cell line overexpressing XSG006 protein, WT, DKO, DKO+CD24 / CD47 cells overexpressing CD24 or CD47 protein, and DKO+CD47+CD24 cells overexpressing CD24 and CD47 protein, detected by RTCA in Example 7. FIG. 1 shows the results of cell killing. FIG. 1 shows a summary of multiple results. [Figure 19] FIG. 13 shows the results of verifying the immune evasion effect of neural cells differentiated from DKO+G6 in Example 8 in mice with a humanized immune system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] The technical solutions of the present invention will be described in more detail below in conjunction with specific examples. It should be understood that the following examples are only intended to illustrate and explain the present invention by way of example only, and should not be construed as limiting the scope of protection of the present invention. All technologies achieved based on the above content of the present invention are included in the scope that the present invention aims to protect.

[0098] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods in the following examples (for which no specific conditions are specified) are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommended conditions.

[0099] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0100] Example 1. Construction of B2M and CIITA double knockout cell line (DKO) 1. Cell culture reagents: [Table 1]

[0101] 2. Methods and Results: In the present invention, human pluripotent stem cell lines H1 (Wicell, WA01) or H9 (Wicell, WA09) were selected to knock out beta-2-microglobulin (B2M) in the endoplasmic reticulum by CRISPR / CAS9, so that MHC-I on the cell surface cannot form functional molecules, thereby inhibiting the expression of allogeneic CD8 + Avoids killing by T cells. CD4 + Evasion of T cell killing was achieved by knocking out CIITA, a positive regulator of MHC-II gene transcription, thereby reducing expression of MHC class II molecules.

[0102] Here, the CRISPR / CAS9 knockout strategy of B2M gene is shown in Figure 1. B2M-gRNA1 and B2M-gRNA2 were used to directly knock out the exon segments of B2M at both ends, and then two pairs of PCR primers B2M-F1 / R1 and B2M-F2 / R2 were used to verify the genome sequence knockout.

[0103] gRNA sequence: B2M-gRNA1:CGTGAGTAAACCTGAATCTT B2M-gRNA2:AGTCACATGGTTCACACGGC Identification primers B2M-F1:TGGGGGCCAAATCATGTAGACTC B2M-R1:TCAGTGGGGGTGAATTCAGTGT B2M-F2+B2M-R2=608bp After knockout: no band B2M-F2:CAGAAGTCCTTGAGAGCCTCC B2M-R2:TGTGCATCAGTATCTCAGCAGG B2M-F2+B2M-R2=812bp After knockout: 569bp.

[0104] Furthermore, the CRISPR / CAS9 knockout strategy of CIITA gene is shown in Figure 2. CIITA-gRNA1 and CIITA-gRNA2 were used to directly knock out the exon segments of CIITA at both ends, and then two pairs of PCR primers, CIITA-F1 / R1 and CIITA-F2 / R2, were used to verify the genomic sequence knockout.

[0105] gRNA sequence: CIITA-gRNA1:GATATTGGCATAAGCCTCCC CIITA-gRNA2:CATCGCTGTTAAGAAGCTCC Identification primers: CIITA-F1:CTGTGCCTCTACCACTTCTATG CIITA-R1:CCTTCCATGTCACACAACAGCC CIITA-F1+CIITA-R1=368bp After knockout: no band CIITA-F2:TGGAATCCACACTTTCCAGTTC CIITA-R2:TGGAGTCTCCGTTCCTCCAG CIITA-F2+CIITA-R2=889bp After knockout: 459bp The specific operations were as follows:

[0106] 1) Human pluripotent stem cells were routinely cultured on Matrigel-coated 6-well plates using mTeSR1 to 80% confluency. After TRYPLE digestion, DMEM / F12 was added for neutralization and counting was performed. 2×10 6 The cells were absorbed into an EP tube, and after centrifugation, the supernatant was discarded.

[0107] 2) Based on the Neon transfection system, i.e., 100 μL electrotransfection system, 15 μg of TrueCut™ Cas9 protein + 3 μg of gRNA (B2MgRNA1 + B2MgRNA2 + CIITA gRNA1 + CIITA gRNA2) was added to form the RNP system, mixed thoroughly, and left at room temperature for 20 minutes.

[0108] 3) The cells were resuspended in 100 μL of RNP electrotransfection system and electrotransfected with Neon transfection system using electrotransfection parameters of 1200 V, 30 ms, and 1 pause. After electrotransfection, the cells were quickly added to pre-warmed medium and evenly seeded onto one of the Matrigel-coated 6-well plates.

[0109] 4) The medium was replaced with fresh mTeSR1 medium every day. When single cells grew, single clones were picked into 48-well plates. After the clones were amplified, genomic samples were collected for PCR to detect gene editing. The PCR results are shown in Figures 1 and 2. PCR-positive clones were sent to the company for Sanger sequencing for further validation.

[0110] 5) Positive clones of B2M / CIITA double allele knockout DKO were identified, amplified, cultured, and cryopreserved.

[0111] The expression of B2M and CIITA at the RNA level in the B2M / CIITA double allele knockout clone DKO was detected by qPCR to confirm the knockout, as shown in Figure 3 .

[0112] B2M-F:AAGATGAGTATGCCTGCCGT B2M-R:ATGCGGCATCTTCAAACCTC CIITA-F:CCTGGAGCTTCTTAACAGCGA CIITA-R: TGTGTCGGGTTCTGAGTAGAG

[0113] The expression of B2M at the protein level in the B2M / CIITA double allele knockout clone DKO was detected by Western blot to confirm the knockout, as shown in Figure 4 .

[0114] Stimulation of WT and DKO with INF-gamma: Cells were plated and medium containing INF-gamma was added to the cells at the next day liquid exchange. After 48 hours, cells were digested and HLA-I / II expression was detected by flow cytometry. The results are shown in Figure 5, where B2M / CIITA double allele knockout stem cell positive clone (DKO) cannot express HLA class I / II molecules in response to INF-gamma stimulation. T cells are the positive control for detecting HLA class I / II molecules.

[0115] Karyotype detection of the resulting positive clones with B2M / CIITA double allele knockout (DKO): The chromosome specimens fixed on slides were treated with trypsin and then stained with Giemsa stain. The chromosome number and morphological structure of metaphase chromosomes were analyzed to determine whether their karyotypes were consistent with normal karyotypes. The results are shown in Figure 6. The karyotype of DKO is normal.

[0116] Example 2. Verification of stemness and immune function of the DKO cell line in Example 1 1. Expression of stemness genes in WT and DKO cells Immunofluorescence detection shows that WT and DKO cells express stemness genes POU5F1 and NANOG at the protein level. Cells were plated in 12-well plates, and after the cells grew to 60-80% density, the medium was pipetted out. 4% paraformaldehyde was added to it for fixation. After the cell membrane was destroyed, primary antibodies for POU5F1 and NANOG were used for overnight incubation at 4°C. After washing the primary antibody, fluorescently labeled secondary antibodies were incubated at room temperature, and then photographed with a fluorescent microscope. The results are shown in Figure 7A. RT-qPCR detection shows that WT and DKO cells express stemness genes POU5F1, NANOG, and SOX2 at the RNA level. The results are shown in Figure 7B. (MSC is a negative control for stemness gene expression)

[0117] Flow cytometry shows that the stemness genes SSEA-4 and Tra1-81 are highly expressed on the surface of both WT and DKO cells, accounting for 100%, 99.98%, 96.75%, and 99.13%, respectively. The results are shown in Figure 8.

[0118] 2. Differentiation ability of positive clones obtained by B2M / CIITA double allele knockout (DKO) 100 μL of the suspension containing 5E+5 DKO cells was injected subcutaneously into immunodeficient mice (SCID beige). Teratomas were 1.5 cm 3 After growing larger, the teratomas were removed, sliced, and stained.

[0119] The obtained B2M / CIITA double allele knockout (DKO) positive clones could form teratomas in vivo and differentiate into cells with three germ layers: endoderm, mesoderm, and ectoderm. The results are shown in Figure 9.

[0120] 3. Verification of immune function of DKO cells The killing experiments of T cells and NK cells were performed using xCELLigence RTCA Instrument. The same number of WT and DKO cell lines were suspended in Essential 8 medium containing IL-2 and seeded on a 96-well E-plate coated with Matrigel, to which activated T cells or NK cells for killing detection were added. The RTCA detection data were analyzed by xCELLigence software to calculate the killing ratio and evasion function. [Table 2] As shown in the RTCA data in Figure 10, WT cells avoid killing by NK cells due to expression of HLA-I, but are killed by T cells, whereas DKO cells are able to avoid killing by T cells, but are more susceptible to killing by NK cells.

[0121] Example 3. Construction and verification of immune function of DKO+CD47 cell line CD47 (NM_198793) was overexpressed in the DKO cells obtained in Example 1 using a lentiviral vector. The amino acid sequence of CD47 is shown in SEQ ID NO: 29. The cDNA of the overexpression sequence (SEQ ID NO: 30) was constructed in a lentiviral plasmid (pGC-EF1a) containing a puromycin screening marker and initiated by EF1a. The structure of the pGC-EF1a plasmid is shown in Figure 11. After the plasmid was digested with BamHI / NheI and successfully ligated, Sanger sequencing was used to verify the accuracy of the inserted sequence and virus packaging was performed. The DKO human pluripotent stem cells constructed in Example 2 were transfected, and the liquid was replaced after 24 hours. After 48 hours, the medium was replaced with one containing puromycin for screening. The results of the constructed stable transfected cell line DKO+CD47 are shown in Figure 12. After confirming that the expression was correct, cell amplification and subsequent function detection were performed.

[0122] Referring to Example 2, RTCA was used to detect whether overexpressing DKO+CD47 cell lines could successfully avoid cell killing by NK cells while avoiding cell killing by T cells. As shown in Figure 13, NK cells can effectively kill DKO cells, and WT and DKO+CD47 overexpressing cells can avoid cell killing by NK cells.

[0123] Example 4. Screening of universal cells expressing a fusion protein containing functional domains of CD47 and CD24 Construction strategy and screening of the fusion proteins of the present invention: CD24 is a precursor protein that contains a signal peptide region and a glycosylphosphatidylinositol (GPI) membrane anchor sequence. After cleavage, CD24 becomes the mature functional CD24 protein (Chen et al., 2014; Pirruccello and LeBien, 1986). CD24 is a glycoprotein with multiple potential O- and N-glycosylation sites and can be sialylated, thus binding to the sialic acid recognition receptor Siglec 10 (sialic acid-binding Ig-like lectin 10) and interacting with macrophages to inhibit phagocytosis (Barkal et al., 2019).

[0124] CD47 is a transmembrane protein with an extracellular N-terminal IgV domain, five transmembrane domains, and an intracellular C-terminus (Logtenberg et al., 2020). The N-terminal IgV domain can interact with the immunoinhibitory receptor SIRPα (signal regulatory protein α) to inhibit immune responses (Jaiswal et al., 2009).

[0125] The strategy of the present invention is to combine the known functional domains of CD24 and CD47 to form various fusion proteins. Each fusion protein must contain at least one membrane anchor sequence or transmembrane sequence selected from CD24 or CD47, while each fusion protein must contain at least one Siglec 10 or SIRPα receptor recognition sequence selected from CD24 or CD47. Each domain is linked by a flexible protein to finally form the fusion proteins XSG01 to XSG20. According to the method of Example 3, the fusion proteins XSG001 to XSG020 were overexpressed by lentivirus infection in the H1 DKO cells prepared in Example 2 to form cell lines DKO+G1 to G20 in which all fusion proteins were stably transfected.

[0126] The following are the construction strategies and specific sequences of the six fusion proteins: 1. Fusion protein XSG006. This fusion protein is constructed by the functional domains of CD47 and CD24, in which the mature peptide sequence of CD24 is connected to the transmembrane domain of CD47, and its amino acid sequence is as shown in SEQ ID NO:1.

[0127] 2. Fusion protein XSG007. This fusion protein is constructed by the functional domains of CD47 and CD24, in which the SIRPα-binding domain of CD47 is connected to the membrane anchor sequence of CD24, and its amino acid sequence is as shown in SEQ ID NO:35.

[0128] 3. Fusion protein XSG009. This fusion protein is constructed by the functional domains of CD47 and CD24, in which the SIRPα-binding domain of CD47 is inserted into the junction of the extracellular sequence and membrane anchor sequence of CD24, and its amino acid sequence is as shown in SEQ ID NO:36.

[0129] 4. Fusion protein XSG010. This fusion protein is constructed by the functional domains of CD47 and CD24, in which CD47 is linked to the extracellular sequence of CD24, and its amino acid sequence is as shown in SEQ ID NO:37.

[0130] 5. Fusion protein XSG011. This fusion protein is constructed by the functional domains of CD47 and CD24, in which the mature peptide of CD24 is linked to the rear of the SIRPα binding domain of CD47, and its amino acid sequence is as shown in SEQ ID NO:38.

[0131] 6. Fusion protein XSG012. This fusion protein is constructed by the functional domains of CD47 and CD24, in which the extracellular mature peptide of CD24 is inserted into the junction of the SIRPα binding domain and the transmembrane domain of CD47, and its amino acid sequence is as shown in SEQ ID NO:39.

[0132] Table 1. Amino acid sequences of some fusion proteins [Table 3]

[0133] The cell lines DKO+G1 to G20 were each subjected to detection of NK cell killing function as shown in Example 2, and the fusion protein with the best degree of NK cell evasion was screened. The results are shown in Figure 14. Only DKO+G6 had a lower average cell killing ratio than the positive control H1 WT many times, making it the best cell to evade NK cell killing.

[0134] Example 5. Construction and verification of immune function of DKO+G6 cell line The nucleic acid sequence encoding XSG006 (the amino acid sequence of XSG006 is shown in SEQ ID NO:1) was directly synthesized and constructed in a lentiviral plasmid (pGC-EF1a) containing a puromycin screening marker and initiated by EF1a. The structure of the pGC-EF1a plasmid is shown in FIG. 11. The plasmid was digested with BamHI / NheI, and after successful ligation, Sanger sequencing was used to verify the accuracy of the inserted sequence and perform viral packaging. The DKO human pluripotent stem cells obtained in Example 2 were transfected, and then the medium was replaced with a medium containing puromycin for screening. XSG006 was a completely exogenous sequence. The insertion of the sequence into the genome of the constructed DKO+G6 cells was detected by PCR, and the DKO cells were used as a negative control. The results are shown in FIG. 15. After confirming that the expression was correct, cell amplification and subsequent functional detection were performed.

[0135] XSG006 F1:CCAGATCTACAGCAGCGAGA XSG006 R1:GTTCTTCAGGCTGTACTCGC XSG006F+XSG006R=352bp XSG006 F2:CCAGATCTACAGCAGCGAGA XSG006 R2:CCAGGATGTAGGGCGATCACC XSG006F+XSG006R=488bp

[0136] Referring to Example 2, in the NK cell killing experiment detected by RTCA, the DKO cells constructed in Example 2 are completely killed by NK cells, while H1WT and DKO+G6 successfully evade. DKO+G6 has a slightly better degree of evasion than H1WT. The results are shown in Figure 16.

[0137] Example 6. Comparison of NK evasion of several cell lines by NK cell killing experiment The cell lines involved in this example are DKO+CD47 and DKO+CD24 in Example 3 and DKO+CD47 in the prior art (WO2020018615A2) (hereinafter referred to as "CD47 prior art") (wherein CD47 in the prior art DKO+CD47 cell line has the amino acid sequence shown in SEQ ID NO: 40 and promotes immune evasion by interacting with signal regulatory protein alpha (SIRPα) on the surface of immune cells). According to the records in WO2020018615A2, the construction of the "CD47 prior art" cell line is completed. Overexpression of CD47 in cells with B2M and CIITA double knockout (DKO) can successfully evade cell killing by T cells and NK cells. (PMID:32433947 / PMID:30778232).

[0138] CD24 promotes immune evasion by interacting with sialic acid-binding Ig-like lectin 10 (Siglec-10), an inhibitory receptor on the surface of immune cells (PMID: 31367043). In this example, the CD24 of cell line DKO+CD24 has the amino acid sequence shown in SEQ ID NO: 41. For the construction of the cell line, see Example 3.

[0139] On the other hand, when the replicates are normalized to the killing rate of each DKO, the summary of the killing results shows that the killing rate of DKO+G6 is the smallest and significantly different from that of WT, DKO+CD24, and "CD47 prior art". The results are shown in Figure 17.

[0140] Example 7. Comparison of NK evasion between DKO+G6 cell line and DKO+CD47+CD24 cell line in NK cell killing experiment DKO+CD47+CD24 cell line was obtained by further overexpressing CD24 through lentivirus transfection based on Example 3. For lentivirus construction and overexpression operation, please refer to Example 3. The amino acid sequence of CD24 is MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKAAGGALQSTASLFVVSLSLLHLYS. Comparing DKO+G6 with DKO+CD47+CD24 through NK cell killing experiment, it can be seen that DKO+G6 cells have a smaller cell killing ratio and better evasion effect. The results are shown in Figure 18.

[0141] Example 8. Verification of immune evasion effect of neural cells differentiated from DKO+G6 in mice with humanized immune systems After passaging, WT and DKO+G6 cells were plated in culture bottles precoated with Matrigel, and after 24 hours of culture, they were switched to pre-differentiation medium to induce the cells to differentiate into mesencephalic cells (Nobrant S, Heuer A, Parmar M, Kirkeby A. Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation. Nat Protoc. 2017 Sep;12(9):1962-1979. doi:10.1038 / nprot.2017.078. Epub 2017 Aug 31. PMID:28858290). After 9 days of differentiation, high-purity mesencephalic cells can be obtained. Then, by adding neural progenitor medium to amplify the obtained mesencephalic cells, a large number of highly pure neural progenitor cells could be obtained. Finally, the neural progenitor cells were further differentiated into neural cells by adding neural progenitor medium. Differentiated WT and DKO+G6 cells were infected with a lentivirus carrying luciferase (luc), and the luc-expressing cells were then transfected with CD34 + HSCs were transplanted into mice with a reconstituted humanized immune system. The survival of the cells in the mice can be shown by intraperitoneal injection of D-fluorescein (A025011, Shanghai Yisheng Biotechnology Co.,Ltd.), a luminescent substrate of luc, and detection of the fluorescence intensity of the cells by iVIS spectrum (PerkinElmer). After continuous detection of the fluorescence at the transplantation site for 51 days, the fluorescence value of the neural cells differentiated from WT tends toward the background value, but after transplantation, a continuously rising fluorescence value of the neural cells differentiated from DKO+G6 can be detected, indicating that the neural cells differentiated from DKO+G6 can effectively avoid the attack of the immune system in vivo compared with the neural cells differentiated from WT. The results are shown in Figure 19.

[0142] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Compared to wild-type cells: 1) Decreased or absent expression of MHC-I and / or MHC-II human leukocyte antigens, 2) Expression of fusion proteins including immunosuppressive checkpoints and A universal cell that includes, The aforementioned cells are able to evade attack by T cells and cell killing by NK cells; The universal cell wherein the fusion protein comprises at least one membrane anchor sequence or transmembrane sequence derived from CD24 or CD47, and at least one Siglec-10 or SIRPα receptor recognition sequence derived from CD24 or CD47.

2. The universal cell according to claim 1, wherein a gene editing tool is used within the cell to target one or more genes encoding one or more transcription factors of MHC-I, or one or more genes encoding one or more transcription factors of MHC-II, in order to achieve reduced or non-expression of MHC-I and / or MHC-II genes.

3. The universal cell according to claim 2, wherein the transcription factor of MHC-I is selected from one or more of B2M, TAP1, TAP2, TAP-related glycoprotein (Tapasin), or NLRC5; and the transcription factor of MHC-II is selected from one or more of CIITA, RFXANK, RFX5, and RFXAP.

4. The universal cell according to claim 3, wherein the transcription factors are B2M and CIITA.

5. The aforementioned cells, Genetic modification targeting the CIITA gene by a rare-cut endonuclease that selectively inactivates the CIITA gene, and Genetic modification targeting the B2M gene by a rarecut endonuclease that selectively inactivates the B2M gene. The universal cell according to claim 4, further comprising:

6. The universal cell according to claim 5, wherein the rare cut end nuclease is selected from CAS protein, TALE nuclease, zinc finger nuclease, large nuclease, and homing nuclease.

7. A gene expression modifying molecule for one or more genes encoding one or more transcriptional control factors of MHC-I, or a gene expression modifying molecule for one or more genes encoding one or more transcriptional control factors of MHC-II is introduced into the cell, whereby a decrease or non-expression of the expression of the MHC-I and / or MHC-II gene is achieved, and the gene expression modifying molecule comprises one selected from siRNA, shRNA, microRNA, antisense RNA, and another RNA-mediated inhibitory molecule. The universal cell according to claim 1.

8. The universal cell according to claim 1, wherein the fusion protein comprises a structural composition selected from the group consisting of: (1) The mature peptide sequence of CD24 is connected to the transmembrane domain of CD47. (2) The SIRPα binding domain of CD47 is connected to the membrane anchor sequence of CD24. (3) The SIRPα binding domain of CD47 is inserted into the connecting site between the extracellular sequence of CD24 and the membrane anchor sequence. (4) The SIRPα binding domain of CD47 is ligated after the extracellular sequence of CD24. (5) The mature peptide of CD24 is ligated after the SIRPα binding domain of CD47. (6) The extracellular mature peptide of CD24 is inserted into the connecting site between the SIRPα binding domain and the transmembrane domain of CD47.

9. The universal cell according to claim 8, wherein the amino acid sequence of the fusion protein has a homology of more than 70%, more than 80%, more than 90%, more than 95%, or more than 98% with the sequence shown in SEQ ID NO:

1.

10. The universal cell according to claim 9, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO:

1.

11. The universal cell according to any one of claims 1 to 10, wherein the cell further comprises a modification that increases the expression of one or more of the following polypeptides, namely CD35, CD27, DUX4, CD26, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, IDO2, TDO, CTLA4-IgG, C1 inhibitor, IL-10, CD46, CCL21, Mfge8, SerpinB9, and IL-35.

12. The universal cell according to any one of claims 1 to 10, wherein the cell is an embryonic stem cell or pluripotent stem cell having low immunogenicity.

13. The universal cell according to claim 12, wherein the cell is a human stem cell.

14. The following steps: 1) A step of knocking out one or more genes of one or more MHC-I transcription regulators in stem cells, and / or 2) The step of knocking out one or more genes of one or more MHC-II transcription regulators of the stem cells; and 3) Step of introducing a nucleic acid sequence encoding a fusion protein containing the functional domains of CD47 and CD24. Includes, A method for preparing universal cells according to any one of claims 1 to 10, wherein the fusion protein comprises at least one membrane anchor sequence or transmembrane sequence derived from CD24 or CD47, and at least one Siglec-10 or SIRPα receptor recognition sequence derived from CD24 or CD47.

15. A method for preparing universal cells according to claim 14, wherein the transcription factor of MHC-I is selected from one or more of B2M, TAP1, TAP2, TAP-related glycoprotein (Tapasin), or NLRC5, and the transcription factor of MHC-II is selected from one or more of CIITA, RFXANK, RFX5, and RFXAP.

16. The method for preparing universal cells according to claim 15, wherein the transcription factor is selected from B2M and CIITA.

17. The preparation method according to claim 14, wherein the knockout in step 1) or 2) is a gene modification targeting the CIITA gene or the B2M gene by a rarecut endonuclease that selectively inactivates the CIITA gene or the B2M gene.

18. The preparation method according to claim 17, wherein the rare-cut endonuclease is selected from CAS protein, TALE nuclease, zinc finger nuclease, large nuclease, and homing nuclease.

19. The preparation method according to claim 14, wherein the knockout in step 1) or 2) involves introducing a gene expression modifying molecule to one or more genes encoding one or more transcription factors of MHC-I, or to one or more genes encoding one or more transcription factors of MHC-II, thereby achieving reduced or non-expression of the MHC-I and / or MHC-II genes, the gene expression modifying molecule comprising one selected from siRNA, shRNA, microRNA, antisense RNA, and another RNA-mediated inhibitory molecule.

20. The preparation method according to claim 14, wherein in step 3), an expression vector is used to introduce a nucleic acid sequence encoding a fusion protein containing the functional domains of CD47 and / or CD24 into stem cells.

21. The preparation method according to claim 20, wherein in step 3), the nucleic acid sequence encoding the fusion protein is introduced into a selected site of the stem cell.

22. The preparation method according to claim 21, wherein the selected site of the stem cell is a safe harbor gene site.

23. The preparation method according to claim 14, wherein the fusion protein comprises a structural configuration selected from the group consisting of the following: (1) The mature peptide sequence of CD24 is linked to the transmembrane domain of CD47. (2) The SIRPα-binding domain of CD47 is connected to the membrane anchor array of CD24. (3) The SIRPα-binding domain of CD47 is inserted into the junction between the extracellular sequence of CD24 and the membrane anchor sequence. (4) The SIRPα-binding domain of CD47 is ligated after the extracellular sequence of CD24. (5) The mature peptide of CD24 is linked after the SIRPα-binding domain of CD47. (6) The extracellular mature peptide of CD24 is inserted into the junction between the SIRPα-binding domain and the transmembrane domain of CD47.

24. The preparation method according to claim 23, wherein the fusion protein has at least 70% homology, 80% homology, 90% homology, 95% homology, or 98% homology with the sequence shown in Sequence ID No.

1.

25. The preparation method according to claim 24, wherein the amino acid sequence of the fusion protein is as shown in Sequence ID No.

1.

26. The preparation method according to claim 14, further comprising a second expression vector containing a polynucleotide sequence encoding one selected from CD35, CD27, DUX4, CD26, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, IDO2, TDO, CTLA4-IgG, Cl inhibitor, IL-10, CD46, CCL21, Mfge8, SerpinB9, and IL-35.

27. A method for preparing differentiated universal cells, comprising culturing the universal cells prepared according to the method of claim 14 under differentiation conditions to prepare differentiated cells with low immunogenicity.

28. The method according to claim 27, wherein the differentiation conditions are suitable for differentiating cells into cell types selected from cardiomyocytes, nerve cells, glial cells, endothelial cells, T cells, NK cells, NKT cells, macrophages, hematopoietic progenitor cells, mesenchymal cells, pancreatic islet cells, chondrocytes, retinal pigment epithelial cells, kidney cells, hepatocytes, thyroid cells, skin cells, blood cells, and epithelial cells.

29. Use of immunogenic differentiated cells prepared according to the method of claim 27 in the manufacture of a pharmaceutical product for treating patients requiring cell therapy.

30. A composition comprising the universal cell according to any one of claims 1 to 10.

31. The composition according to claim 30, further comprising one or more therapeutic agents.

32. A fusion protein comprising functional domains of CD47 and CD24, wherein the fusion protein comprises at least one membrane anchor sequence or transmembrane sequence derived from CD24 or CD47, and at least one Siglec-10 or SIRPα receptor recognition sequence derived from CD24 or CD47.

33. The fusion protein according to claim 32, wherein the fusion protein comprises a structural configuration selected from the group consisting of: (1) The mature peptide sequence of CD24 is linked to the transmembrane domain of CD47. (2) The SIRPα-binding domain of CD47 is linked to the aforementioned membrane anchor array of CD24. (3) The SIRPα-binding domain of CD47 is inserted into the junction between the extracellular sequence of CD24 and the membrane anchor sequence. (4) The SIRPα-binding domain of CD47 is ligated after the extracellular sequence of CD24. (5) The mature peptide of CD24 is ligated after the SIRPα-binding domain of CD47, (6) The extracellular mature peptide of CD24 is inserted into the junction between the SIRPα-binding domain and the transmembrane domain of CD47.

34. The fusion protein according to claim 32, wherein the amino acid sequence of the fusion protein has more than 70% homology, more than 80% homology, more than 90% homology, more than 95% homology, or more than 98% homology with the sequence shown in Sequence ID No.

1.

35. The fusion protein according to claim 34, wherein the amino acid sequence of the fusion protein is as shown in Sequence ID No.

1.

36. A nucleic acid sequence encoding the fusion protein described in Claim 33.

37. An expression vector or expression cassette comprising the nucleic acid sequence described in claim 36.

38. Use of universal cells according to any one of claims 1 to 10 in the preparation of a product for cell therapy.

39. Use of universal cells according to any one of claims 1 to 10 in the preparation of products for organ transplantation.

40. Use of universal cells according to any one of claims 1 to 10 in the construction of a universal PSC cell library.

41. Use of the universal cell according to any one of claims 1 to 10 as a gene drug carrier.

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