WO2023179772A1 - 一种通用型细胞及其制备方法 - Google Patents

一种通用型细胞及其制备方法 Download PDF

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WO2023179772A1
WO2023179772A1 PCT/CN2023/083761 CN2023083761W WO2023179772A1 WO 2023179772 A1 WO2023179772 A1 WO 2023179772A1 CN 2023083761 W CN2023083761 W CN 2023083761W WO 2023179772 A1 WO2023179772 A1 WO 2023179772A1
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Prior art keywords
fusion protein
cells
cell
sequence
mhc
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French (fr)
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WO2023179772A9 (zh
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李翔
朱珉喆
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Xellsmart Biomedical Suzhou Co Ltd
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Xellsmart Biomedical Suzhou Co Ltd
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Priority to US18/849,429 priority Critical patent/US20250222039A1/en
Priority to AU2023239641A priority patent/AU2023239641A1/en
Priority to IL315137A priority patent/IL315137A/en
Priority to EP23774016.2A priority patent/EP4502148A4/en
Priority to CA3254656A priority patent/CA3254656A1/en
Priority to KR1020247031484A priority patent/KR20250017200A/ko
Application filed by Xellsmart Biomedical Suzhou Co Ltd filed Critical Xellsmart Biomedical Suzhou Co Ltd
Priority to JP2024556369A priority patent/JP2025510093A/ja
Publication of WO2023179772A1 publication Critical patent/WO2023179772A1/zh
Publication of WO2023179772A9 publication Critical patent/WO2023179772A9/zh
Priority to MX2024011407A priority patent/MX2024011407A/es
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Definitions

  • the invention requires that the patent application number submitted to the State Intellectual Property Office of China on March 25, 2022 is 202210307870.8, the name of the invention is "A universal cell and its preparation method" and the patent application number submitted to the State Intellectual Property Office of China on July 8, 2022 The patent application number is 202210806871.7, and the invention title is “a universal cell and its preparation method”. The entire contents of these two earlier applications are incorporated into this application by reference.
  • the invention belongs to the intersection field of genetic engineering and stem cell technology, and specifically relates to a universal cell and a preparation method thereof.
  • Stem cells are a type of "seed" cells that have the ability to self-renew and differentiate into specific functional somatic cells. Based on the differences in stem cell characteristics, stem cells are mainly divided into: totipotent stem cells (Totipotent stem cells) and pluripotent stem cells (Pluripotent stem cells) , PSCs) and adult stem cells.
  • Totipotent stem cells totipotent stem cells
  • PSCs pluripotent stem cells
  • hESC Human embryonic stem cells
  • iPSC induced pluripotent stem cells
  • Immunosuppressive drugs, HLA matching, and gene editing can be used to reduce immunogenicity or reduce the host immune system's rejection of allogeneic transplanted cells.
  • Immunosuppressive drugs have serious side effects and can cause bone marrow suppression, liver toxicity, hair loss and gastrointestinal adverse reactions.
  • the United States, Japan, and China are now establishing HLA-matched iPSC libraries, but library construction and maintenance require high costs because HLA antigen genes are the most polymorphic observed in the human genome. genes.
  • iPSC libraries in various places cannot provide matches for most people in their respective countries and can only cover specific groups of people (Solomon et al., 2015; Turner et al., 2013). Allogeneic cell therapy for large patient groups may have very obvious advantages over matching libraries in terms of economics and construction and operation costs, but allogeneic cell therapy will be subject to strong immune rejection. Therefore, it is urgent to construct allogeneic immune-compatible universal PSCs.
  • MHC Human major histocompatibility complex
  • HLA human leukocyte antigen
  • the HLA complex is composed of a series of genes and can be divided into classes I, II and III.
  • MHC-I genes are expressed in almost all tissue cell types, and transplanted cells expressing "non-self" MHC-I class molecules will stimulate the activation of CD8+ T cells and be eliminated.
  • CD4+ helper T cells recognize the MHC-II genes of "non-self” cells, thereby performing immune rejection, while class III molecules do not participate in immune activities.
  • Using gene editing methods to alter immunogenic elements to produce cells with low immunogenicity enables large-scale manufacturing of immune-privileged "off-the-shelf" cell therapy products.
  • the present invention provides a new method for transforming cells to obtain low immunogenicity.
  • a new gene strategy of domain fusion is adopted, and a method of transforming human stem cells based on CD47 and CD24 to obtain low immunogenicity is carried out.
  • This new fusion gene can significantly reduce or evade recognition and attack by the immune system, especially natural killer cells, To attack macrophages, etc., the present invention proposes a feasible strategy to achieve immune immunity of cells by developing new genes with domain fusion.
  • the present invention successfully constructs B2M/CIITA biallelic knockout positive clones by knocking out ⁇ -2-microglobulin (B2M) in the endoplasmic reticulum of human pluripotent stem cells and knocking out CIITA, a positive regulator of MHC-II gene transcription. (DKO cells); and then use lentiviral vectors to overexpress the new fusion genes identified in the present invention in DKO cells.
  • the human pluripotent stem cells thus obtained can further escape the killing of NK cells on the basis of escaping T cell attack.
  • CD47 prior art an unexpectedly excellent immune evasion effect compared to DKO+CD47 (hereinafter referred to as "CD47 prior art”) can be obtained.
  • CD47 prior art an unexpectedly excellent immune evasion effect compared to DKO+CD47 (hereinafter referred to as "CD47 prior art”) can be obtained.
  • CD47 prior art an unexpectedly excellent immune evasion effect compared to DKO+CD47 (hereinafter referred to as
  • the present invention provides a universal cell, which, relative to a wild-type cell, includes:
  • MHC-I and/or MHC-II human leukocyte antigen are reduced or not expressed;
  • the cells can escape T cell attack and NK cell killing.
  • the fusion protein containing an immunosuppressive checkpoint includes two or more types of PD-L1, CTLA4-Ig, CD47, and CD24.
  • a fusion protein containing two of the immunosuppressive checkpoints is expressed.
  • fusion proteins comprising functional domains of CD47 and/or CD24 are expressed.
  • the cells include reduced or no expression of MHC-I and MHC-II human leukocyte antigens.
  • one or more genes encoding one or more transcriptional regulators of MHC-I are targeted in the cell using gene editing tools (such as TALEN and/or CRISPR systems), as well as one or more genes encoding MHC-II.
  • gene editing tools such as TALEN and/or CRISPR systems
  • the transcription regulator of MHC-I may be preferably selected from: B2M, TAP1, TAP2, TAP-related glycoprotein (Tapasin) Or one or more of NLRC5;
  • the MHC-II transcription regulator may preferably be selected from: one or more of CIITA, RFXANK, RFX5, and RFXAP;
  • the transcription regulators are preferably B2M and CIITA.
  • the cells further comprise a genetic modification that targets the CIITA gene by a rare-cutting endonuclease that selectively inactivates the CIITA gene.
  • the cells further comprise rare cleaved nucleic acids by selectively inactivating B2M genes.
  • Dicer targets genetic modification of the B2M gene.
  • the genetic modification wherein the CIITA gene or B2M gene is targeted by a rare cutting endonuclease includes a CAS protein or a polynucleotide encoding a CAS protein, and at least one for specifically targeting the CIITA gene or B2M Gene guide RNA sequence.
  • the CRISPR/CAS9 system is used to directly knock out both ends of the B2M and CIITA exon segments respectively, wherein the target sequences of the gRNA for the B2M gene are SEQ ID NO: 2 and SEQ ID NO: 3.
  • the target sequences of gRNA targeting the CIITA gene are SEQ ID NO:4 and SEQ ID NO:5.
  • the cells are modified by introducing one or more genes targeting one or more transcriptional regulators encoding MHC-I, or one or more genes encoding one or more transcriptional regulators encoding MHC-II.
  • the functional domain of CD47 in the fusion protein comprising the functional domain of CD47 and/or CD24 is the CD47 transmembrane domain; preferably, the amino acid sequence of the CD47 transmembrane domain is such as SEQ ID NO: shown in any one of 6-10.
  • the functional domain of CD24 in the fusion protein comprising the functional domain of CD47 and/or CD24 is the signal peptide sequence of CD24, CD24 mature peptide, CD24 extracellular peptide, CD24 membrane anchoring sequence, CD24 Extracellular mature peptide;
  • the amino acid sequence of the functional domain of CD24 is as shown in any one of SEQ ID NO: 11-16.
  • the fusion protein constructed from the functional domains of CD47 and CD24 connects the SIRP ⁇ binding domain of CD47 and the membrane anchoring sequence of CD24.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is a linkage site in which the SIRP ⁇ binding domain of CD47 is inserted into the extracellular sequence and membrane anchoring sequence of CD24.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is linked to the SIRP ⁇ binding domain of CD47CD47 to the extracellular sequence of CD24.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is a mature peptide of CD24 linked to the SIRP ⁇ binding domain of CD47.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is to insert the extracellular mature peptide of CD24 into the linking point between the SIRP ⁇ binding domain and the transmembrane domain of CD47.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is a combination of CD24
  • the mature peptide sequence is connected to the CD47 transmembrane domain
  • the amino acid sequence of the fusion protein constructed from the functional domains of CD47 and CD24 has more than 70% homology, such as more than 80% homology, with the sequence shown in SEQ ID NO: 1, and then For example, more than 90%, more than 95%, more than 98% homology;
  • amino acid sequence of the fusion protein constructed from the functional domains of CD47 and CD24 is shown in SEQ ID NO: 1.
  • nucleic acid sequence encoding the fusion protein constructed from the functional domains of CD47 and CD24 can be obtained based on the amino acid sequence of the fusion protein.
  • the cells further include 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.
  • the cells are embryonic stem cells.
  • the cells are pluripotent stem cells.
  • the cells are low immunogenic stem cells.
  • the cells are human stem cells or human somatic cells.
  • the present invention provides a method for preparing universal cells according to the first aspect, comprising the following steps:
  • the immunosuppressive checkpoint fusion protein includes multiple types of PD-L1, CTLA4-Ig, CD47, and CD24.
  • a nucleic acid sequence encoding a fusion protein containing two of the immunosuppressive checkpoints is introduced.
  • nucleic acid sequence of the fusion protein constructed including the functional domains of CD47 and/or CD24 is introduced.
  • the transcriptional regulator of MHC-I can be preferably selected from: one or more of B2M, TAP1, TAP2, TAP-related glycoprotein (Tapasin) or NLRC5; the transcriptional regulator of MHC-II can be Preferably, one or more of CIITA, RFXANK, RFX5, and RFXAP.
  • the transcriptional regulator is selected from B2M and CIITA.
  • the knockout described in steps 1) and 2) is a genetic modification that targets the CIITA gene or B2M gene by selectively inactivating a rare cutting endonuclease of the CIITA gene or B2M gene.
  • the rare cutting endonuclease is selected from the group consisting of CAS proteins, TALE-nucleases, zinc finger nucleases, large nucleases and homing nucleases.
  • the genetic modification wherein the CIITA gene or the B2M gene is targeted by a rare cutting endonuclease includes a CAS protein or a polynucleotide encoding a CAS protein, and at least one guide for specifically targeting the CIITA gene or the B2M gene.
  • RNA sequence RNA sequence.
  • steps 1) and 2) use the CRISPR system to directly knock out both ends of the B2M and CIITA exon segments respectively, wherein the target sequence of the gRNA targeting the B2M gene is SEQ ID NO: 2 , 3.
  • the target sequence of the gRNA targeting the CIITA gene is SEQ ID NO: 4, 5.
  • the knockout described in step 1) or 2) is performed by introducing one or more genes targeting one or more transcriptional regulators encoding MHC-I, or one or more genes encoding MHC-II.
  • step 3) uses an expression vector to introduce a nucleic acid sequence encoding a fusion protein comprising a functional domain of CD47 and/or CD24 into the cell.
  • the expression vector used in step 3 is a viral vector.
  • the viral vector used in step 3 is lentivirus.
  • step 3) introduces 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 is a safe harbor gene locus point.
  • the functional domain of CD47 in the fusion protein comprising the functional domain of CD47 and/or CD24 is the CD47 transmembrane domain; preferably, the amino acid sequence of the CD47 transmembrane domain is such as SEQ ID NO: shown in any one of 6-10.
  • the functional domain of CD24 in the fusion protein comprising the functional domain of CD47 and/or CD24 is the signal peptide sequence of CD24, CD24 mature peptide, CD24 extracellular peptide, CD24 membrane anchoring sequence, CD24 Extracellular mature peptide;
  • the amino acid sequence of the functional domain of CD24 is as shown in any one of SEQ ID NO: 11-16.
  • the fusion protein constructed from the functional domains of CD47 and CD24 connects the SIRP ⁇ binding domain of CD47 and the membrane anchoring sequence of CD24.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is a linkage site in which the SIRP ⁇ binding domain of CD47 is inserted into the extracellular sequence and membrane anchoring sequence of CD24.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is linked to the SIRP ⁇ binding domain of CD47CD47 to the extracellular sequence of CD24.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is a mature peptide of CD24 linked to the SIRP ⁇ binding domain of CD47.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is to insert the extracellular mature peptide of CD24 into the linking point between the SIRP ⁇ binding domain and the transmembrane domain of CD47.
  • the fusion protein constructed from the functional domains of CD47 and CD24 connects the mature peptide sequence of CD24 and the transmembrane domain of CD47;
  • the fusion protein constructed from the functional domains of CD47 and CD24 has at least 70% or more homology with the sequence shown in SEQ ID NO: 1, such as more than 80% homology, and for example, 90% or more. % or more, 95% or more, 98% or more homology; further preferably, the amino acid sequence of the fusion protein constructed from the functional domains of CD47 and CD24 is shown in SEQ ID NO: 1.
  • the universal cell further includes a second expression vector comprising coding selected from the group consisting of CD35, CD27, DUX4, CD26, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, The polynucleotide sequence of one of PD-L1, IDO1, IDO2, TDO, CTLA4-IgG, Cl-inhibitor, IL-10, CD46, CD55, CD59, CCL21, Mfge8, SerpinB9 and IL-35.
  • a second expression vector comprising selected from the group consisting of CD35, CD27, DUX4, CD26, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, The polynucleotide sequence of one of PD-L1, IDO1, IDO2, TDO, CTLA4-IgG, Cl-inhibitor, IL-10, CD46, CD55, CD59, CCL21, Mfge8, SerpinB9 and IL
  • the second expression vector is an inducible expression vector; preferably, the second expression vector is a viral vector.
  • the present invention provides a method for preparing differentiated universal cells, which includes culturing the universal cells prepared according to the method of the second aspect under differentiation conditions, thereby preparing differentiated cells with low immunogenicity.
  • the differentiation conditions are suitable for differentiating cells into cells selected from the group consisting of cardiomyocytes, neural cells, glial cells, endothelial cells, T cells, NK cells, NKT cells, macrophages, hematopoietic progenitor cells, mesenchymal cells, Cell types include cytoplasmic cells, islet cells, chondrocytes, retinal pigment epithelial cells, kidney cells, liver cells, thyroid cells, skin cells, blood cells, and epithelial cells.
  • the present invention provides a method of treating a patient in need of cell therapy, comprising administering a differentiated low-immunogenic cell population prepared according to the method of the third aspect.
  • the present invention provides a composition comprising the universal cell described in the first aspect.
  • the composition includes the universal cells described in the first aspect and one or more therapeutic agents, and the therapeutic agents include peptides, cytokines, small molecule compounds, macromolecules, ADCs, antibodies, nanoparticles, etc. Particles, biosimilars, mRNA, traditional Chinese medicine, proteins, vaccines, checkpoint inhibitors, mitogens, growth factors, small RNA, double stranded RNA (dsRNA), mononuclear blood cells, feeder cells, feeder cell groups or replacement factors thereof, vectors containing one or more polynucleic acids of interest, antibodies, etc.
  • the therapeutic agents include peptides, cytokines, small molecule compounds, macromolecules, ADCs, antibodies, nanoparticles, etc. Particles, biosimilars, mRNA, traditional Chinese medicine, proteins, vaccines, checkpoint inhibitors, mitogens, growth factors, small RNA, double stranded RNA (dsRNA), mononuclear blood cells, feeder cells, feeder cell groups or replacement factors thereof, vectors containing one
  • the present invention provides a fusion protein comprising functional domains of CD47 and/or CD24.
  • the functional domain of CD47 in the fusion protein comprising the functional domain of CD47 and/or CD24 is the CD47 transmembrane domain; preferably, the amino acid sequence of the CD47 transmembrane domain is such as SEQ ID NO: shown in any one of 6-10.
  • the functional domain of CD24 in the fusion protein comprising the functional domain of CD47 and/or CD24 is the signal peptide sequence of CD24, CD24 mature peptide, CD24 extracellular peptide, CD24 membrane anchoring sequence, CD24 Extracellular mature peptide;
  • the amino acid sequence of the functional domain of CD24 is as shown in any one of SEQ ID NO: 11-16.
  • the fusion protein constructed from the functional domains of CD47 and CD24 connects the SIRP ⁇ binding domain of CD47 and the membrane anchoring sequence of CD24.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is a linkage site in which the SIRP ⁇ binding domain of CD47 is inserted into the extracellular sequence and membrane anchoring sequence of CD24.
  • the fusion protein constructed from the functional domains of CD47 and CD24 links CD47 to the extracellular sequence of CD24.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is a mature peptide of CD24 linked to the SIRP ⁇ binding domain of CD47.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is to insert the extracellular mature peptide of CD24 into the linking point between the SIRP ⁇ binding domain and the transmembrane domain of CD47.
  • the fusion protein constructed from the functional domains of CD47 and CD24 connects the mature peptide sequence of CD24 and the transmembrane domain of CD47;
  • the fusion protein constructed from the functional domains of CD47 and CD24 has at least 70% or more homology with the sequence shown in SEQ ID NO: 1, such as more than 80% homology, and for example, 90% or more. % or more, 95% or more, 98% or more homology; further preferably, the amino acid sequence of the fusion protein constructed from the functional domains of CD47 and CD24 is shown in SEQ ID NO: 1.
  • the present invention provides a nucleic acid sequence encoding the fusion protein of the sixth aspect, and the nucleic acid sequence can be obtained according to the amino acid sequence of the fusion protein.
  • the present invention provides an expression vector or expression cassette, said expression cassette or expression vector comprising the nucleic acid sequence of the seventh aspect.
  • the present invention provides a method that expresses the fusion protein described in the sixth aspect and has reduced expression or no expression.
  • MHC class I and/or MHC class II human leukocyte antigen cells are provided.
  • the present invention provides a cell that does not express CIITA, expresses the fusion protein described in the sixth aspect, and has reduced or no expression of MHC class I and/or MHC class II human leukocyte antigens.
  • the present invention provides a cell that does not express B2M, expresses the fusion protein described in the sixth aspect, and has reduced or no expression of MHC class I and/or MHC class II human leukocyte antigens.
  • the present invention provides a cell that does not express CIITA and B2M, expresses the fusion protein described in the sixth aspect, and has reduced or no expression of MHC class I and/or MHC class II human leukocyte antigens.
  • the present invention provides a method for expressing the fusion protein described in the sixth aspect and at least one selected from: DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, Cl-inhibitor, peptides of CD46, CD55, CD59 and IL-35, and cells with reduced or no expression of MHC class I and/or MHC class II human leukocyte antigens.
  • the present invention provides a method that does not express CIITA but expresses the fusion protein described in the sixth aspect and at least 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, CD55, CD59, CCL21, Mfge8, SerpinB9 and IL-35 polypeptides and having cells that express reduced or no expression of MHC class I and/or MHC class II human leukocyte antigens.
  • the present invention provides a product that does not express B2M and expresses the fusion protein described in the sixth aspect and at least one selected from: DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, Cl - Inhibitors, polypeptides of CD46, CD55, CD59 and IL-35, and cells with reduced or no expression of MHC class I and/or MHC class II human leukocyte antigens.
  • the present invention provides a product that does not express CIITA and B2M but expresses the fusion protein described in the sixth aspect and at least one selected from the group consisting of: 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 have cells expressing reduced or no expression of MHC class I and/or MHC class II human leukocyte antigens.
  • the present invention provides a cell according to the ninth aspect to the sixteenth aspect, wherein the cell is selected from the group consisting of stem cells, differentiated cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, and progenitor cells. , somatic cells, primary T cells and chimeric antigen receptor T cells.
  • the present invention provides the use of the universal cells described in the first aspect, the composition described in the fifth aspect, or the expression vector or expression cassette described in the eighth aspect in the preparation of products for cell therapy.
  • the present invention provides the universal cell described in the first aspect, the composition described in the fifth aspect, or the Use of the expression vector or expression cassette described in the eighth aspect in preparing products for organ transplantation.
  • the present invention provides the use of the universal cells described in the first aspect, the composition described in the fifth aspect, or the expression vector or expression cassette described in the eighth aspect in constructing a universal PSCs cell bank.
  • the present invention provides the use of the universal cell described in the first aspect, the composition described in the fifth aspect, or the expression vector or expression cassette described in the eighth aspect as a gene drug vector.
  • the present invention inactivates the major histocompatibility complex MHC-I and II genes in stem cells, and then overexpresses the fusion protein XSG006 constructed from the functional domains of CD47 and CD24.
  • the obtained human pluripotent stem cells can escape T cells. On the basis of attack, it further escapes the killing of NK cells, and its effect is even better than that of the reported positive targets CD47 and CD24.
  • these low-immunogenic pluripotent stem cells retain their stemness and differentiation capabilities.
  • neural cells differentiated from DKO+G6 cells overexpressing the fusion protein XSG006 can effectively escape the attack of the immune system in vivo.
  • Example 1 INF-gamma was used to stimulate wild-type H1 (WT) and DKO, and the expression diagram of HLA-I/II in various cells was detected by flow cytometry;
  • T cells are the positive control for detecting HLA-I/II class molecules
  • MSC is the negative control
  • FIG. 8 immunofluorescence was used to detect the expression of stemness genes SSEA-4 and Tra1-81 on the surface of WT and DKO cells.
  • FIG. 9 Immunohistochemistry shows that DKO cells can form teratomas with three germ layers: inner, middle and outer germ layers;
  • FIG. 1 Schematic diagram of the pGC-EF1a plasmid structure
  • RTCA detects the killing results of NK cells against the DKO+CD47 cell line, WT, and DKO cells that overexpress CD47;
  • RTCA detects the killing results of NK cells against cell lines DKO+G1-G20, H1WT (positive control), and H1DKO cells (negative control) overexpressing XSG1-XSG20 fusion protein.
  • Figure 16 The results of RTCA detection of NK cell killing of DKO+G6 cell line, WT, and DKO cells overexpressing XSG006 protein in Example 5.
  • RTCA detects the killing results of NK cells against DKO+G6 cell lines that overexpress XSG006 protein, WT, DKO, and DKO+CD24/CD47 cells that overexpress CD24 or CD47 protein;
  • FIG. 18 RTCA detection of NK cells in Example 7 on DKO+G6 cell lines overexpressing XSG006 protein, WT, DKO, DKO+CD24/CD47 cells overexpressing CD24 or CD47 proteins, and DKO+ overexpressing CD24 and CD47 proteins. Picture of killing results of CD47+CD24 cells;
  • the present invention uses human pluripotent stem cell lines H1 (Wicell, WA01) or H9 (Wicell, WA09), and uses CRISPR/CAS9 to knock out ⁇ -2-microglobulin (B2M) in the endoplasmic reticulum, so that MHC-I on the cell surface It cannot form functional molecules, thereby escaping the killing of allogeneic CD8 + T cells; escaping the killing of CD4 + T cells is by knocking out the positive regulator CIITA of MHC-II gene transcription, thereby reducing the expression of MHC-II class molecules.
  • H1 Human pluripotent stem cell lines
  • H9 Wicell, WA09
  • B2M ⁇ -2-microglobulin
  • B2M's CRISPR/CAS9 gene knockout strategy is shown in Figure 1.
  • B2M-gRNA1 and B2M-gRNA2 are used to directly knock out both ends of the B2M exon segment, and then B2M-F1/R1 and B2M- are used respectively.
  • Two pairs of PCR primers F2/R2 were used for genome sequence knockout verification.
  • B2M-gRNA1 CGTGAGTAAACCTGAATCTT
  • B2M-gRNA2 AGTCACATGGTTCACACGGC
  • CIITA's CRISPR/CAS9 gene knockout strategy is shown in Figure 2.
  • CIITA-gRNA1 and CIITA-gRNA2 are used to directly knock out both ends of the CIITA exon segment, and then CIITA-F1/R1 and CIITA- Two pairs of PCR primers F2/R2 were used for genome sequence knockout verification.
  • CIITA-R1 CCTTCCATGTCACACAACAGCC
  • INF-gamma to stimulate WT and DKO: cells are plated, and the medium containing INF-gamma is added to the cells when the medium is changed the next day. After 48 hours of action, the cells are digested and flow cytometry is used to detect the expression of HLA-I/II. .
  • the results shown in Figure 5 show that B2M/CIITA biallelic knockout stem cell positive clones (DKO) cannot express HLA-I/II class molecules in response to INF-gamma stimulation. T cells are positive controls for detecting HLA-I/II class molecules.
  • Example 2 Verification of the stemness and immune function of the DKO cell line in Example 1
  • Immunofluorescence detection shows that WT and DKO cells express stemness genes POU5F1 and NANOG at the protein level: Plate the cells in a 12-well plate. After the cells grow to a density of 60-80%, aspirate the medium and add 4% paraformaldehyde. To fix. After the cells were ruptured, they were incubated overnight at 4°C with primary antibodies of POU5F1 and NANOG. After washing away the primary antibodies, they were incubated with fluorescently labeled secondary antibodies at room temperature, and then photographed using a fluorescence microscope. The results are shown in Figure 7A. RT-qPCR detection showed that WT and DKO cells expressed stemness genes POU5F1, NANOG and SOX2 at the RNA level: the results are shown in Figure 7B shown. (MSC is a negative control for stemness gene expression)
  • Immunodeficient mice (SCID Beige) were injected subcutaneously with 100 ⁇ L of suspension containing 5E+5DKO cells. When the teratoma volume was greater than 1.5 cm 3 , the cells were removed and slices were stained.
  • the obtained B2M/CIITA biallelic knockout positive clones can form teratomas in vivo and differentiate into cells of the inner, middle and outer germ layers. As shown in Figure 9.
  • T cell and NK cell killing experiments were performed using xCELLigence RTCA Instrument.
  • the same number of WT and DKO cell lines were resuspended in Essential 8 medium containing IL-2 and seeded on 96-well E-plates coated with Matrigel.
  • Activated T cells or NK cells were added for killing detection.
  • RTCA detection data is analyzed using xCELLigence software to calculate kill rate and escape function.
  • WT cells escape the killing of NK cells because of the expression of HLA-I, but will be killed by T cells.
  • DKO cells can escape T cell killing and are more sensitive to NK cell killing.
  • the lentiviral vector was used to overexpress CD47 (NM_198793) in the DKO cells obtained in Example 1.
  • the amino acid sequence of CD47 is such as SEQ ID NO.29, and the cDNA of the overexpression sequence (SEQ ID NO.30) was constructed in EF1a
  • pGC-EF1a lentiviral plasmid
  • the plasmid was digested with BamHI/NheI. After successful ligation, Sanger sequencing was used to verify the correctness of the inserted sequence and perform virus packaging.
  • the DKO human pluripotent stem cells constructed in Example 2 were transfected, and the medium was changed after 24 hours. After 48 hours, the medium was replaced with puromycin-containing medium for screening.
  • the results of the DKO+CD47 cells of the constructed stably transfected cell line are shown in Figure 12. After confirming that the expression is correct, cell amplification and subsequent functional testing are performed.
  • Example 2 use RTCA to detect whether the overexpressed DKO+CD47 cell line can successfully escape the killing of NK cells while escaping T cell killing.
  • NK cells can effectively kill DKO cells, WT and DKO+ CD47 overexpressing cells can escape NK killing.
  • CD24 is a precursor protein that contains a signal peptide region and glycosylphosphatidylinositol (GPI) membrane anchoring sequence, which becomes a mature and functional CD24 protein after cleavage (Chen et al., 2014; Pirruccello and LeBien, 1986).
  • CD24 is a glycoprotein with multiple potential O- and N-glycosylation sites, which can be sialylated to bind to the sialic acid recognition receptor Siglec 10 (sialic acid-binding Ig-like lectin 10) and interact with macrophages. interact to inhibit phagocytosis (Barkal et al., 2019).
  • CD47 is a transmembrane protein with an extracellular N-terminal IgV domain, 5 transmembrane domains and an intracellular C-terminal (Logtenberg et al., 2020).
  • the N-terminal IgV domain can interact with the immunosuppressant receptor SIRPA (signal regulatory protein ⁇ ) to inhibit the immune response (Jaiswal et al., 2009).
  • the strategy of the present invention is to combine known functional domains of CD24 and CD47 to form multiple fusion proteins.
  • Each fusion protein must contain at least one membrane anchoring or transmembrane sequence selected from CD24 or CD47; at the same time, each fusion protein must contain at least one Siglec 10 or SIRP ⁇ receptor recognition sequence, The receptor recognition sequence is selected from CD24 or CD47.
  • Each domain is linked through a flexible protein to finally form the fusion protein XSG01-XSG20.
  • the fusion protein XSG001-XSG020 was overexpressed in the H1DKO cells prepared in Example 2 through lentiviral infection to form all The fusion protein was stably transfected into the cell line DKO+G1-G20.
  • Fusion protein XSG006. The fusion protein constructed from the functional domains of CD47 and CD24 connects the mature peptide sequence of CD24 and the transmembrane domain of CD47. Its amino acid sequence is shown in SEQ ID NO.1.
  • the fusion protein constructed from the functional domains of CD47 and CD24 connects the SIRP ⁇ binding domain of CD47 and the membrane anchoring sequence of CD24. Its amino acid sequence is shown in SEQ ID NO. 35.
  • the fusion protein constructed from the functional domains of CD47 and CD24 is to insert the SIRP ⁇ binding domain of CD47 into the linking site of the extracellular sequence and membrane anchoring sequence of CD24. Its amino acid sequence is as SEQ ID Shown in NO.36.
  • Fusion protein XSG010 The fusion protein constructed from the functional domains of CD47 and CD24 links CD47 to the extracellular sequence of CD24, and its amino acid sequence is as shown in SEQ ID NO. 37.
  • Fusion protein XSG011 The fusion protein constructed from the functional domains of CD47 and CD24 links the CD24 mature peptide to the SIRP ⁇ binding domain of CD47, and its amino acid sequence is as shown in SEQ ID NO. 38.
  • Fusion protein XSG012. The fusion protein constructed from the functional domains of CD47 and CD24 is to insert the extracellular mature peptide of CD24 into the linking point of the SIRP ⁇ binding domain and the transmembrane domain of CD47. Its amino acid sequence is as SEQ ID NO. 39 shown.
  • the cell lines DKO+G1-G20 were respectively subjected to the NK cell killing function test as shown in Example 2, and the fusion protein with the optimal degree of NK cell escape was intended to be screened. The results are shown in Figure 14. The multiple killing rates of DKO+G6 were lower than the average killing rate of the positive control H1WT, indicating that they were the optimal cells to escape NK killing.
  • the nucleic acid sequence encoding XSG006 (the amino acid sequence of As shown in Figure 11.
  • the plasmid was digested with BamHI/NheI. After successful ligation, Sanger sequencing was used to verify the correctness of the inserted sequence and perform virus packaging.
  • the DKO human pluripotent stem cells obtained in Example 2 were transfected and then replaced with a medium containing puromycin for screening.
  • XSG006 is a completely foreign sequence.
  • the constructed DKO+G6 cells used PCR to detect the insertion of the sequence into the genome. DKO cells were used as negative controls. The results are shown in Figure 15. After confirming that the expression is correct, cell amplification and subsequent functional testing were performed. .
  • XSG006F2 CCAGATCTACAGCAGCGAGA
  • Example 2 in the NK cell killing experiment detected by RTCA, the DKO cells constructed in Example 2 were completely killed by NK cells, and H1WT and DKO+G6 successfully escaped. The degree of escape is slightly better for DKO+G6 than H1WT. As shown in Figure 16.
  • Example 6 NK cell killing experiment to compare the degree of NK escape of several cell lines
  • CD47 prior art DKO+CD47, DKO+CD24 in Example 3, DKO+CD47 in the prior art (WO2020018615A2) (hereinafter referred to as “CD47 prior art”), among which DKO+ in the prior art
  • the amino acid sequence of CD47 in the CD47 cell line is shown in SEQ ID NO.40, which promotes immune evasion by interacting with signal regulatory protein ⁇ (SIRP ⁇ ) on the surface of immune cells.
  • SIRP ⁇ signal regulatory protein ⁇
  • the construction of the "CD47 existing technology” cell line is in accordance with WO2020018615A2
  • Overexpression of CD47 in B2M and CIITA double knockout cells (DKO) can successfully escape killing by T and NK cells. (PMID:32433947/PMID:30778232).
  • CD24 promotes immune evasion by interacting with the inhibitory receptor sialic acid-binding Ig-like lectin 10 (Siglec-10) on the surface of immune cells (PMID: 31367043).
  • the amino acid sequence of CD24 in the cell line DKO+CD24 in this example is shown in SEQ ID NO. 41, and its construction method is as described in Example 3.
  • Example 7 NK cell killing experiment comparing the degree of NK escape of DKO+G6 and DKO+CD47+CD24 cell lines
  • the DKO+CD47+CD24 cell line further overexpresses CD24 through lentiviral transfection based on Example 3.
  • the lentivirus construction and overexpression operations refer to Example 3.
  • the amino acid sequence of CD24 is MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKAAGGALQSTASLFVVSLSLLHLYS.
  • DKO+G6 and DKO+CD47+CD24 were compared. The killing ratio of DKO+G6 cells was smaller and the escape effect was better. As shown in Figure 18.
  • Example 8 Verification of immune evasion effect of DKO+G6 differentiated neural cells in mice with human immune system
  • WT and DKO+G6 cells were spread into culture bottles pre-coated with Matrigel. After 24 hours of culture, they were changed to pre-differentiation medium to induce the cells to differentiate into midbrain cells.
  • Nolbrant S Heuer A, Parmar M, Kirkeby A. Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation a nd intracerebral transplantation. Nat Protoc. 2017 Sep; 12(9):1962-1979.doi:10.1038 /nprot.2017.078.Epub 2017 A ⁇ g 31.PMID:28858290)
  • highly pure midbrain cells can be obtained.
  • neural progenitor cell culture medium to expand the obtained midbrain cells, a large number of highly pure neural progenitor cells can be obtained; finally, by adding neural precursor cell culture medium, the neural progenitor cells are further differentiated into neural progenitor cells.
  • Differentiated WT and DKO+G6 cells were infected with lentivirus carrying luciferase (luc), and then the luc-expressing cells were transplanted into CD34 + HSC-reconstituted human immune system mice.

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Abstract

本发明公开了一种通用型细胞的制备方法。通过在细胞中失活主要组织相容性复合体MHC-I和II类基因后，过表达包含CD47和/或CD24的功能结构域的融合蛋白，获得的人多能干细胞或人类细胞系可在逃逸T细胞攻击的基础上，进一步逃逸NK细胞的杀伤，其效果甚至优于已经报道的明星靶点CD47。同时这些低免疫原性的多能干细胞保留了其干性和分化能力。

Description

一种通用型细胞及其制备方法
相关申请的交叉引用
本发明要求2022年03月25日向中国国家知识产权局提交的专利申请号为202210307870.8，发明名称为“一种通用型细胞及其制备方法”和2022年07月08日向中国国家知识产权局提交的专利申请号为202210806871.7，发明名称为“一种通用型细胞及其制备方法”的在先申请的优先权。该两件在先申请的全文通过引用的方式结合于本发明中。
技术领域
本发明属于基因工程与干细胞技术交叉领域，具体涉及一种通用型细胞及其制备方法。
背景技术
通过细胞的体外培养或干细胞的诱导分化，能够在体外大量的再生健康的功能细胞，能够通过异体功能细胞移植治疗疾病，但是，免疫不相容和移植细胞遭受的免疫排斥仍然是其临床应用的关键障碍。干细胞是一类具备自我更新能力及向特定功能体细胞分化能力的“种子”细胞，依据干细胞特性的程度差异，主要将干细胞分为：全能干细胞(Totipotent stem cells)、多能干细胞(Pluripotent stem cells，PSCs)和成体干细胞(Adult stem cell)。人类胚胎干细胞(hESC)和诱导多能干细胞(iPSC)具有无限增殖、自我更新和分化到各种类型细胞的潜力，在治疗癌症、神经相关、心血管等疾病具有重要应用前景。
移植自体细胞治疗可以回避免疫排斥问题，但是从病人身上制造自体细胞成本高昂且制备流程周期长(Khera et al.，2013)，并且个体来源的细胞产品的质量和有效性不确定。有数据表明病人体内的细胞与正常人的存在差异，治疗效果可能受影响。
可通过免疫抑制药物、HLA匹配和基因编辑来降低免疫原性，或降低宿主免疫系统对异体移植细胞的排斥反应。免疫抑制药物副作用大，会导致骨髓抑制、肝毒性、脱发和胃肠道不良反应。现在美国、日本、中国都在建立HLA匹配的iPSC库，但建库和维护都需要高额的成本，因为HLA抗原的基因是在人类基因组中观察到的最具多态性 的基因。目前各地的iPSC库无法为各自国家的大多数人提供匹配，覆盖的只能是特定人群(Solomon et al.，2015；Turner et al.，2013)。针对大患者群体的异体细胞治疗可能在经济上和建设及运行成本上相对配型库具有非常明显的优势，但异体细胞治疗会受到强烈的免疫排斥。于是，构建同种异体免疫兼容的通用型PSCs迫在眉睫。
人类的主要组织相容性复合体(MHC)，即人白细胞抗原(HLA)，是导致免疫不相容的主要原因。HLA复合体由一系列基因组成，可分为I类、II类和III类。MHC-I基因在几乎所有的组织细胞类型中表达，表达“非己”MHC-I类分子的移植细胞将刺激CD8+T细胞的活化而被消除。CD4+辅助T细胞识别“非己”细胞的MHC-II基因，从而进行免疫排斥，而III类分子不参与免疫活动。使用基因编辑的方法改变免疫原性元件，以产生低免疫原性细胞，使得具备免疫豁免性的“off-the-shelf”细胞治疗产品的大规模制造成为可能。
近年已有先驱报道通过敲除B2M、CIITA等基因，实现MHC-I和MHC-II细胞表面或本身基因的缺失表达，进而使细胞具备免疫耐受或逃逸T/B细胞特异性免疫应答，产生免疫兼容的通用型PSCs，为更广泛的通用型PSCs源细胞、组织、器官应用奠定了重要的基础。然而，HLA分子是自然杀伤细胞(NK细胞)的主要抑制性配体，MHC-I类阴性细胞易受天然杀伤(NK)细胞裂解。体内外数据均显示宿主NK细胞可消除植入的B2M-/-供体细胞(Flahou et al.，2021)。因此，需要改善先前的方法以产生可以避免免疫反应的通用供体细胞。已报道在破坏MHC-I和MHC-II类基因表达的基础上使细胞表达HLA-E/G等非经典HLA-I类分子，或表达PD-L1、CTLA4-Ig、CD47、CD24等免疫抑制检查点蛋白，可有效逃逸NK细胞的杀伤(Zhao，W.et al.，2020；Ye，Q.et al.，2020；WO2021041316A1)。
这些方案存在仅基于领域已知常识明星分子的直接应用，但存在免疫兼容性不彻底、不明确或缺乏持久性、有效剂量窗口窄等技术问题，由此表明需要更创新和更优化的策略来实现更优化的改造干细胞获得更优的免疫豁免性方案。
发明内容
针对现有技术所存在的不足，本发明提供了改造细胞获得低免疫原性的新方法，首次采用了结构域融合的新基因策略，开展了基于CD47和CD24改造人类干细胞获得低免疫原性的策略实践，通过多轮筛选和序列组合测试，最终鉴定出了代表性的融合新基因。该融合新基因能够显著减少或逃逸免疫系统的识别和攻击，尤其是自然杀伤细胞、 巨噬细胞等的攻击，本发明提出了通过开发结构域融合的新基因实现细胞的免疫豁免性的可行策略。
本发明通过敲除人多能干细胞内质网中β-2-微球蛋白(B2M)和敲除MHC-II基因转录的正调节因子CIITA，成功构建B2M/CIITA双等位基因敲除阳性克隆(DKO细胞)；然后使用慢病毒载体在DKO细胞中过表达本发明鉴定出的融合新基因，由此获得的人多能干细胞可在逃逸T细胞攻击的基础上，进一步逃逸NK细胞的杀伤，从而能够获得相对DKO+CD47(下文简称“CD47现有技术”)的出人意料的优异的免疫逃逸效果。同时这些低免疫原性的多能干细胞保留了其干性和分化能力等多能干细胞的关键生物学功能。
为了实现上述目的，本发明采用如下技术方案：
第一方面，本发明提供一种通用型细胞，其相对于野生型细胞，包含：
1)MHC-I和/或MHC-II人类白细胞抗原的表达降低或不表达；
2)表达包含免疫抑制检查点的融合蛋白；
所述细胞能逃逸T细胞攻击及NK细胞的杀伤。
在某些方面，所述包含免疫抑制检查点的融合蛋白包括PD-L1、CTLA4-Ig、CD47、CD24中的两种及两种以上。优选地，表达包含2种所述免疫抑制检查点的融合蛋白。
根据本发明，表达包含CD47和/或CD24的功能结构域的融合蛋白。
在某些方面，所述细胞包括MHC-I和MHC-II人类白细胞抗原的表达降低或不表达。
在某些方面，所述细胞中使用基因编辑工具(诸如TALEN和/或CRISPR系统)靶向编码MHC-I的一种或多种转录调节因子的一个或多个基因，以及编码MHC-II的一种或多种转录调节因子的一个或多个基因，来实现MHC-I和MHC-II基因的表达降低或不表达。
在某些实施方式中，为实现MHC-I和MHC-II基因的表达降低或不表达，所述MHC-I的转录调节因子可优选自：B2M、TAP1、TAP2、TAP相关糖蛋白(Tapasin)或NLRC5中的一个或多个；所述MHC-II的转录调节因子可优选自：CIITA、RFXANK、RFX5、RFXAP中的一个或多个；
所述转录调节因子优选B2M和CIITA。
在某些实施方式中，所述细胞还包括通过选择性灭活CIITA基因的稀有切割核酸内切酶靶向CIITA基因的遗传修饰。
在某些实施方式中，所述细胞还包括通过选择性灭活B2M基因的稀有切割核酸内 切酶靶向B2M基因的遗传修饰。
在某些实施方式中，其中通过稀有切割内切核酸酶靶向CIITA基因或B2M基因的遗传修饰包括CAS蛋白或编码CAS蛋白的多核苷酸，和至少一个用于特异性靶向CIITA基因或B2M基因的导向核糖核酸序列。
在具体的实施方式中，使用CRISPR/CAS9系统分别对B2M和CIITA外显子区段进行两端直接敲除，其中，针对B2M基因的gRNA的靶序列为SEQ ID NO:2和SEQ ID NO:3，针对CIITA基因的gRNA的靶序列为SEQ ID NO:4和SEQ ID NO:5。
在某些方面，所述细胞中通过引入针对编码MHC-I的一种或多种转录调节因子的一个或多个基因，或编码MHC-II的一种或多种转录调节因子的一个或多个基因的基因表达修饰分子，来实现MHC-I和/或MHC-II基因的表达降低或不表达，其中所述基因表达修饰分子包括选自siRNA，shRNA，microRNA，反义RNA和另一种RNA介导的抑制分子中的一种。
在某些方面，所述包含CD47和/或CD24的功能结构域的融合蛋白中的CD47的功能结构域为CD47跨膜结构域；优选地，所述CD47跨膜结构域的氨基酸序列如SEQ ID NO：6-10任一项所示。
在某些方面，所述包含CD47和/或CD24的功能结构域的融合蛋白中的CD24的功能结构域为CD24的信号肽序列、CD24成熟肽、CD24胞外肽、CD24膜锚定序列、CD24胞外成熟肽；优选地，所述CD24的功能结构域的氨基酸序列如SEQ ID NO：11-16任一项所示。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域和CD24的膜锚定序列连接。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域插入至CD24的胞外序列和膜锚定序列的链接位点。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47CD47的SIRPα结合域链接到CD24的胞外序列后。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24成熟肽链接到CD47的SIRPα结合域后。
在某些实施例方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的胞外成熟肽插入到CD47的SIRPα结合域和跨膜结构域链接点。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24 的成熟肽序列和CD47跨膜结构域连接；
优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列与如SEQ ID NO:1所示的序列具有70％以上的同源性，例如80％以上的同源性，再例如90％以上、95％以上、98％以上的同源性；
进一步优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列如SEQ ID NO:1所示。
在某些方面，所述编码上述由CD47和CD24的功能结构域构建的融合蛋白核酸序列可以根据所述融合蛋白的氨基酸序列得到。
在某些方面，所述细胞还包括修饰以增加以下一种或多种多肽的表达：DUX4，CD27，CD35，CD46，CD55，CD59，CD200，HLA-C，HLA-E，HLA-E重链，HLA-G，PD-L1，IDOl，CTLA4-Ig，Cl-抑制剂，IL-10，IL-35，，CCL21，Mfge8和SerpinB9。
在某些方面，所述细胞是胚胎干细胞。
在某些方面，所述细胞是多能干细胞。
在某些实施方案中，所述细胞是低免疫原性干细胞。
在某些实施方案中，所述细胞是人类干细胞或人类体细胞。
第二方面，本发明提供一种第一方面通用型细胞的制备方法，包含如下步骤：
1)敲除细胞的MHC-I的一种或多种转录调节因子的一个或多个基因；和/或，
2)敲除细胞的MHC-II的一种或多种转录调节因子的一个或多个基因；
3)在细胞中引入编码包含免疫抑制检查点的融合蛋白的核酸序列。
在某些方面，所述免疫抑制检查点的融合蛋白包括PD-L1、CTLA4-Ig、CD47、CD24中的多种。优选地，引入编码包含2种所述免疫抑制检查点的融合蛋白的核酸序列。
根据本发明，引入包含CD47和/或CD24的功能结构域构建的融合蛋白的核酸序列。
在某些方面，所述MHC-I的转录调节因子可优选自：B2M、TAP1、TAP2、TAP相关糖蛋白(Tapasin)或NLRC5中的一个或多个；所述MHC-II的转录调节因子可优选自：CIITA、RFXANK、RFX5、RFXAP中的一个或多个。
在某些实施例方式中，所述转录调节因子选自B2M和CIITA。
在某些方面，步骤1)和2)所述的敲除为通过选择性灭活CIITA基因或B2M基因的稀有切割核酸内切酶靶向CIITA基因或B2M基因的遗传修饰。
优选地，所述稀有切割核酸内切酶选自CAS蛋白，TALE-核酸酶，锌指核酸酶，大核酸酶和归巢核酸酶。
进一步优选地，其中通过稀有切割内切核酸酶靶向CIITA基因或B2M基因的遗传修饰包括CAS蛋白或编码CAS蛋白的多核苷酸，和至少一个用于特异性靶向CIITA基因或B2M基因的导向核糖核酸序列。
在某些实施例方式中，步骤1)和2)使用CRISPR系统分别对B2M和CIITA外显子区段进行两端直接敲除，其中，针对B2M基因的gRNA的靶序列为SEQ ID NO:2、3，针对CIITA基因的gRNA的靶序列为SEQ ID NO:4、5。
在某些方面，步骤1)或2)所述的敲除为通过引入针对编码MHC-I的一种或多种转录调节因子的一个或多个基因，或编码MHC-II的一种或多种转录调节因子的一个或多个基因的基因表达修饰分子，来实现MHC-I和/或MHC-II基因的表达降低或不表达，其中所述基因表达修饰分子包括选自siRNA，shRNA，microRNA，反义RNA和另一种RNA介导的抑制分子中的一种。
在某些方面，步骤3)采用表达载体将编码包含CD47和/或CD24的功能结构域的融合蛋白的核酸序列引入细胞中。
优选地，所述步骤3)采用的表达载体为病毒载体。
在某些实施例方式中，步骤3)采用的病毒载体为慢病毒。
在某些方面，步骤3)将编码包含CD47和/或CD24的功能结构域的融合蛋白的核酸序列导入所述细胞的选定位点；优选地，所述细胞的选定位点是安全港基因位点。
在某些方面，所述包含CD47和/或CD24的功能结构域的融合蛋白中的CD47的功能结构域为CD47跨膜结构域；优选地，所述CD47跨膜结构域的氨基酸序列如SEQ ID NO：6-10任一项所示。
在某些方面，所述包含CD47和/或CD24的功能结构域的融合蛋白中的CD24的功能结构域为CD24的信号肽序列、CD24成熟肽、CD24胞外肽、CD24膜锚定序列、CD24胞外成熟肽；优选地，所述CD24的功能结构域的氨基酸序列如SEQ ID NO：11-16任一项所示。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域和CD24的膜锚定序列连接。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域插入至CD24的胞外序列和膜锚定序列的链接位点。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47CD47的SIRPα结合域链接到CD24的胞外序列后。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24成熟肽链接到CD47的SIRPα结合域后。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的胞外成熟肽插入到CD47的SIRPα结合域和跨膜结构域链接点。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的成熟肽序列和CD47跨膜结构域连接；
优选地，所述由CD47和CD24的功能结构域构建的融合蛋白与如SEQ ID NO:1所示的序列具有至少70％以上的同源性，例如80％以上的同源性，再例如90％以上、95％以上、98％以上的同源性；进一步优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列如SEQ ID NO:1所示。
在某些方面，所述通用型细胞还包括第二表达载体，其包含编码选自CD35，CD27，DUX4，CD26，CD200,HLA-C，HLA-E，HLA-E重链，HLA-G，PD-L1，IDOl，IDO2,TDO,CTLA4-IgG，Cl-抑制剂，IL-10，CD46，CD55，CD59，CCL21，Mfge8，SerpinB9和IL-35中的一种的多核苷酸序列。
在某些实施方案中，所述第二表达载体是诱导型表达载体；优选地，所述第二表达载体是病毒载体。
第三方面，本发明提供一种制备分化的通用型细胞的方法，包括在分化条件下培养根据第二方面的方法制备的通用型细胞，从而制备分化的低免疫原性细胞。
在某些方面，其中所述分化条件适于将细胞分化为选自心肌细胞，神经细胞，胶质细胞，内皮细胞，T细胞，NK细胞，NKT细胞，巨噬细胞，造血祖细胞，间充质细胞，胰岛细胞，软骨细胞，视网膜色素上皮细胞，肾细胞，肝细胞，甲状腺细胞，皮肤细胞，血细胞和上皮细胞的细胞类型。
第四方面，本发明提供一种治疗需要细胞治疗的患者的方法，包括给予根据第三方面的方法制备的分化的低免疫原性细胞群。
第五方面，本发明提供一种组合物，所述组合物包含第一方面所述的通用型细胞。
在某些方面，所述组合物包含第一方面所述的通用型细胞和一种或多种治疗剂，所述治疗剂包含肽、细胞因子、小分子化合物、大分子、ADC、抗体、纳米粒子、生物类似物、mRNA、中药、蛋白、疫苗、检查点抑制剂、丝裂原、生长因子、小RNA、双链RNA(double stranded RNA；dsRNA)、单核血细胞、饲养细胞、饲养细胞组分或其置换因子、包含一种或多种所关注多核酸的载体、抗体等。
第六方面，本发明提供一种融合蛋白，所述融合蛋白包含CD47和/或CD24的功能结构域。
在某些方面，所述包含CD47和/或CD24的功能结构域的融合蛋白中的CD47的功能结构域为CD47跨膜结构域；优选地，所述CD47跨膜结构域的氨基酸序列如SEQ ID NO：6-10任一项所示。
在某些方面，所述包含CD47和/或CD24的功能结构域的融合蛋白中的CD24的功能结构域为CD24的信号肽序列、CD24成熟肽、CD24胞外肽、CD24膜锚定序列、CD24胞外成熟肽；优选地，所述CD24的功能结构域的氨基酸序列如SEQ ID NO：11-16任一项所示。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域和CD24的膜锚定序列连接。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域插入至CD24的胞外序列和膜锚定序列的链接位点。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47链接到CD24的胞外序列后。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24成熟肽链接到CD47的SIRPα结合域后。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的胞外成熟肽插入到CD47的SIRPα结合域和跨膜结构域链接点。
在某些实施方式中，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的成熟肽序列和CD47跨膜结构域连接；
优选地，所述由CD47和CD24的功能结构域构建的融合蛋白与如SEQ ID NO:1所示的序列具有至少70％以上的同源性，例如80％以上的同源性，再例如90％以上、95％以上、98％以上的同源性；进一步优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列如SEQ ID NO:1所示。
第七方面，本发明提供编码第六方面融合蛋白的核酸序列，所述核酸序列可以根据所述融合蛋白的氨基酸序列得到。
第八方面，本发明提供一种表达载体或表达盒，所述表达盒或表达载体包含第七方面的核酸序列。
第九方面，本发明提供一种表达第六方面所述融合蛋白，并具有表达降低或不表达 的MHC I类和/或MHC II类人白细胞抗原的细胞。
第十方面，本发明提供一种不表达CIITA，表达第六方面所述融合蛋白，并且具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
第十一方面，本发明提供一种不表达B2M，表达第六方面所述融合蛋白，并具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
第十二方面，本发明提供一种不表达CIITA和B2M，表达第六方面所述融合蛋白，并具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
第十三方面，本发明提供一种表达第六方面所述融合蛋白和至少一种选自：DUX4，HLA-C，HLA-E，HLA-G，PD-L1，CTLA4，Cl-抑制剂，CD46，CD55，CD59和IL-35的多肽，并具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的的细胞。
第十四方面，本发明提供一种不表达CIITA，表达第六方面所述融合蛋白和至少一种选自：CD35，CD27，DUX4，CD26，CD55，CD59，CD200,HLA-C，HLA-E，HLA-E重链，HLA-G，PD-L1，IDOl，IDO2,TDO,CTLA4-IgG，Cl-抑制剂，IL-10，CD46，CD55，CD59，CCL21，Mfge8，SerpinB9和IL-35中的多肽，并且具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
第十五方面，本发明提供一种不表达B2M，表达第六方面所述融合蛋白和至少一种选自：DUX4，HLA-C，HLA-E，HLA-G，PD-L1，CTLA4，Cl-抑制剂，CD46，CD55，CD59和IL-35的多肽，并且具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
第十六方面，本发明提供一种不表达CIITA和B2M，表达第六方面所述融合蛋白和至少一种选自：CD35，CD27，DUX4，CD26，CD55，CD59，CD200,HLA-C，HLA-E，HLA-E重链，HLA-G，PD-L1，IDOl，IDO2,TDO,CTLA4-IgG，Cl-抑制剂，IL-10，CD46，CD55，CD59，CCL21，Mfge8，SerpinB9和IL-35中的多肽，并且具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
第十七方面，本发明提供一种上述第九方面～第十六方面所述的细胞，其中所述细胞选自干细胞，分化细胞，多能干细胞，诱导的多能干细胞，成体干细胞，祖细胞，体细胞，原代T细胞和嵌合抗原受体T细胞。
第十八方面，本发明提供第一方面所述的通用型细胞、第五方面所述的组合物或第八方面所述的表达载体或表达盒在制备用于细胞治疗的产品中的应用。
第十九方面，本发明提供第一方面所述的通用型细胞、第五方面所述的组合物或第 八方面所述的表达载体或表达盒在制备用于器官移植的产品中的应用。
第二十方面，本发明提供第一方面所述的通用型细胞、第五方面所述的组合物或第八方面所述的表达载体或表达盒在构建通用型PSCs细胞库中的应用。
第二十一方面，本发明提供第一方面所述的通用型细胞、第五方面所述的组合物或第八方面所述的表达载体或表达盒作为基因药物载体的应用。
本发明的有益效果：
本发明在干细胞中失活主要组织相容性复合体MHC-I和II类基因后，过表达由CD47和CD24的功能结构域构建的融合蛋白XSG006，获得的人多能干细胞可在逃逸T细胞攻击的基础上，进一步逃逸NK细胞的杀伤，其效果甚至优于已经报道的阳性靶点CD47和CD24。同时这些低免疫原性的多能干细胞保留了其干性和分化能力。融合蛋白XSG006过表达的DKO+G6细胞分化的神经细胞相较于WT分化的神经细胞，可在体内有效逃逸免疫系统的攻击。
附图说明
图1.人胚胎干细胞系H1中B2M基因敲除策略及结果图；
图2.人胚胎干细胞系H1中CIITA基因敲除策略及结果图；
图3.实施例1中使用RT-qPCR检测DKO细胞中B2M和CIITA在RNA水平的表达图；
图4.实施例1中使用Western-blot检测B2M蛋白水平的结果图；
图5.实施例1中用INF-gamma刺激野生型H1(WT)及DKO，通过流式细胞计数仪检测各种细胞中HLA-I/II的表达图；
其中，T细胞为检测HLA-I/II类分子的阳性对照；
图6.实施例1中获得的B2M/CIITA双等位基因敲除阳性克隆(DKO)核型图；
图7.实施例2中利用免疫荧光和RT-qPCR检测WT和DKO细胞在RNA和蛋白水平干性基因POU5F1/NANOG/SOX2的表达图；
其中MSC为阴性对照；
图8.实施例2中利用免疫荧光检测WT和DKO细胞表面干性基因SSEA-4和Tra1-81的表达图。
图9.使用免疫组化显示DKO细胞可形成具有内、中、外三个胚层的畸胎瘤；
图10.实施例2中采用RTCA检测对DKO细胞免疫功能进行验证的结果图；
图11.pGC-EF1a质粒结构示意图；
图12.流式细胞计数仪检测实施例3中构建的DKO+CD47细胞系CD47的表达图；
图13.实施例3中RTCA检测NK细胞对过表达CD47的DKO+CD47细胞株、WT、DKO细胞的杀伤结果图；
图14.实施例4中RTCA检测NK细胞对过表达XSG1-XSG20融合蛋白的细胞株DKO+G1-G20、H1WT(阳性对照)、H1DKO细胞(阴性对照)的杀伤结果图。
图15.实施例5中对构建的DKO+G6细胞株基因组进行序列PCR鉴定。
图16.实施例5中RTCA检测NK细胞对过表达XSG006蛋白的DKO+G6细胞株、WT、DKO细胞的杀伤结果图。
图17.实施例6中RTCA检测NK细胞对过表达XSG006蛋白的DKO+G6细胞株、WT、DKO、过表达CD24或CD47蛋白的DKO+CD24/CD47细胞的杀伤结果图；
A为杀伤结果图；B为多次结果汇总图。
图18.实施例7中RTCA检测NK细胞对过表达XSG006蛋白的DKO+G6细胞株、WT、DKO、过表达CD24或CD47蛋白的DKO+CD24/CD47细胞、过表达CD24和CD47蛋白的DKO+CD47+CD24细胞的杀伤结果图；
A为杀伤结果图；B为多次结果汇总图。
图19.实施例8中DKO+G6分化的神经细胞在人源免疫系统小鼠中的免疫逃逸作用验证结果图；
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。
除非另外定义或由背景清楚指示，否则在本公开中的全部技术与科学术语具有如本 公开所属领域的普通技术人员通常理解的相同含义。
实施例1.构建B2M及CIITA双敲除细胞系(DKO)
1、细胞培养试剂：
2、方法及结果：
本发明选用了人多能干细胞系H1(Wicell，WA01)或H9(Wicell，WA09)，使用CRISPR/CAS9敲除内质网中β-2-微球蛋白(B2M)，使细胞表面MHC-I不能形成功能性分子，从而逃逸同种异体CD8+T细胞的杀伤；逃逸CD4+T细胞的杀伤则是通过敲除MHC-II基因转录的正调节因子CIITA，而降低MHC-II类分子表达。
其中，B2M的CRISPR/CAS9基因敲除策略如图1所示，使用B2M-gRNA1和B2M-gRNA2对B2M外显子区段进行两端直接敲除，然后分别使用B2M-F1/R1和B2M-F2/R2两对PCR引物进行基因组序列敲除验证。
gRNA序列:
B2M-gRNA1：CGTGAGTAAACCTGAATCTT
B2M-gRNA2：AGTCACATGGTTCACACGGC
鉴定引物
B2M-F1:TGGGGCCAAATCATGTAGACTC
B2M-R1:TCAGTGGGGGTGAATTCAGTGT
B2M-F2+B2M-R2＝608bp
敲除后：无条带
B2M-F2:CAGAAGTCCTTGAGAGCCTCC
B2M-R2:TGTGCATCAGTATCTCAGCAGG
B2M-F2+B2M-R2＝812bp
敲除后：569bp。
另外，CIITA的CRISPR/CAS9基因敲除策略如图2所示，使用CIITA-gRNA1和CIITA-gRNA2对CIITA外显子区段进行两端直接敲除，然后分别使用CIITA-F1/R1和CIITA-F2/R2两对PCR引物进行基因组序列敲除验证。
gRNA序列:
CIITA-gRNA1:GATATTGGCATAAGCCTCCC
CIITA-gRNA2:CATCGCTGTTAAGAAGCTCC
鉴定引物：
CIITA-F1:CTGTGCCTCTACCACTTCTATG
CIITA-R1:CCTTCCATGTCACACAACAGCC
CIITA-F1+CIITA-R1＝368bp
敲除后：无条带
CIITA-F2:TGGAATCCACACTTTCCAGTTC
CIITA-R2:TGGAGTCTCCGTTCCTCCAG
CIITA-F2+CIITA-R2＝889bp
敲除后：459bp
具体操作如下：
1)在Matrigel包被的6孔板上正常使用mTeSR1培养人类多能干细胞至80％密度。使用TRYPLE消化后加入DMEM/F12中和，计数。吸取2×106细胞于EP管中，离心后弃上清。
2)根据Neon转染系统100μL电转体系，加入15μg TrueCutTM Cas9 Protein+3μg gRNA(B2MgRNA1+B2MgRNA2+CIITA gRNA1+CIITA gRNA2)组成RNP体系，混匀室温下放置20min。
3)100μL RNP电转体系重悬细胞，Neon转染系统进行电转，电转参数为1200V， 30ms，1pause。电转后细胞迅速加入提前预热的培养基，均匀接种于1孔包被了Matrigel的6孔板中。
4)每天更换新鲜mTeSR1培养基。待单细胞生长起来，挑取单个克隆于48孔板内，待克隆扩增后，收取基因组样品进行PCR检测基因编辑情况，PCR结果如图1和2所示。PCR阳性克隆送公司做Sanger测序进一步验证。
5)鉴定阳性的B2M/CIITA双等位基因敲除克隆DKO扩增培养及冻存。
使用qPCR检测B2M/CIITA双等位基因敲除克隆DKO的B2M和CIITA在RNA水平的表达情况，如图3所示，确定敲除。
B2M-F:AAGATGAGTATGCCTGCCGT
B2M-R:ATGCGGCATCTTCAAACCTC
CIITA-F:CCTGGAGCTTCTTAACAGCGA
CIITA-R:TGTGTCGGGTTCTGAGTAGAG
使用Western-Blot检测B2M/CIITA双等位基因敲除克隆DKO的B2M蛋白水平的表达情况，如图4所示，确定敲除。
使用INF-gamma刺激WT及DKO：细胞铺板，第二天换液时将含有INF-gamma的培养基加入到细胞中，作用48h后将细胞消化后使用流式检测HLA-I/II的表达情况。结果如图5显示B2M/CIITA双等位基因敲除的干细胞阳性克隆(DKO)不能响应INF-gamma的刺激而表达HLA-I/II类分子。T细胞为检测HLA-I/II类分子的阳性对照。
对获得的B2M/CIITA双等位基因敲除阳性克隆(DKO)进行核型检测：用胰蛋白酶处理固定于载玻片上的染色体标本，再用Giemsa染液染色。将分裂中期染色体进行染色体数目、形态结构分析，确定其核型是否与正常核型一致。结果如图6所示，DKO核型正常。
实施例2.验证实施例1中DKO细胞系的干性和免疫功能
1、WT和DKO细胞中干性基因的表达
免疫荧光检测显示WT和DKO细胞在蛋白水平表达干性基因POU5F1和NANOG：将细胞铺板在12孔板中，待细胞长到60-80％的密度后吸去培养基，加入4％多聚甲醛进行固定。细胞破膜后使用POU5F1和NANOG的一抗在4℃过夜孵育，洗去一抗后室温孵育带有荧光标记的二抗，随后使用荧光显微镜进行拍照。结果如图7A所示。RT-qPCR检测显示WT和DKO细胞在RNA水平表达干性基因POU5F1、NANOG和SOX2：结果如图7B 所示。(MSC为干性基因表达的阴性对照)
流式结果检测显示WT和DKO细胞表面均高表达干性基因SSEA-4和Tra1-81占比分别为100％，99.98％和96.75％，99.13％。结果如图8所示。
2、获得的B2M/CIITA双等位基因敲除阳性克隆(DKO)的分化能力
在免疫缺陷小鼠(SCID Beige)皮下注射100μL含5E+5DKO细胞的悬液，待畸胎瘤体积大于1.5cm3后取出，并进行切片染色。
获得的B2M/CIITA双等位基因敲除阳性克隆(DKO)可以在体内形成畸胎瘤并分化出内中外三胚层的细胞。如图9所示。
3、DKO细胞免疫功能验证
T细胞及NK细胞的杀伤性实验使用xCELLigence RTCA Instrument。将相同数量WT和DKO细胞系使用含有IL-2的Essential 8培养基重悬，并接种于用基质胶包被的96-well E-plates，加入活化的T细胞或NK细胞进行杀伤检测。RTCA检测数据使用xCELLigence软件进行分析，计算杀伤率和逃逸功能。
如图10的RTCA数据显示，WT细胞因为有HLA-I的表达而逃逸NK细胞的杀伤，但会被T细胞杀伤。DKO细胞能逃逸T细胞杀伤，同时对NK细胞的杀伤更为敏感。
实施例3.DKO+CD47细胞系的构建及免疫功能验证
在实施例1获得的DKO细胞中使用慢病毒载体过表达CD47(NM_198793)，所述CD47的氨基酸序列如SEQ ID NO.29，将过表达序列的cDNA(SEQ ID NO.30)构建在由EF1a启动，且带puromycin筛选标记的慢病毒质粒中(pGC-EF1a)，pGC-EF1a质粒的结构如图11所示。质粒用BamHI/NheI酶切，连接成功后采用Sanger测序验证插入序列正确性并进行病毒包装。对实施例2构建的DKO人类多能干细胞进行转染，24h换液，48h后换成带puromycin的培养基进行筛选。构建的稳转株细胞DKO+CD47的结果如图12所示，确认表达无误后进行细胞扩增和后续功能性检测。
参照实施例2，使用RTCA检测过表达的DKO+CD47细胞株是否可以在逃逸T细胞杀伤的同时成功逃逸NK细胞的杀伤，如图13所示，NK细胞可有效杀伤DKO细胞，WT和DKO+CD47过表达细胞可逃逸NK杀伤。
实施例4.表达包含CD47和CD24的功能结构域的融合蛋白的通用型细胞的筛选
本发明融合蛋白的构建策略及筛选：
CD24是一种前体蛋白，包含一个信号肽区域和糖基磷脂酰肌醇(GPI)膜锚定序列，通过切割后成为成熟有功能的CD24蛋白(Chen et al.,2014；Pirruccello and LeBien,1986)。CD24有多个潜在O-和N-糖基化位点的糖蛋白，其可以被唾液酸化，从而与唾液酸识别受体Siglec 10(唾液酸结合Ig样凝集素10)结合，与巨噬细胞相互作用以抑制吞噬作用(Barkal et al.,2019)。
CD47是一个具有胞外N-末端IgV结构域、5个跨膜结构域和胞内C-末端的跨膜蛋白(Logtenberg et al.,2020)。N-末端IgV结构域可与免疫抑制剂受体SIRPΑ(信号调节蛋白α)相互作用，抑制免疫反应(Jaiswal et al.,2009)。
本发明的策略是组合已知的CD24及CD47功能结构域形成多种融合蛋白。每种融合蛋白必须包含至少一个膜锚定或跨膜序列，所述膜锚定或跨膜序列选自CD24或CD47；同时，每种融合蛋白必须包含至少一个Siglec 10或SIRPα受体识别序列，所述受体识别序列选自CD24或CD47。各结构域均通过柔性蛋白进行链接最终形成融合蛋白XSG01-XSG20，依照实施例3的方式，在实施例2制备的H1DKO细胞中通过慢病毒感染的方式进行过表达融合蛋白XSG001-XSG020，形成所有融合蛋白稳转细胞株DKO+G1-G20。
以下列举6种融合蛋白的构建策略和具体序列：
1、融合蛋白XSG006，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的成熟肽序列和CD47跨膜结构域连接，其氨基酸序列如SEQ ID NO.1所示。
2、融合蛋白XSG007，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域和CD24的膜锚定序列连接，其氨基酸序列如SEQ ID NO.35所示。
3、融合蛋白XSG009，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域插入至CD24的胞外序列和膜锚定序列的链接位点，其氨基酸序列如SEQ ID NO.36所示。
4、融合蛋白XSG010，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47链接到CD24的胞外序列后，其氨基酸序列如SEQ ID NO.37所示。
5、融合蛋白XSG011，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24成熟肽链接到CD47的SIRPα结合域后，其氨基酸序列如SEQ ID NO.38所示。
6、融合蛋白XSG012，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的胞外成熟肽插入到CD47的SIRPα结合域和跨膜结构域链接点其氨基酸序列如SEQ ID NO.39所示。
表1.部分融合蛋白的氨基酸序列

对细胞株DKO+G1-G20分别进行如实施例2所示的NK细胞杀伤功能检测，拟筛选出对NK细胞的逃逸程度最优的的融合蛋白。结果如图14所示，有且只有DKO+G6多次杀伤均小于阳性对照H1WT的平均杀伤率，为逃逸NK杀伤的最优细胞。
实施例5.DKO+G6细胞系的构建及免疫功能验证
直接合成编码XSG006(XSG006的氨基酸序列如SEQ ID NO：1所示)的核酸序列并构建在由EF1a启动，且带puromycin筛选标记的慢病毒质粒中(pGC-EF1a)，pGC-EF1a质粒的结构如图11所示。质粒用BamHI/NheI酶切，连接成功后采用Sanger测序验证插入序列正确性并进行病毒包装。对实施例2获得的DKO人类多能干细胞进行转染后换成带puromycin的培养基进行筛选。XSG006为完全外源序列，构建的DKO+G6细胞使用PCR检测序列在基因组中的插入情况，DKO细胞为阴性对照，结果如图15所示，确认表达无误后进行细胞扩增和后续功能性检测。
XSG006F1：CCAGATCTACAGCAGCGAGA
XSG006R1：GTTCTTCAGGCTGTACTCGC
XSG006F+XSG006R＝352bp
XSG006F2：CCAGATCTACAGCAGCGAGA
XSG006R2：CCAGGATGTAGGCGATCACC
XSG006F+XSG006R＝488bp
参照实施例2，在RTCA检测的NK细胞杀伤性实验中，实施例2构建的DKO细胞被NK细胞完全杀伤，H1WT、DKO+G6成功逃逸。逃逸程度为DKO+G6略优于H1WT。如图16所示。
实施例6.NK细胞杀伤性实验比较几种细胞系NK逃逸程度
本实施例涉及细胞系：实施例3中的DKO+CD47、DKO+CD24、现有技术(WO2020018615A2)中的DKO+CD47(以下简称“CD47现有技术”)，其中现有技术中的DKO+CD47细胞系中的CD47的氨基酸序列如SEQ ID NO.40所示，通过与免疫细胞表面的信号调节蛋白α(SIRPα)相互作用来促进免疫逃逸，“CD47现有技术”细胞系的构建按照WO2020018615A2中的记载完成。在B2M和CIITA双敲的细胞(DKO)中过表达CD47可以成功逃逸T及NK细胞的杀伤。(PMID:32433947/PMID:30778232)。
CD24通过与免疫细胞表面的抑制性受体唾液酸结合Ig样凝集素10(Siglec-10)相互作用来促进免疫逃(PMID:31367043)。本实施例中的细胞系DKO+CD24中的CD24的氨基酸序列如SEQ ID NO.41所示，其构建方法参照实施例3。
同时将多次重复实验归一至每次DKO杀伤，杀伤结果汇总后显示DKO+G6杀伤比例最小，且与WT、DKO+CD24、“CD47现有技术”相比具有显著性差异。如图17所示。
实施例7.NK细胞杀伤性实验比较DKO+G6和DKO+CD47+CD24细胞系NK逃逸程度
DKO+CD47+CD24细胞系为实施例3的基础上通过慢病毒转染的方式进一步过表达CD24，慢病毒构建及过表达操作参考实施例3，CD24的氨基酸序列为MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKAAGGALQSTASLFVVSLSLLHLYS。通过NK细胞杀伤性实验比较DKO+G6与DKO+CD47+CD24，DKO+G6细胞杀伤比例小，逃逸效果更佳。如图18所示。
实施例8.DKO+G6分化的神经细胞在人源免疫系统小鼠中的免疫逃逸作用验证
将WT及DKO+G6细胞传代后铺至已预包被Matrigel的培养瓶中，培养24h后换至预分化培养基，诱导细胞向中脑细胞分化 (Nolbrant S,Heuer A,Parmar M,Kirkeby A.Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation a nd intracerebral transplantation.Nat Protoc.2017 Sep；12(9):1962-1979.doi:10.1038/nprot.2017.078.Epub 2017 Aμg 31.PMID:28858290)；在分化9d后，可获得高纯度的中脑细胞。其后，通过加入神经祖细胞培养基对获得的中脑细胞进行扩增，可获得大量的高纯度的神经祖细胞；最后，再通过加入神经前体细胞培养基，神经祖细胞进一步分化成为神经细胞。将携带荧光素酶(luc)的慢病毒感染分化的WT和DKO+G6细胞，然后将表达luc的细胞移植到CD34+HSC重建的人源免疫系统小鼠上。通过腹腔注射luc的发光底物D-荧光素(A025011,上海翊圣生物科技有限公司)，并使用活体成像仪(iVIS spectrum,PerkinElmer)检测细胞的荧光强度，可指示细胞在小鼠体内的存活情况。持续检测移植部位荧光51天，WT分化的神经细胞荧光值趋于背景值，而DKO+G6分化的神经细胞移植后可以检测到持续上升的荧光值，说明DKO+G6分化的神经细胞相较于WT分化的神经细胞，可在体内有效逃逸免疫系统的攻击。如图19所示。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (63)

  1. 一种通用型细胞，其特征在于，相对于野生型细胞，包含：
    1)MHC-I和/或MHC-II人类白细胞抗原的表达降低或不表达；
    2)表达包含免疫抑制检查点的融合蛋白；
    所述细胞能逃逸T细胞攻击及NK细胞的杀伤；
    优选地，所述包含免疫抑制检查点的融合蛋白包括PD-L1、CTLA4-Ig、CD47、CD24中的两种及两种以上；优选地，表达包含2种所述免疫抑制检查点的融合蛋白；更优选，表达包含CD47和/或CD24的功能结构域的融合蛋白。
  2. 根据权利要求1所述的通用型细胞，其特征在于，所述细胞包括MHC-I和MHC-II人类白细胞抗原的表达降低或不表达。
  3. 根据权利要求1或2所述的通用型细胞，其特征在于，所述细胞中使用基因编辑工具靶向编码MHC-I的一种或多种转录调节因子的一个或多个基因，或编码MHC-II的一种或多种转录调节因子的一个或多个基因，来实现MHC-I和/或MHC-II基因的表达降低或不表达；
    优选地，所述MHC-I的转录调节因子选自：B2M、TAP1、TAP2、TAP相关糖蛋白(Tapasin)或NLRC5中的一个或多个；所述MHC-II的转录调节因子选自：CIITA、RFXANK、RFX5、RFXAP中的一个或多个；
    进一步优选地，所述转录调节因子为B2M和CIITA。
  4. 根据权利要求3所述的通用型细胞，其特征在于，所述细胞还包括通过选择性灭活CIITA基因的稀有切割核酸内切酶靶向CIITA基因的遗传修饰。
  5. 根据权利要求1-4任一项所述的通用型细胞，其特征在于，所述细胞还包括通过选择性灭活B2M基因的稀有切割核酸内切酶靶向B2M基因的遗传修饰。
  6. 根据权利要求4或5所述的通用型细胞，其特征在于，所述稀有切割核酸内切酶选自CAS蛋白，TALE-核酸酶，锌指核酸酶，大核酸酶和归巢核酸酶。
  7. 根据权利要求6所述的通用型细胞，其特征在于，其中通过稀有切割内切核酸酶靶向CIITA基因或B2M基因的遗传修饰包括CAS蛋白或编码CAS蛋白的多核苷酸，和至少一个用于特异性靶向CIITA基因或B2M基因的导向核糖核酸序列。
  8. 根据权利要求7所述的通用型细胞，其特征在于，使用CRISPR/CAS9系统分别 对B2M和CIITA外显子区段进行两端直接敲除，其中，针对B2M基因的导向核糖核酸序列gRNA的靶序列为SEQ ID NO:2和3，针对CIITA基因的导向核糖核酸序列gRNA的靶序列为SEQ ID NO:4和5。
  9. 根据权利要求1或2所述的通用型细胞，其特征在于，所述细胞中通过引入针对编码MHC-I的一种或多种转录调节因子的一个或多个基因的基因表达修饰分子，或编码MHC-II的一种或多种转录调节因子的一个或多个基因的基因表达修饰分子，来实现MHC-I和/或MHC-II基因的表达降低或不表达，其中所述基因表达修饰分子包括选自siRNA，shRNA，microRNA，反义RNA和另一种RNA介导的抑制分子中的一种。
  10. 根据权利要求1-9任一项所述的通用型细胞，其特征在于，所述包含CD47和/或CD24的功能结构域的融合蛋白中的CD47的功能结构域为CD47跨膜结构域；优选地，所述CD47跨膜结构域的氨基酸序列如SEQ ID NO：6-10任一项所示。
  11. 根据权利要求1-10任一项所述的通用型细胞，其特征在于，所述包含CD47和/或CD24的功能结构域的融合蛋白中的CD24的功能结构域为CD24的信号肽序列、CD24成熟肽、CD24胞外肽、CD24膜锚定序列、CD24胞外成熟肽，优选地，所述CD24的功能结构域的氨基酸序列如SEQ ID NO：11-16任一项所示。
  12. 根据权利要求11所述的通用型细胞，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域和CD24的膜锚定序列连接。
  13. 根据权利要求11所述的通用型细胞，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域插入至CD24的胞外序列和膜锚定序列的链接位点。
  14. 根据权利要求11所述的通用型细胞，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域链接到CD24的胞外序列后。
  15. 根据权利要求11所述的通用型细胞，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24成熟肽链接到CD47的SIRPα结合域后。
  16. 根据权利要求11所述的通用型细胞，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的胞外成熟肽插入到CD47的SIRPα结合域和跨膜结构域链接点。
  17. 根据权利要求11所述的通用型细胞，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的成熟肽序列和CD47跨膜结构域连接；
    优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列与如 SEQ ID NO:1所示的序列具有70％以上的同源性，例如80％以上的同源性，再例如90％以上、95％以上、98％以上的同源性；
    进一步优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列如SEQ ID NO:1所示。
  18. 根据权利要求1-17任一项所述的通用型细胞，其特征在于，所述细胞还包括修饰以增加以下一种或多种多肽的表达：CD35，CD27，DUX4，CD26，CD55，CD59，CD200,HLA-C，HLA-E，HLA-E重链，HLA-G，PD-L1，IDOl，IDO2,TDO,CTLA4-IgG，Cl-抑制剂，IL-10，CD46，CD55，CD59，CCL21，Mfge8，SerpinB9和IL-35。
  19. 根据权利要求1-18任一项所述的通用型细胞，其特征在于，所述细胞是胚胎干细胞或多能干细胞；优选为低免疫原性干细胞；进一步优选为人类干细胞或人类体细胞。
  20. 一种权利要求1-19任一项所述的通用型细胞的制备方法，其特征在于，包含如下步骤：
    1)敲除干细胞的MHC-I的一种或多种转录调节因子的一个或多个基因；和/或，
    2)敲除干细胞的MHC-II的一种或多种转录调节因子的一个或多个基因；
    3)在干细胞中引入编码包含免疫抑制检查点蛋白的核酸序列；
    优选地，所述免疫抑制检查点蛋白包括PD-L1、CTLA4-Ig、CD47、CD24中的一种或多种；优选地，引入编码包含2种所述免疫抑制检查点的融合蛋白的核酸序列；更优选地，引入包含CD47和/或CD24的功能结构域构建的融合蛋白的核酸序列。
  21. 根据权利要求20所述的通用型细胞的制备方法，其特征在于，所述MHC-I的转录调节因子选自：B2M、TAP1、TAP2、TAP相关糖蛋白(Tapasin)或NLRC5中的一个或多个；所述MHC-II的转录调节因子选自：CIITA、RFXANK、RFX5、RFXAP中的一个或多个；优选地，所述转录调节因子选自B2M和CIITA。
  22. 根据权利要求20或21所述的制备方法，其特征在于，步骤1)或2)所述的敲除为通过选择性灭活CIITA基因或B2M基因的稀有切割核酸内切酶靶向CIITA基因或B2M基因的遗传修饰；
    优选地，所述稀有切割核酸内切酶选自CAS蛋白，TALE-核酸酶，锌指核酸酶，大核酸酶和归巢核酸酶；
    进一步优选地，其中通过稀有切割内切核酸酶靶向CIITA基因或B2M基因的遗传修饰包括CAS蛋白或编码CAS蛋白的多核苷酸，和至少一个用于特异性靶向CIITA基因或B2M基因的导向核糖核酸序列；
    更进一步优选地，步骤1)和2)使用CRISPR系统分别对B2M和CIITA外显子区段进行两端直接敲除，其中，针对B2M基因的gRNA的靶序列为SEQ ID NO:2和3，针对CIITA基因的gRNA的靶序列为SEQ ID NO:4和5。
  23. 根据权利要求20或21所述的制备方法，其特征在于，步骤1)或2)所述的敲除为通过引入针对编码MHC-I的一种或多种转录调节因子的一个或多个基因，或编码MHC-II的一种或多种转录调节因子的一个或多个基因的基因表达修饰分子，来实现MHC-I和/或MHC-II基因的表达降低或不表达，其中所述基因表达修饰分子包括选自siRNA，shRNA，microRNA，反义RNA和另一种RNA介导的抑制分子中的一种。
  24. 根据权利要求20-23任一项所述的制备方法，其特征在于，所述步骤3)采用表达载体将编码包含CD47和/或CD24的功能结构域的融合蛋白的核酸序列引入干细胞中；
    优选地，所述步骤3)采用的表达载体为病毒载体；
    进一步优选地，所述病毒载体为慢病毒。
  25. 根据权利要求20-24任一项所述的制备方法，其特征在于，所述步骤3)将编码包含CD47和/或CD24的功能结构域的融合蛋白的核酸序列导入所述干细胞的选定位点；优选地，所述干细胞的选定位点是安全港基因位点。
  26. 根据权利要求20-25任一项所述的制备方法，其特征在于，所述包含CD47和/或CD24的功能结构域的融合蛋白中的CD47的功能结构域为CD47跨膜结构域；优选地，所述CD47跨膜结构域的氨基酸序列如SEQ ID NO：6-10任一项所示。
  27. 根据权利要求20-26任一项所述的制备方法，其特征在于，所述包含CD47和/或CD24的功能结构域的融合蛋白中的CD24的功能结构域为CD24的信号肽序列、CD24成熟肽、CD24胞外肽、CD24膜锚定序列、CD24胞外成熟肽，优选地，所述CD24的功能结构域的氨基酸序列如SEQ ID NO：11-16任一项所示。
  28. 根据权利要求27所述的制备方法，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域和CD24的膜锚定序列连接。
  29. 根据权利要求27所述的制备方法，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域插入至CD24的胞外序列和膜锚定序列的链接位点。
  30. 根据权利要求27所述的制备方法，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域链接到CD24的胞外序列后。
  31. 根据权利要求27所述的制备方法，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24成熟肽链接到CD47的SIRPα结合域后。
  32. 根据权利要求27所述的制备方法，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的胞外成熟肽插入到CD47的SIRPα结合域和跨膜结构域链接点。
  33. 根据权利要求27所述的制备方法，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47跨膜结构域和CD24的成熟肽序列连接；优选地，所述由CD47和CD24的功能结构域构建的融合蛋白与如SEQ ID NO:1所示的序列具有至少70％以上的同源性，例如80％以上的同源性，再例如90％以上、95％以上、98％以上的同源性；进一步优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列如SEQ ID NO:1所示。
  34. 根据权利要求20-33任一项所述的制备方法，其特征在于，所述通用型干细胞还包括第二表达载体，其包含编码选自CD35，CD27，DUX4，CD26，CD55，CD59，CD200,HLA-C，HLA-E，HLA-E重链，HLA-G，PD-L1，IDOl，IDO2,TDO,CTLA4-IgG，Cl-抑制剂，IL-10，CD46，CD55，CD59，CCL21，Mfge8，SerpinB9和IL-35中的一种的多核苷酸序列。
  35. 根据权利要求34所述的制备方法，其特征在于，所述第二表达载体是诱导型表达载体；优选地，所述第二表达载体是病毒载体。
  36. 一种制备分化的通用型细胞的方法，包括在分化条件下培养根据权利要求20-35中任一项的方法制备的通用型细胞，从而制备分化的低免疫原性细胞。
  37. 如权利要求36所述的方法，其中所述分化条件适于将细胞分化为选自心肌细胞，神经细胞，胶质细胞，内皮细胞，T细胞，NK细胞，NKT细胞，巨噬细胞，造血祖细胞，间充质细胞，胰岛细胞，软骨细胞，视网膜色素上皮细胞，肾细胞，肝细胞，甲状腺细胞，皮肤细胞，血细胞和上皮细胞的细胞类型。
  38. 一种治疗需要细胞治疗的患者的方法，包括给予根据权利要求36或37的方法制备的分化的低免疫原性细胞群。
  39. 一种组合物，其特征在于，所述组合物包含权利要求1-29任一项所述的通用型细胞；优选地，所述组合物还包含一种或多种治疗剂；优选地，所述治疗剂包含肽、细胞因子、检查点抑制剂、丝裂原、生长因子、小RNA、双链RNA(double stranded RNA；dsRNA)、单核血细胞、饲养细胞、饲养细胞组分或其置换因子、包含一种或多种所关 注多核酸的载体、抗体等。
  40. 一种融合蛋白，其特征在于，所述融合蛋白包含CD47和/或CD24的功能结构域。
  41. 根据权利要求40所述的融合蛋白，其特征在于，所述包含CD47和/或CD24的功能结构域的融合蛋白中的CD47的功能结构域为CD47跨膜结构域；优选地，所述CD47跨膜结构域的氨基酸序列如SEQ ID NO：6-10任一项所示。
  42. 根据权利要求40所述的融合蛋白，其特征在于，所述包含CD47和/或CD24的功能结构域的融合蛋白中的CD24的功能结构域为CD24的信号肽序列、CD24成熟肽、CD24胞外肽、CD24膜锚定序列、CD24胞外成熟肽；优选地，所述CD24的功能结构域的氨基酸序列如SEQ ID NO：11-16任一项所示。
  43. 根据权利要求42所述的融合蛋白，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域和CD24的膜锚定序列连接。
  44. 根据权利要求42所述的融合蛋白，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域插入至CD24的胞外序列和膜锚定序列的链接位点。
  45. 根据权利要求42所述的融合蛋白，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47的SIRPα结合域链接到CD24的胞外序列后。
  46. 根据权利要求42所述的融合蛋白，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24成熟肽链接到CD47的SIRPα结合域后。
  47. 根据权利要求42所述的融合蛋白，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD24的胞外成熟肽插入到CD47 SIRPα结合域和跨膜结构域链接点。
  48. 根据权利要求42所述的融合蛋白，其特征在于，所述由CD47和CD24的功能结构域构建的融合蛋白是将CD47跨膜结构域插入到CD24的信号肽序列和前体蛋白序列链接位点而获得；或者，将CD47跨膜结构域和CD24的信号肽序列和成熟肽序列连接；优选地，所述融合蛋白的氨基酸序列与如SEQ ID NO:1所示的序列具有70％以上的同源性，例如80％以上的同源性，再例如90％以上、95％以上、98％以上的同源性；进一步优选地，所述由CD47和CD24的功能结构域构建的融合蛋白的氨基酸序列如SEQ ID NO:1所示。
  49. 一种编码权利要求40-48任一项所述融合蛋白的核酸序列，其特征在于，所述 核酸序列可以根据权利要求40-48任一项所述融合蛋白的氨基酸序列得到。
  50. 一种表达载体或表达盒，其特征在于，所述表达盒或表达载体包含权利要求49所述的核酸序列。
  51. 一种表达权利要求40-48任一项所述融合蛋白，并具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
  52. 一种不表达CIITA，表达权利要求40-48任一项所述融合蛋白，并且具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
  53. 一种不表达B2M，表达权利要求40-48任一项所述融合蛋白，并具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
  54. 一种不表达CIITA和B2M，表达权利要求40-48任一项所述融合蛋白，并具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
  55. 表达权利要求40-48任一项所述融合蛋白和至少一种选自：CD35，CD27，DUX4，CD26，CD55，CD59，CD200,HLA-C，HLA-E，HLA-E重链，HLA-G，PD-L1，IDOl，IDO2,TDO,CTLA4-IgG，Cl-抑制剂，IL-10，CD46，CD55，CD59，CCL21，Mfge8，SerpinB9和IL-35的多肽，并具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的的细胞。
  56. 一种不表达CIITA，表达权利要求40-48任一项所述融合蛋白和至少一种选自：DUX4，HLA-C，HLA-E，HLA-G，PD-L1，CTLA4，Cl-抑制剂，CD46，CD55，CD59和IL-35的多肽，并且具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
  57. 一种不表达B2M，表达权利要求40-48任一项所述融合蛋白和至少一种选自：DUX4，HLA-C，HLA-E，HLA-G，PD-L1，CTLA4，Cl-抑制剂，CD46，CD55，CD59和IL-35的多肽，并且具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
  58. 一种不表达CIITA和B2M，表达权利要求40-489任一项所述融合蛋白和至少一种选自：DUX4，HLA-C，HLA-E，HLA-G，PD-L1，CTLA4，Cl-抑制剂，CD46，CD55，CD59和IL-35的多肽，并且具有表达降低或不表达的MHC I类和/或MHC II类人白细胞抗原的细胞。
  59. 权利要求52-58任一项所述的细胞，其中所述细胞选自干细胞，分化细胞，多能干细胞，诱导的多能干细胞，成体干细胞，祖细胞，体细胞，原代T细胞和嵌合抗原 受体T细胞。
  60. 权利要求1-19任一项所述的通用型细胞、权利要求39所述的组合物、权利要求40-48任一项所述融合蛋白或权利要求50所述的表达载体或表达盒、权利要求52-58所述的细胞在制备用于细胞治疗的产品中的应用。
  61. 权利要求1-19任一项所述的通用型细胞、权利要求39所述的组合物、权利要求40-48任一项所述融合蛋白或权利要求50所述的表达载体或表达盒、权利要求52-58所述的细胞在制备用于器官移植的产品中的应用。
  62. 权利要求1-19任一项所述的通用型干细胞、权利要求39所述的组合物、权利要求40-48任一项所述融合蛋白或权利要求50所述的表达载体或表达盒、权利要求52-58所述的细胞在构建通用型PSCs细胞库中的应用。
  63. 权利要求1-19任一项所述的通用型干细胞、权利要求39所述的组合物、权利要求40-48任一项所述融合蛋白或权利要求50所述的表达载体或表达盒、权利要求52-58所述的细胞作为基因药物载体的应用。
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