Universal cells expressing faslg and methods of making the same

Abstract

The application discloses universal FASLG-expressing cells and a preparation method and application thereof. After major histocompatibility complex (MHC) class I and II genes are inactivated in cells and FASLG protein is overexpressed, the obtained human pluripotent stem cells or human cell lines or human induced pluripotent stem cells can further escape the killing of NK cells on the basis of escaping T cell attack, and the effect is even better than that of the reported star target CD47 and CD24. Meanwhile, the pluripotent stem cells with low immunogenicity retain stemness and differentiation ability.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefits to Chinese Patent Application No. 202210813518.1, filed on July 11, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the interdisciplinary field of genetic engineering and stem cell technology, specifically relating to a universal cell expressing FASLG and its preparation method. Background Technology

[0004] Through in vitro cell culture or induced differentiation of stem cells, large quantities of healthy functional cells can be regenerated in vitro, enabling the treatment of diseases through allogeneic functional cell transplantation. However, immunoincompatibility and immune rejection of transplanted cells remain key obstacles to their clinical application. Stem cells are a type of "seed" cell possessing self-renewal capacity and the ability to differentiate into specific functional somatic cells. Based on the degree of difference in stem cell characteristics, stem cells are mainly divided into: totipotent stem cells (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 for unlimited proliferation, self-renewal, and differentiation into various cell types, showing significant application prospects in the treatment of cancer, neurological diseases, and cardiovascular diseases.

[0005] Autologous cell transplantation can avoid the problem of immune rejection, but producing autologous cells from patients is costly and time-consuming (Khera et al., 2013), and the quality and efficacy of cell products derived from individuals are uncertain. Data suggests that cells in patients differ from those in healthy individuals, potentially affecting treatment efficacy.

[0006] Immunogenicity can be reduced by immunosuppressive drugs, HLA matching, and gene editing, or to reduce the rejection of allogeneic transplanted cells by the host immune system. Immunosuppressive drugs have large side effects, causing myelosuppression, hepatotoxicity, alopecia, and gastrointestinal adverse reactions. Now the United States, Japan, and China are building HLA-matched iPSC banks, but the cost of building and maintaining the bank is high, because the gene of HLA antigen is the most polymorphic gene observed in the human genome. The iPSC banks in various places cannot provide matching for the majority of people in their respective countries, and can only cover specific populations (Solomon et al., 2015; Turner et al., 2013). Allogeneic cell therapy for a large patient population can have a very obvious advantage over matching banks in terms of economy 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.

[0007] The major histocompatibility complex (MHC) of humans, i.e., human leukocyte antigen (HLA), is the main cause of immune incompatibility. The HLA complex is composed of a series of genes, which can be divided 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 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 causing immune rejection, and III class molecules are not involved in immune activity. Using gene editing methods to change immunogenic elements to produce low immunogenic cells makes it possible to mass-produce “off-the-shelf” cell therapy products with immune privilege.

[0008] In recent years, there have been pioneer reports that by knocking out B2M, CIITA, etc., MHC-I and MHC-II cell surface or itself gene expression is deleted, thereby making cells immune tolerant or escaping T / B cell-specific immune response, and producing immune-compatible universal PSCs, which lays an important foundation for more extensive universal PSCs source cells, tissues, and organs applications. However, HLA molecules are the main inhibitory ligands of natural killer cells (NK cells), and MHC-I class negative cells are susceptible to natural killer (NK) cell lysis. In vivo and in vitro data show that host NK cells can eliminate implanted B2M - / - Donor cells (Flahou et al., 2021). Therefore, it is necessary to improve previous methods to produce universal donor cells that can avoid immune response.

[0009] The human immune system can recognize and attack non-self antigens, thereby resisting infection. Therefore, in the process of allogeneic organ transplantation, the immune system will attack the transplanted organ, leading to immune rejection and transplantation failure. Although the embryo and placenta are semi-allogeneic transplants, similar to transplanted organs, they can induce maternal tolerance and do not produce a strong immune response. The high expression of HLA-G in the placenta regulates maternal-fetal tolerance by interacting with decidual natural killer cells, while high expression of CD47 is also detected in the placenta (Liu et al., 2021; Than et al., 2019). On the other hand, it is well known that tumor cells can escape immune surveillance by reducing the expression of HLA, reducing antigen presentation, thereby preventing them from being recognized by immune cells; and increasing the expression of immunosuppressive components such as HLA-G, PD-L1, and CTLA-4, etc. (de Charette and Houot, 2018).

[0010] Some studies have explored the relationship between maternal-fetal immune tolerance, tumor immune escape, and organ transplantation. It has been reported that simulating the mechanisms of maternal-fetal tolerance and tumor escape, such as expressing HLAG and other non-classical HLA-I class molecules based on the destruction of MHC-I and MHC-II class gene expression, or expressing immune checkpoint proteins such as PD-L1, CTLA4-Ig, CD47, CD24, etc., exhibit certain immune tolerance protection to NK cells (Zhao, W. et al., 2020; Ye, Q. et al., 2020).

[0011] However, these schemes exist only based on the direct application of field-known star molecules, and immune escape and tolerance are mostly dependent on the binding of immunosuppressive receptors and ligands, and have specific microenvironments in the tumor state or maternal-fetal interface. There is uncertainty in the cross-field direct application of low immune factors to allogeneic transplantation. Do the paired receptors or ligands exist on the transplanted cells and immune cells? Does the transplanted environment meet the escape conditions? What is the state in the pregnant maternal-fetal interface, which may be different from the state at the time of organ transplantation. Therefore, the prior art has the technical problems of incomplete, unclear, or lack of durability of immune compatibility, narrow effective dose window, etc. And so far, the potential molecular mechanisms of maternal-fetal immune tolerance and tumor escape are still being explored, and new genes are continuously discovered, which can further develop low-immunogenic cells. This indicates the need to screen more new molecules to achieve more optimized modified stem cells to obtain a more optimal immune exemption scheme.

[0012] References:

[0013] De Charette, M., and Houot, R. (2018). Hide or defend, the two strategies of lymphoma immune evasion: potential implications for immunotherapy. Haematologica 103, 1256-1268. Liu, Y., Gao, S., Zhao, Y., Wang, H., Pan, Q., and Shao, Q. (2021). Decidual Natural Killer Cells: A Good Nanny at the Maternal-Fetal Interface During Early Pregnancy. Front Immunol 12, 663660.

[0014] Than, N.G., Hahn, S., Rossi, S.W., and Szekeres-Bartho, J. (2019). Editorial: Fetal-Maternal Immune Interactions in Pregnancy. Front Immunol 10, 2729.

[0015] Zhao, W., Lei, A., Tian, L., Wang, X., Correia, C., Weiskittel, T., Li, H., Trounson, A., Fu, Q., Yao, K., et al. (2020). Strategies for Genetically Engineering Hypoimmunogenic Universal Pluripotent Stem Cells. iScience 23, 101162. SUMMARY

[0016] In view of the deficiencies of the prior art, the present application provides a new method for obtaining low immunogenicity by modifying cells. For the first time, a strategy of screening a large number of molecules related to maternal-fetal tolerance and tumor escape is adopted. Through multiple rounds of functional detection, a representative new gene FASLG is finally identified. This gene can significantly reduce or escape the recognition and attack of the immune system, especially the attack of natural killer cells, macrophages, etc. The present application proposes a feasible strategy for realizing the immunological exemption of cells by transforming new genes in an unpredictable field.

[0017] The present application successfully constructs a B2M / CIITA double-allele knockout positive clone DKO cell by knocking out the beta-2-microglobulin (B2M) in the endoplasmic reticulum of human pluripotent stem cells and knocking out the positive regulator CIITA of MHC-II gene transcription; then overexpressing the new gene identified by the present application in the DKO cell using a lentiviral vector, whereby the human pluripotent stem cell obtained can further escape the killing of NK cells on the basis of escaping T cell attack, so as to obtain an unexpectedly superior immune escape effect relative to DKO+CD47 (hereinafter referred to as "CD47 prior art"). At the same time, these low immunogenic pluripotent stem cells retain the key biological functions of pluripotent stem cells such as stemness and differentiation ability.

[0018] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0019] In a first aspect, the present application provides a universal cell, which comprises, relative to a wild-type cell:

[0020] 1) reduced or no expression of MHC-I and / or MHC-II human leukocyte antigens;

[0021] 2) an expression sequence of FASLG;

[0022] The cell can escape T cell attack and killing by NK cells.

[0023] In some aspects, the cell comprises reduced or no expression of MHC-I and MHC-II human leukocyte antigens.

[0024] In some aspects, the cell further comprises a modification to increase expression of one or more polypeptides selected from the group consisting of: DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9.

[0025] In some aspects, the reduced or no expression of MHC-I and MHC-II genes is achieved by targeting 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 in the cell using a gene editing tool such as a TALEN and / or a CRISPR system.

[0026] In certain embodiments, to achieve reduced expression or no expression of MHC-I and MHC-II genes, the transcriptional regulator of MHC-I can be preferably selected from one or more of B2M, TAP1, TAP2, Tapasin or NLRC5; the transcriptional regulator of MHC-II can be preferably selected from one or more of CIITA, RFXANK, RFX5, RFXAP.

[0027] The transcriptional regulator is preferably B2M and CIITA.

[0028] In certain embodiments, the cell further comprises a genetic modification targeting the CIITA gene by a rare-cutting endonuclease that selectively inactivates the CIITA gene.

[0029] In certain embodiments, the cell further comprises a genetic modification targeting the B2M gene by a rare-cutting endonuclease that selectively inactivates the B2M gene.

[0030] In certain embodiments, the rare-cutting endonuclease is selected from a CAS protein, a TALE-nuclease, a zinc finger nuclease, a meganuclease and a homing nuclease.

[0031] In certain embodiments, wherein the genetic modification targeting the CIITA gene or the 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 the B2M gene.

[0032] In a specific embodiment, a CRISPR / CAS9 system is used to perform a two-end direct knockout on the exon segments of B2M and CIITA, respectively, wherein the target sequence of gRNA against the B2M gene is SEQ ID NO: 2 and SEQ ID NO: 3, and the target sequence of gRNA against the CIITA gene is SEQ ID NO: 4 and SEQ ID NO: 5.

[0033] In certain aspects, the reduced expression or no expression of MHC-I and / or MHC-II genes in the cell is achieved by introducing a gene expression modification molecule against 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, wherein the gene expression modification molecule comprises one selected from siRNA, shRNA, microRNA, antisense RNA and another RNA-mediated inhibition molecule.

[0034] In some aspects, the amino acid sequence of the FASLG has more than 70% homology to the sequence as set forth in SEQ ID NO: 1, for example, more than 80% homology, for another example, more than 90%, more than 95%, more than 98% homology.

[0035] Further preferably, the amino acid sequence of the FASLG is as set forth in SEQ ID NO: 1.

[0036] In some aspects, the cell is an embryonic stem cell.

[0037] In some aspects, the cell is a pluripotent stem cell.

[0038] In some aspects, the cell is an induced pluripotent stem cell.

[0039] In some embodiments, the cell is a hypoimmunogenic stem cell.

[0040] In some embodiments, the cell is a human stem cell or a human somatic cell.

[0041] In some specific embodiments, the cell is a human induced pluripotent stem cell or a human pluripotent stem cell.

[0042] In a second aspect, the present application provides a method for preparing the universal cell of the first aspect, comprising the following steps:

[0043] 1) knocking out one or more genes of one or more transcriptional regulators of MHC-I of the cell; and / or,

[0044] 2) knocking out one or more genes of one or more transcriptional regulators of MHC-II of the cell;

[0045] 3) introducing a nucleic acid sequence encoding a FASLG protein into the cell.

[0046] In some aspects, the transcriptional regulator of MHC-I can be preferably selected from one or more of B2M, TAP1, TAP2, Tapasin or NLRC5; the transcriptional regulator of MHC-II can be preferably selected from one or more of CIITA, RFXANK, RFX5, RFXAP.

[0047] In some embodiments, the transcriptional regulator is selected from B2M and CIITA.

[0048] In some aspects, the knocking out of steps 1) and 2) is a genetic modification targeting the CIITA gene or the B2M gene by a rare-cutting endonuclease that selectively inactivates the CIITA gene or the B2M gene.

[0049] Preferably, the rare-cutting endonuclease is selected from the group consisting of a CAS protein, a TALE-nuclease, a zinc-finger nuclease, a meganuclease and a homing nuclease.

[0050] Further preferably, wherein the genetic modification of the CIITA gene or the B2M gene by the rare-cutting endonuclease comprises a CAS protein or a polynucleotide encoding the CAS protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene or the B2M gene.

[0051] In certain embodiments, steps 1) and 2) use the CRISPR system to perform two-end direct knockout on the B2M and CIITA exon segments, respectively, wherein the target sequence of the gRNA for the B2M gene is SEQ ID NO: 2, 3, and the target sequence of the gRNA for the CIITA gene is SEQ ID NO: 4, 5.

[0052] In certain aspects, the knockout of step 1) or 2) is achieved by introducing a gene expression modification 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, wherein the gene expression modification molecule comprises one selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA and another RNA-mediated inhibition molecule.

[0053] In certain aspects, step 3) employs an expression vector to introduce a nucleic acid sequence encoding a FASLG protein into the cell.

[0054] Preferably, the expression vector employed in step 3) is a viral vector.

[0055] In certain embodiments, the viral vector employed in step 3) is a lentivirus.

[0056] In certain aspects, step 3) introduces the nucleic acid sequence encoding the FASLG protein into a selected site of the cell; preferably, the selected site of the cell is a safe harbor gene site.

[0057] In certain aspects, the amino acid sequence of the FASLG has more than 70% homology, for example more than 80% homology, for example more than 90%, more than 95%, more than 98% homology with the sequence as shown in SEQ ID NO: 1.

[0058] Further preferably, the amino acid sequence of the FASLG is as shown in SEQ ID NO: 1.

[0059] In some aspects, the universal cell further comprises a second expression vector comprising a polynucleotide sequence encoding one selected from the group consisting of DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9.

[0060] In some embodiments, the second expression vector is an inducible expression vector; preferably, the second expression vector is a viral vector.

[0061] In a third aspect, the present application provides a method for preparing a differentiated universal cell, comprising culturing the universal cell prepared according to the method of the second aspect under differentiation conditions, thereby preparing a differentiated hypoimmunogenic cell.

[0062] In some aspects, wherein the differentiation conditions are suitable for differentiating the cell into a cell type selected from the group consisting of a cardiomyocyte, a neural cell, a glial cell, an endothelial cell, a T cell, an NK cell, an NKT cell, a macrophage, a hematopoietic progenitor cell, a mesenchymal cell, an islet cell, a chondrocyte, a retinal pigment epithelial cell, a kidney cell, a hepatocyte, a thyroid cell, a skin cell, a blood cell, and an epithelial cell.

[0063] In a fourth aspect, the present application provides a method for treating a patient in need of cell therapy, comprising administering a population of differentiated hypoimmunogenic cells prepared according to the method of the third aspect.

[0064] In a fifth aspect, the present application provides a composition comprising the universal cell of the first aspect.

[0065] In some aspects, the composition comprises the universal cell of the first aspect and one or more therapeutic agents, which comprises a peptide, a cytokine, a small molecule compound, a macromolecule, an ADC, an antibody, a nanoparticle, a biosimilar, an mRNA, a traditional Chinese medicine, a protein, a vaccine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a double stranded RNA (dsRNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a replacement factor thereof, a vector comprising one or more polynucleic acids of interest, an antibody, etc.

[0066] In a sixth aspect, the present application provides a cell expressing FASLG protein, and having reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0067] In a seventh aspect, the present application provides a cell not expressing CIITA, expressing FASLG protein, and having reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0068] In an eighth aspect, the present application provides a cell that does not express B2M, expresses FASLG protein, and has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0069] In a ninth aspect, the present application provides a cell that does not express CIITA and B2M, expresses FASLG protein, and has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0070] In a tenth aspect, the present application provides a cell that expresses FASLG protein and at least one polypeptide selected from the group consisting of: DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9, and has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0071] In an eleventh aspect, the present application provides a cell that does not express CIITA, expresses FASLG protein and at least one polypeptide selected from the group consisting of: DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9, and has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0072] In a twelfth aspect, the present application provides a cell that does not express B2M, expresses FASLG protein and at least one polypeptide selected from the group consisting of: DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9, and has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0073] In a thirteenth aspect, the present application provides a cell that does not express CIITA and B2M, expresses FASLG protein and at least one polypeptide selected from the group consisting of: DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9, and has reduced or no expression of MHC class I and / or MHC class II human leukocyte antigens.

[0074] In a fourteenth aspect, the present application provides a cell according to the sixth to thirteenth aspects described above, wherein the cell is selected from the group consisting of 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.

[0075] In a fifteenth aspect, the present application provides use of the universal cell of the first aspect, the composition of the fifth aspect, or the cell of the sixth aspect to the thirteenth aspect in the preparation of a product for cell therapy.

[0076] In a sixteenth aspect, the present application provides use of the universal cell of the first aspect, the composition of the fifth aspect, or the cell of the sixth aspect to the thirteenth aspect in the preparation of a product for organ transplantation.

[0077] In a seventeenth aspect, the present application provides use of the universal cell of the first aspect, the composition of the fifth aspect, or the cell of the sixth aspect to the thirteenth aspect in the construction of a universal PSC cell bank.

[0078] In an eighteenth aspect, the present application provides use of the universal cell of the first aspect, the composition of the fifth aspect, or the cell of the sixth aspect to the thirteenth aspect as a gene drug carrier.

[0079] Advantages of the present application:

[0080] 1. After inactivating the major histocompatibility complex (MHC) class I and II genes in stem cells and overexpressing FASLG, the obtained human pluripotent stem cells or human induced pluripotent stem cells can further escape the killing of NK cells on the basis of escaping T cell attack, and the effect is even better than the reported positive target CD47 and CD24.

[0081] 2. The obtained human induced pluripotent stem cells DKO+FASLG can significantly escape the killing of PBMC, and the escape effect is better than that of WT, DKO+CD47 and DKO+CD24 cells; the obtained human induced pluripotent stem cells DKO+FASLG can significantly escape the killing of macrophages (MAC), and the escape effect is better than that of WT cells.

[0082] 3. At the same time, these low-immunogenic pluripotent stem cells retain their stemness and differentiation ability. The immune-exempted hiPSC-DKO+FASLG cells differentiated into iMSCs from mesoderm can significantly escape the killing of NK and PBMC.

[0083] The immune-exempted hiPSC-DKO+FASLG cells differentiated into iDAPs from ectoderm can significantly escape the killing of NK, MAC and PBMC.

[0084] 5. In a non-human primate (NHP) model, the in vivo and in vitro immunogenicity of universal monkey stem cells (iPSC) was explored, and it was found that the constructed NHP DKO+FASLG can significantly escape the killing of PBMC and still cannot activate the immune system; the survival time of NHP DKO+FASLG cells in vivo is the longest. BRIEF DESCRIPTION OF DRAWINGS

[0085] Figure 1 . Strategy and results of B2M gene knockout in human embryonic stem cell line H1;

[0086] Figure 2 . Strategy and results of CIITA gene knockout in human embryonic stem cell line H1;

[0087] Figure 3 . Figure of using RT-qPCR to detect the expression of B2M and CIITA at RNA level in DKO cells in Example 1;

[0088] Figure 4 . Figure of using Western-blot to detect the protein level of B2M in Example 1;

[0089] Figure 5 . Figure of using flow cytometry to detect the expression of HLA-I / II in various cells stimulated by INF-gamma in Example 1;

[0090] Wherein, T cells are positive control for detecting HLA-I / II molecules;

[0091] Figure 6 . Karyotype of B2M / CIITA double allele knockout positive clone (DKO) obtained in Example 1;

[0092] Figure 7 . Figure of using immunofluorescence and RT-qPCR to detect the expression of sternness genes POU5F1 / NANOG / SOX2 at RNA and protein levels in WT and DKO cells in Example 2;

[0093] Wherein, MSC is negative control;

[0094] Figure 8 . Figure of using immunofluorescence to detect the expression of sternness genes SSEA-4 and Tra1-81 on the surface of WT and DKO cells in Example 2.

[0095] Figure 9 . Figure of using immunohistochemistry to show that DKO cells can form teratoma with three germ layers of endoderm, mesoderm and ectoderm;

[0096] Figure 10 . Figure of using RTCA to detect the results of verifying the immune function of DKO cells in Example 2;

[0097] Figure 11 . Schematic diagram of pGC-EF1a plasmid structure;

[0098] Figure 12Figure of DKO+CD47 cell line constructed in Example 3 detected by flow cytometry for expression of CD47;

[0099] Figure 13 Figure of killing results of NK cells on DKO+CD47 cell line overexpressing CD47, WT, DKO cells detected by RTCA in Example 3;

[0100] Figure 14 Figure of killing results of NK cells on cell line overexpressing candidate protein, H1 WT (positive control), H1 DKO cells (negative control) detected by RTCA in Example 4.

[0101] Figure 15 Figure of overexpression level of mRNA of DKO+FASLG cell line constructed in Example 5 compared with DKO detected by qPCR.

[0102] Figure 16 Figure of expression of sternness genes of DKO+FASLG cell line constructed in Example 5 detected by immunofluorescence (A) and flow cytometry (B).

[0103] Figure 17 Figure of killing results of NK cells on DKO+FASLG cell line overexpressing FASLG protein, WT, DKO cells detected by RTCA in Example 5.

[0104] Figure 18 Figure of killing results of NK cells on DKO+FASLG cell line overexpressing FASLG protein, WT, DKO, DKO+CD24 / CD47 overexpressing CD24 or CD47 protein, and CD24 and CD47 co-expressing cells detected by RTCA in Example 6;

[0105] A is a killing result figure; B is a summary figure of multiple results.

[0106] Explanation: the labels WT, DKO, DKO+CD47, DKO+CD24, DKO+FASLG in the following Figures 19-22 correspond to hiPSC-WT, hiPSC-DKO, hiPS-CDKO+CD47, hiPSC-DKO+CD24, hiPSC-DKO+FASLG;

[0107] Figure 19 Figure of killing results of NK cells on hiPSC-DKO+FASLG cell line overexpressing FASLG protein, hiPSC-WT, hiPSC-DKO, hiPSC-DKO+CD24 / CD47 cells overexpressing CD24 or CD47 protein detected by RTCA in Example 7.

[0108] Figure 20 Figure of killing results of PBMC cells on hiPSC-DKO+FASLG cell line overexpressing FASLG protein, hiPSC-WT, hiPSC-DKO, hiPSC-DKO+CD24 / CD47 cells overexpressing CD24 or CD47 protein detected by RTCA in Example 7.

[0109] Figure 21 Figure of killing results of MAC cells on hiPSC-DKO+FASLG cell line overexpressing FASLG protein, hiPSC-WT and hiPSC-DKO cells detected by RTCA in Example 7.

[0110] Figure 22 Figure of killing results of MAC cells on hiPSC-DKO+FASLG cell line overexpressing FASLG protein, hiPSC-WT and hiPSC-DKO cells detected by RTCA in Example 7.

[0111] Legend: the labels WT, DKO, DKO+FASLG in the following Figure 23 , Figure 32 correspond to hiMSC-WT, hiMSC-DKO, hiMSC-DKO+FASLG; Figure 23 Figure of killing results of NK cells on hiMSC-DKO+FASLG cell line overexpressing FASLG protein, hiMSC-WT and hiMSC-DKO cells detected by RTCA in Example 8.

[0112] Figure 24 Figure of killing results of PBMC cells on hiMSC-DKO+FASLG cell line overexpressing FASLG protein, hiMSC-WT and hiMSC-DKO cells detected by RTCA in Example 8.

[0113] Legend: the labels WT, DKO, DKO+FASLG in the following Figures 25-27 correspond to hiDAP WT, hiDAP DKO, hiDAP DKO+FASLG;

[0114] Figure 25 Figure of killing results of MAC cells on hiDAP DKO+FASLG cell line overexpressing FASLG protein, hiDAP WT and hiDAP DKO cells detected by RTCA in Example 8.

[0115] Figure 26Figure of RTCA detection of PBMC cell killing of hiDAP DKO+FASLG cell line overexpressing FASLG protein, hiDAP WT and hiDAP DKO cells in Example 8.

[0116] Figure 27 Figure of RTCA detection of NK cell killing of hiDAP DKO+FASLG cell line overexpressing FASLG protein, hiDAP WT and hiDAP DKO cells in Example 8.

[0117] Figure 28 Figure of flow cytometry detection of HLA-I / II expression in NHP iPSC-WT and NHP iPSC-DKO cells stimulated with INF-gamma in Example 9.

[0118] Legend: The labels NHP WT, NHP DKO1, NHP DKO2, NHP DKO+FASLG in the following Figures 29-32 correspond to NHP iPSC-WT, NHP iPSC-DKO1, NHP iPSC-DKO2, NHP iPSC-DKO+FASLG, respectively.

[0119] Figure 29 Figure of RTCA detection of T cell killing of NHP iPSC-WT and iPSC-DKO cells in Example 9.

[0120] Figure 30 Figure of RTCA detection of PBMC cell killing of NHP iPSC-WT and iPSC-DKO cells and NHP iPSC-DKO+FASLG cell line overexpressing FASLG protein in Example 9.

[0121] Figure 31 Figure of Elispot detection of IFN-gamma secretion by PBMC cells stimulated after 24 hours of co-culture with NHP iPSC-WT and iPSC-DKO cells and NHP iPSC-DKO+FASLG cell line overexpressing FASLG protein in Example 9.

[0122] Figure 32 Survival rate of transplanted cells in NHP (A) and NOG mice (B) in Example 9. DETAILED DESCRIPTION

[0123] The technical solutions of the present application will be further described in detail below in combination with specific embodiments. It should be understood that the following embodiments are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present application. Any technology implemented based on the above description of the present application is covered within the scope of protection intended by the present application.

[0124] 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 without specific conditions are generally carried out according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0125] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0126] Example 1. Construction of B2M and CIITA double knockout cell line (DKO)

[0127] 1. Cell culture reagents:

[0128] Table 1

[0129]

[0130] 2. Methods and results:

[0131] The present application selects human pluripotent stem cell line H1 (Wicell, WA01) or H9 (Wicell, WA09), and uses CRISPR / CAS9 to knock out β-2-microglobulin (B2M) in the endoplasmic reticulum, so that the cell surface MHC-I cannot form functional molecules, thereby escaping the killing of allogeneic CD8 + T cells; the escape of CD4+T cell killing is achieved by knocking out the positive regulator CIITA of MHC-II gene transcription to reduce the expression of MHC-II class molecules.

[0132] Among them, the CRISPR / CAS9 gene knockout strategy of B2M is as 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-F2 / R2 are used to verify the knockout of the genomic sequence.

[0133] gRNA sequence:

[0134] B2M-gRNA1: CGTGAGTAAACCTGAATCTT

[0135] B2M-gRNA2: AGTCACATGGTTCACACGGC

[0136] Identification primer:

[0137] B2M-F1: TGGGGCCAAATCATGTAGACTC

[0138] B2M-R1: TCAGTGGGGGTGAATTCAGTGT

[0139] B2M-F1 + B2M-R1 = 608 bp

[0140] After knockout: no band

[0141] B2M-F2: CAGAAGTCCTTGAGAGCCTCC

[0142] B2M-R2: TGTGCATCAGTATCTCAGCAGG

[0143] B2M-F2 + B2M-R2 = 812 bp

[0144] After knockout: 569 bp.

[0145] In addition, the CRISPR / CAS9 gene knockout strategy of CIITA is shown as Figure 2 CIITA-gRNA1 and CIITA-gRNA2 are used to directly knockout both ends of the CIITA exon segment, and then CIITA-F1 / R1 and CIITA-F2 / R2 are used to verify the knockout of the genomic sequence.

[0146] gRNA sequence:

[0147] CIITA-gRNA1: GATATTGGCATAAGCCTCCC

[0148] CIITA-gRNA2: CATCGCTGTTAAGAAGCTCC

[0149] Identification primer:

[0150] CIITA-F1: CTGTGCCTCTACCACTTCTATG

[0151] CIITA-R1: CCTTCCATGTCACACAACAGCC

[0152] CIITA-F1 + CIITA-R1 = 368 bp

[0153] Post knockout: no band

[0154] CIITA-F2: TGGAATCCACACTTTCCAGTTC

[0155] CIITA-R2: TGGAGTCTCCGTTCCTCCAG

[0156] CIITA-F2 + CIITA-R2 = 889 bp

[0157] Post knockout: 459 bp

[0158] The specific operation is as follows:

[0159] 1) Culture human pluripotent stem cells to 80% density on Matrigel-coated 6-well plates with mTeSR1 normally. After digestion with TRYPLE, neutralize in DMEM / F12, count. Take 2 x 107cells in an EP tube, centrifuge, and discard the supernatant. 6 Cells in EP tube, centrifuge, and discard the supernatant.

[0160] 2) According to the 100 μL electroporation system of the Neon transfection system, add 15 μg TrueCut Cas9 Protein + 3 μg gRNA (B2M gRNA1 + B2M gRNA2 + CIITA gRNA1 + CIITA gRNA2) to form an RNP system, mix, and place at room temperature for 20 min. TM

[0161] 3) Resuspend the cells in 100 μL RNP electroporation system, and perform electroporation with the Neon transfection system. The electroporation parameters are 1200 V, 30 ms, and 1 pause. After electroporation, quickly add preheated culture medium to the cells, and evenly inoculate in a 6-well plate coated with Matrigel.

[0162] 4) Replace the fresh mTeSR1 culture medium every day. When the single cells grow up, pick single clones in a 48-well plate. After the clones are expanded, collect the genomic samples to detect the gene editing, and the PCR results are shown in Figure 1 and 2 Sanger sequencing is performed on the PCR positive clones to further verify.

[0163] 5) Identify the positive B2M / CIITA double-allele knockout clone DKO for expansion, culture, and cryopreservation.

[0164] Use qPCR to detect the expression of B2M and CIITA at the RNA level of the B2M / CIITA double-allele knockout clone DKO, as shown in Figure 3 , to determine the knockout.​

[0165] B2M-F:AAGATGAGTATGCCTGCCGT

[0166] B2M-R:ATGCGGCATCTTCAAACCTC

[0167] CIITA-F:CCTGGAGCTTCTTAACAGCGA

[0168] CIITA-R:TGTGTCGGGTTCTGAGTAGAG

[0169] The expression level of B2M protein in the B2M / CIITA biallelic knockout clone DKO was detected using Western blotting. Figure 4 As shown, the removal is confirmed.

[0170] WT and DKO cells were stimulated with INF-gamma: Cells were plated, and on the second day, medium containing INF-gamma was added to the cells during medium change. After 48 hours of incubation, the cells were digested and HLA-I / II expression was detected by flow cytometry. Results are as follows: Figure 5 The B2M / CIITA biallelic knockout stem cell positive clone (DKO) was found to be unable to express HLA-I / II molecules in response to INF-gamma stimulation. T cells served as a positive control for detecting HLA-I / II molecules.

[0171] Karyotype analysis was performed on the obtained B2M / CIITA biallelic knockout positive clones (DKO): chromosome specimens fixed on glass slides were treated with trypsin and then stained with Giemsa stain. Chromosomes in metaphase were analyzed for chromosome number and morphological structure to determine whether their karyotype was consistent with the normal karyotype. Results are as follows: Figure 6 As shown, the DKO karyotype is normal.

[0172] Example 2. Verification of the stemness and immune function of the DKO cell line in Example 1

[0173] 1. Expression of stemness genes in WT and DKO cells

[0174] Immunofluorescence assays showed that WT and DKO cells expressed the stemness genes POU5F1 and NANOG at the protein level. Cells were seeded in 12-well plates, and after reaching a density of 60-80%, the culture medium was removed, and the cells were fixed with 4% paraformaldehyde. After cell lysis, the cells were incubated overnight at 4°C with primary antibodies against POU5F1 and NANOG. After washing away the primary antibody, the cells were incubated at room temperature with fluorescently labeled secondary antibody, followed by imaging using a fluorescence microscope. Results are shown below. Figure 7RT-qPCR detection showed that WT and DKO cells expressed the sternness genes POU5F1, NANOG and SOX2 at the RNA level: results as shown in Figure Figure 7 B. (MSCs are negative controls for sternness gene expression)

[0175] Flow cytometry results showed that WT and DKO cells both highly expressed the sternness genes SSEA-4 and Tra1-81 on the cell surface, with percentages of 100%, 99.98% and 96.75%, 99.13% respectively. Results as shown in Figure Figure 8 .

[0176] 2. Differentiation ability of the obtained B2M / CIITA double allele knockout positive clone (DKO)

[0177] The immunodeficient mice (SCID Beige) were subcutaneously injected with 100 μL of a suspension containing 5E+5 DKO cells. When the teratoma volume was greater than 1.5 cm 3 , it was removed and sectioned for staining.

[0178] The obtained B2M / CIITA double allele knockout positive clone (DKO) can form teratomas in vivo and differentiate into cells of the endoderm, mesoderm and ectoderm. As shown in Figure Figure 9 .

[0179] 3. Verification of the immune function of DKO cells

[0180] The killing experiments of T cells and NK cells used xCELLigence RTCA Instrument. The same number of WT and DKO cell lines were resuspended using Essential 8 medium containing IL-2 and inoculated in 96-well E-plates coated with Matrigel, and activated T cells or NK cells were added for killing detection. The RTCA detection data were analyzed using xCELLigence software to calculate the killing rate and escape function.

[0181] Table 2

[0182] Name Cat No. Size Manufacturer PE anti-human CD4 980804 500ul / tube Biolegend APC anti-human CD8 980904 500ul / tube Biolegend FITC anti-human CD3 300440 500tests Biolegend APC anti-human CD16 301012 100tests Biolegend PE anti-human CD56(NCAM) 318306 100tests Biolegend human IL-2 202-1L-050 / CF 50ug R&D Y-27632 2HCl S1049 5mg Selleck Matrigel 354277 5ml Gibco Essential 8 TM culture medium A1517001 500ml Gibco E-Plate VIEW 96PET 300601030 6wells / box Agilent

[0183] As shown in the RTCA data of Figure 10 , WT cells escaped the killing of NK cells due to the expression of HLA-I, but were killed by T cells. DKO cells could escape the killing of T cells, while being more sensitive to the killing of NK cells.

[0184] Example 3. Construction of DKO+CD47 cell lines and verification of immune function

[0185] DKO cells obtained in Example 1 were used to overexpress CD47 (NM_198793) using a lentiviral vector, the amino acid sequence of which is shown as SEQ ID NO. 18, and the cDNA (SEQ ID NO. 19) of the overexpression sequence was constructed in a lentiviral plasmid (pGC-EF1a) with an EF1a promoter and a puromycin screening marker. The structure of the pGC-EF1a plasmid is shown in Figure 11 . The plasmid was digested with BamHI / NheI, and after successful ligation, Sanger sequencing was used to verify the correctness of the inserted sequence and virus packaging. The DKO human pluripotent stem cells constructed in Example 2 were transfected, the medium was changed 24 h later, and after 48 h, the medium containing puromycin was changed for screening. The results of the constructed stable DKO+CD47 cell line are shown in Figure 12 , and after confirming the correct expression, the cells were expanded and subjected to subsequent functional detection.

[0186] Referring to Example 2, RTCA was used to detect whether the overexpressed DKO+CD47 cell line could successfully escape the killing of NK cells while escaping the killing of T cells, as shown in Figure 13 , NK cells can effectively kill DKO cells, and WT and DKO+CD47 overexpressed cells can escape NK killing.

[0187] Example 4. Screening of universal cell lines expressing molecules involved in maternal-fetal tolerance and tumor immune escape

[0188] Construction strategy and screening of each molecule protein of the present application:

[0189] A total of 22 candidate targets were screened, and their amino acid and cDNA sequences are shown in Table 3. The candidate targets are related to maternal-fetal tolerance or tumor immune escape, among which:

[0190] Molecules related to maternal-fetal tolerance: HLA-E, HLA-G, CTLA4-Ig.

[0191] Inhibitory immune receptors (immune checkpoints) expressed by tumors: MICA, MICB, ULBP1, ULBP2, ULBP3, C1-Inhibitor, FASLG, CD46, CD55, CD59, CD20, HER2.

[0192] Regulatory factors in the tumor microenvironment: TDO, IDO1, IDO2, IL-10, IL37, IL-12A, IL-35B.

[0193] Table 3

[0194]

[0195]

[0196] Reengineering pluripotent stem cells with natural immune suppression mechanisms to confer hypoimmunogenicity, but these types of approaches rely on immune inhibitory receptor-ligand pairs or specific microenvironments, and whether they are present on the transplanted cells and immune cells, so introducing these genes alone can not necessarily lead to complete hypoimmunogenicity, which needs to be functionally confirmed by actual detection (Zhao et al., 2020).

[0197] Referring to Example 3, all target points were reengineered on the basis of H1 DKO, and multiple NK in vitro killing experiments (n≥5) were performed on the stable transfection strains of 22 candidate target points to screen the candidate new target points with the best escape ability from NK cells. The relative killing ratio was normalized to the negative control H1 DKO cells, and was summarized at high (high: E:T = 2:1) and low (low: E:T = 1:1 or 0.5:1) NK ratios (E:T). The killing ratio less than 1 indicates that there is an escape ability relative to DKO, and the lower the ratio, the stronger the escape ability. The positive control is H1 WT and H1 DKO+CD47. The results are shown in Figure 14 , and only DKO+FASLG has multiple ratios and multiple killing rates less than the average killing rate of the positive controls H1 WT and DKO+CD47, which is the optimal cell for escaping NK killing. The effect is even better than the common sense star molecules HLA-G, CTLA4-Ig, etc., achieving an unexpected effect.

[0198] Example 5. Construction of DKO+FASLG cell lines and verification of immune function

[0199] 1. Construction of DKO+FASLG cells and overexpression detection

[0200] The nucleic acid sequence (NM_000639.3) encoding FASLG (the amino acid sequence of FASLG is shown in NM_000639.3) was directly synthesized and constructed in a lentiviral plasmid (pGC-EF1a) with EF1a promoter and puromycin selection marker. The structure of the pGC-EF1a plasmid is shown in Figure 11 . The plasmid was digested with BamHI / NheI, and after successful ligation, Sanger sequencing was used to verify the correctness of the inserted sequence and virus packaging. After transfection of the DKO human pluripotent stem cells obtained in Example 2, the medium containing puromycin was used for screening. The DKO+FASLG cells constructed were used to detect the overexpression level of mRNA by qPCR, and the DKO cells were used as negative controls. The results are shown in Figure 15 .

[0201] FASLG F1 : TGCCTTGGTAGGATTGGGC

[0202] FASLG R1 : GCTGGTAGACTCTCGGAGTTC

[0203] 2. Expression of stemness genes in DKO+FASLG cells

[0204] Referring to Example 2, immunofluorescence detection showed that DKO+FASLG cells expressed stemness genes OCT4, NANOG, SOX2, TRA-1-60, and TRA-1-81 at the protein level, and the results are shown in FIG. A. Flow cytometry results showed that DKO+FASLG cells highly expressed stemness genes SSEA-4, OCT4, TRA-1-60, and Tra1-81 on the cell surface, accounting for 98.70%, 99.51%, and 94.34%, respectively. The results are shown in FIG. B. Figure 16 Figure 16

[0205] 3. Detection of immune function of DKO+FASLG cells

[0206] After confirming the construction of DKO+FASLG cells and the correct expression of stemness, the cells were expanded and subjected to subsequent functional detection. Referring to 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 H1 WT, H1+CD47, and DKO+FASLG successfully escaped. The degree of escape of DKO+FASLG was better than that of H1 WT and H1+CD47. As shown in FIG. C. Figure 17

[0207] Example 6. Comparison of NK escape degrees of DKO+FASLG and several cell lines

[0208] This example relates to cell lines: DKO+CD47, DKO+CD24 in Example 3, DKO+CD47 in the prior art (WO2020018615A2) (hereinafter referred to as “CD47 prior art”), and DKO+CD47+CD24. The amino acid sequence of CD47 in the DKO+CD47 cell line in the prior art is shown in SEQ ID NO. 22, which promotes immune escape by interacting with signal regulatory protein alpha (SIRPα) on the surface of immune cells. The construction of the “CD47 prior art” cell line was completed according to the description in WO2020018615A2. Overexpression of CD47 in B2M and CIITA double-knockout cells (DKO) can successfully escape the killing of T and NK cells. (PMID:32433947 / PMID:30778232).

[0209] ​​​CD24 promotes immune escape 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 embodiment is shown as SEQ ID NO. 23, and the construction method thereof is referred to Example 3.

[0210] The DKO+CD47+CD24 cell line is further overexpressed with CD24 based on Example 3 by means of lentiviral transfection. The lentivirus construction and overexpression operation are referred to Example 3, and the amino acid sequence of CD24 is shown as SEQ ID NO. 23.

[0211] At the same time, the multiple repeated experiments are normalized to each DKO killing, and the killing results after being summarized show that the killing ratio of DKO+FASLG is the smallest, and has a significant difference compared with the "CD47 prior art". As shown in Figure 18 .

[0212] Example 7. In vitro detection of hiPSC-DKO+FASLG escaping killing of the immune system

[0213] In this embodiment, according to the method described above, human induced pluripotent stem cells (hiPSC cells) are selected to prepare hiPSC-DKO and hiPSC-DKO+FASLG (hiPSC cells are reprogrammed by CTS TM CytoTune TM -iPS 2.1 Sendai virus reprogramming kit (item number: A34546) prepared).

[0214] This embodiment relates to comparative cell lines: hiPSC-DKO+CD47, hiPSC-DKO+CD24.

[0215] hiPSC-DKO+CD47 is constructed by the method in Example 3, and hiPSC-DKO+CD24 is overexpressed with CD24 by means of lentiviral transfection based on hiPSC-DKO. The lentivirus construction and overexpression operation are referred to Example 3. The amino acid sequence of CD24 is MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKAAGGALQSTASLFVVSLSLLHLYS.

[0216] CD47 promotes immune escape by interacting with signal regulatory protein alpha (SIRPa) on the surface of immune cells. Overexpression of CD47 in B2M and CIITA double-knockout cells (DKO) can successfully escape the killing of T and NK cells. (Method reference PMID: 32433947 / PMID: 30778232). CD24 promotes immune escape by interacting with the inhibitory receptor sialic acid-binding Ig-like lectin 10 (Siglec-10) on the surface of immune cells. (Method reference PMID: 31367043).

[0217] 1. Verification of NK cell killing function escape

[0218] NK cell killing assay was performed on the XCelligence platform (ACEA BioSciences). Each type of hiPSC cell was resuspended in 100 μl cell-specific medium and plated on a 96-well E-plate coated with Matrigel (Sigma-Aldrich) (ACEA BioSciences). After the cell index value reached 1, NK cells were added at an E:T ratio of 1:1. The data were standardized and analyzed using RTCA software (ACEA). As shown in Figure 19 , hiPSC-DKO+FASLG cells can significantly escape the killing of NK cells, and the escape effect is better than that of hiPSC-WT, hiPSC-DKO+CD47 and hiPSC-DKO+CD24 cells.

[0219] 2. Verification of PBMC killing function escape

[0220] NK activating factors were added in advance in PBMC to improve the proportion of NK cells in PBMC and the killing performance of T cells. Activated mixed lymphocytes (PBMC) were used as effector cells for RTCA experiments to comprehensively evaluate the immune escape ability of hiPSC-DKO+FASLG cells. (Method reference PMID: 33309274). PBMC cell killing assay was performed on the XCelligence platform (ACEA BioSciences). Each type of hiPSC cell was resuspended in 100 μl cell-specific medium and plated on a 96-well E-plate coated with Matrigel (Sigma-Aldrich) (ACEA BioSciences). After the cell index value reached 1, PBMC cells were added at an E:T ratio of 2:1. The data were standardized and analyzed using RTCA software (ACEA). As shown in Figure 20As shown, hiPSC-DKO+FASLG cells can significantly escape the killing of PBMCs, with better escape effect than hiPSC-WT, hiPSC-DKO+CD47 and hiPSC-DKO+CD24 cells.

[0221] 3. Macrophage (MAC) killing escape detection

[0222] MAC cell killing assay was performed on XCelligence platform (ACEA BioSciences). Each type of hiPSC cells was resuspended in 100 μl cell-specific medium and plated on 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index reached 1, MAC cells were added at an E:T ratio of 3:1. Data were normalized and analyzed by RTCA software (ACEA). Results are shown in Figure 21 As shown, hiPSC-DKO+FASLG cells can significantly escape the killing of MACs, with better escape effect than WT cells.

[0223] 4. Immune system activation detection

[0224] Cellular activation of the immune system was measured by Elispot assay, referring to the method disclosed in “Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle” (Method reference PMID: 26275095), PBMC cell IFN-γ spot secretion was detected by Elispot to determine the immune escape function of DKO+FASLG cells. The specific operation method is as follows:

[0225] hiPSC-WT, hiPSC-DKO, hiPSC-DKO+FASLG cells were plated in 12-well plates, and after 24 h, the culture medium was discarded and PBMC cells were added for culture. PBMC cells were collected after 24 h for subsequent IFN-γ secretion detection, and at the same time, the remaining hiPSC-WT, hiPSC-DKO, hiPSC-DKO+FASLG cells after removal of PBMC cells were observed, and the results are shown in Figure 22 As shown in A, compared with hiPSC-WT and hiPSC-DKO cells, hiPSC-DKO+FASLG cells were less killed by PBMC cells, indicating that the ability of hiPSC-DKO+FASLG cells to escape PBMC cell killing was significantly higher than that of WT and DKO cells.

[0226] Elispot assay results Figure 22 As shown in FIG. 8B, the number of spots formed by IFN-γ secreted by PBMC cells stimulated by hiPSC-DKO+FASLG cells co-cultured with PBMC was significantly lower than that of hiPSC-WT and hiPSC-DKO cells, indicating that overexpression of FASLG not only avoids the activation of T cells, but also offsets the activation of NK cells caused by B2M / CIITA knockout.

[0227] Example 8. In vitro detection of hiPSC-derived cells escaping killing by the immune system

[0228] This example describes the differentiation of immune-exempt hiPSC cells into iMSCs of mesoderm and iDAPs of ectoderm, to study whether the hiPSC-derived cells still have immune exemption. The method comprises using various immune effector cells: PBMC, NK, MAC, etc. to verify the low immunogenicity of the derived cells.

[0229] 1. In vitro detection of hiPSC-derived iMSCs escaping killing by the immune system

[0230] hiPSC-WT, hiPSC-DKO or hiPSC-DKO+FASLG cells were plated on Matrigel, and when the confluence reached 40%, the medium was replaced with differentiation medium (Retinoic Acid-induce) and the liquid was changed daily. After covering, they were transferred to 0.1% gelatin-coated dishes, and passaged every 3 days. During this period, the liquid still needed to be changed daily, and the differentiation period was 10 days (iMSC cell differentiation method reference: PMID: 22865887).

[0231] a) NK

[0232] NK cell killing assay was performed on the XCelligence platform (ACEA BioSciences). Each type of iMSC cell was resuspended in 100 μl of cell-specific medium and plated on a 96-well E-plate coated with Matrigel (Sigma-Aldrich) (ACEA BioSciences). After the cell index value reached 1, NK cells were added at an E:T ratio of 1:1. The data were standardized and analyzed using RTCA software (ACEA). As shown in FIG. 9A, hiPSC-DKO+FASLG-derived iMSC cells can significantly escape NK killing. Figure 23

[0233] b) PBMC

[0234] ​PBMC cell killing assay was performed on XCelligence platform (ACEA BioSciences). Each type of iMSC cells were resuspended in 100 μΐ cell-specific medium and plated on 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After cell index reached 1, PBMC cells were added at 1 : 1 E:T ratio. Data were normalized and analyzed by RTCA software (ACEA). Results are shown in Figure 24 Figure 6, hiPSC-DKO+FASLG derived iMSC cells can significantly escape killing by PBMC.

[0235] 2. hiPSC derived iDAP cells escape killing by immune system in vitro

[0236] Each type of hiPSC-WT, hiPSC-DKO or hiPSC-DKO+FASLG cells were passaged and plated into culture flasks pre-coated with Matrigel, and after 24h incubation, cells were switched to pre-differentiation medium to induce differentiation into midbrain cells (Nolbrant 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 midbrain cells were obtained. Thereafter, the obtained midbrain cells were expanded by addition of neural progenitor cell medium, and a large number of high purity neural progenitor cells were obtained; finally, the neural progenitor cells were further differentiated into dopaminergic neural precursor (DAP) cells by addition of neural precursor cell medium.

[0237] a) NK

[0238] NK cell killing assays were performed on the XCelligence platform (ACEA BioSciences). Each type of iDAP cells were resuspended in 100 μΐ cell-specific medium and plated on 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, NK cells were added at a 1:1 E:T ratio. Data were normalized and analyzed using RTCA software (ACEA). Results are shown in Figure 25 Figure 6, hiPSC-DKO+FASLG-derived iDAP cells can significantly escape NK killing.

[0239] b) MAC

[0240] MAC cell killing assays were performed on the XCelligence platform (ACEA BioSciences). Each type of iDAP cells were resuspended in 100 μΐ cell-specific medium and plated on 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, MAC cells were added at a 1:1 E:T ratio. Data were normalized and analyzed using RTCA software (ACEA). Results are shown in Figure 26 Figure 7, hiPSC-DKO+FASLG-derived iDAP cells can significantly escape MAC killing.

[0241] c) PBMC

[0242] PBMC cell killing assays were performed on the XCelligence platform (ACEA BioSciences). Each type of iDAP cells were resuspended in 100 μΐ cell-specific medium and plated on 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, PBMC cells were added at a 1:1 E:T ratio. Data were normalized and analyzed using RTCA software (ACEA). Results are shown in Figure 27 Figure 8, hiPSC-DKO+FASLG-derived iDAP cells can significantly escape PBMC killing.

[0243] Example 9. Construction and validation of universal NHP cells

[0244] This example describes an in vitro and in vivo immunogenicity study of universal monkey stem cells (iPSC) in a non-human primate (NHP) model.

[0245] 1. Construction of universal NHP cells

[0246] a) Construction of NHP iPSC-DKO cells

[0247] In this study, monkey iPSC-DKO (monkey iPSC cells, by CTS TM CytoTune TM -iPS 2.1 Sendai virus reprogramming kit (Cat No: A34546) were prepared) cells. NHP-B2M-gRNA1 and NHP-B2M-gRNA2 were used to knock out the B2M exon segments at both ends, and then NHP-B2M-F / R pair of PCR primers were used for genome sequence knockout verification. NHP-CIITA-gRNA1 and NHP-CIITA-gRNA2 were used to knock out the CIITA exon segments at both ends, and NHP-CIITA-F / R pair of PCR primers were used for genome PCR verification of genome knockout, and 2 NHP iPSC-DKO were selected for subsequent verification work.

[0248] NHP-B2M-gRNA1 CGTGAGTAAACCTGAATCTT

[0249] NHP-B2M-gRNA2 AGTCACATGGTTCACACGGC

[0250] NHP-CIITA-gRNA1 CATCGCTGTTGAGAAGCTCC

[0251] NHP-CIITA-gRNA2 GATATTGGCATAAGCCTCCC

[0252] B2M identification primer

[0253] NHP-B2M-F CATTTGGCCAGAGTGGAAATG

[0254] NHP-B2M-R TGGGACTCATTCAGGGTAGTA

[0255] CIITA identification primer

[0256] NHP-CIITA-F CTGTGAGGTGACTGAGCATATC

[0257] NHP-CIITA-R GGCCAGCAATGAGCATACTA

[0258] NHP iPSC-WT and NHP iPSC-DKO1 and NHP iPSC-DKO2 cells were stimulated with INF-gamma: cells were plated in well plates and the next day INF-gamma containing media was added to the cells at the time of media change. After 48h cells were trypsinized and flow cytometry was used to detect HLA-I / II expression. Results are shown in Figure 28 Figure 6B2M / CIITA double allele knockout NHP iPSC-DKO1 and NHP iPSC-DKO2 cells do not express HLA-I / II and fail to upregulate HLA-I / II in response to INF-gamma stimulation.

[0259] b) Functional validation of NHP iPSC-DKO cell escape from T cell killing

[0260] T cell killing assays were performed on the XCelligence platform (ACEA BioSciences). NHP iPSC-WT and iPSC-DKO cells were resuspended in 100 μΐ cell specific medium and plated on 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index reached 1, T cells were added at a 2:1 E:T ratio. Data were normalized and analyzed using RTCA software (ACEA). Results are shown in Figure 29 Figure 7A NHP DKO cells significantly escape from T cell killing, while NHP iPSC-WT cells are killed.

[0261] 2. In vitro detection of universal NHP cell escape from immune system killing

[0262] NHP iPSC-DKO+FASLG (gene sequence of monkey FASLG is shown in XM_045392855.1) cells were prepared according to the methods described above and confirmed to be overexpressing.

[0263] a) PBMC killing

[0264] PBMC cell killing assays were performed on the XCelligence platform (ACEA BioSciences). NHP iPSC-WT and NHP iPSC-DKO and NHP iPSC-DKO+FASLG cells were resuspended in 100 μΐ cell specific medium and plated on 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index reached 1, T cells were added at a 1:1 E:T ratio. Data were normalized and analyzed using RTCA software (ACEA). Results are shown in Figure 30As shown, NHP iPSC-DKO+FASLG cells can significantly escape the killing of PBMCs.

[0265] b) Immune system activation detection

[0266] Referring to the operation of Example 7, the Elispot detection results are as shown in Figure 31 As shown, Neg. Ctrl is the negative control of PBMCs without co-culturing with target cells. The number of spots formed by the secreted IFN-γ of PBMCs co-cultured with NHP iPSC-DKO+FASLG cells is significantly lower than that of NHP iPSC-WT and NHP iPSC-DKO cells. It is indicated that overexpression of FASLG can still not activate the immune system in NHP cells.

[0267] 3. In vivo detection of universal NHP cells escaping the killing of the immune system - survival rate of transplanted cells

[0268] The survival rate of universal NHP cells was monitored by observing the bioluminescence imaging (BLI) after the NHPs were injected with cells throughout the experiment. For BLI, D-luciferin Firefly potassium salt (375 mg / kg) (Biosynth AG) dissolved in sterile PBS (pH 7.4) (Gibco, Invitrogen) was intravenously injected into anesthetized monkeys (intraperitoneally injected for anesthetized mice). The animals were imaged using Largo (Spectral Instruments Imaging, Tucson, AZ). The bioluminescence of the region of interest (ROI) was quantified in units of maximum photons per second per square centimeter per steradian (p / s / cm2 / sr).

[0269] Specifically, NHP iPSC-WT, NHP iPSC-DKO and NHP iPSC-DKO+FASLG cells infected with luciferase (luc)-carrying lentivirus were subcutaneously injected, respectively, and the fluorescence intensity of the cells was detected by intravenous injection of the luminescent substrate D-luciferin Firefly potassium salt of luc and using a live imaging instrument (iVIS spectrum, PerkinElmer), which can indicate the survival of the cells in the NHPs. The survival time of NHP iPSC-DKO+FASLG cells in vivo is the longest, which is lower than the detection lower limit until 21d, as shown in Figure 32 As shown in FIG. A, the cells of the same batch were injected (subcutaneously injected) and observed for fluorescence (luminescent substrate intraperitoneally injected) on immunodeficient NOG mice. NHP iPSC-WT and NHP iPSC-DKO+FASLG cells can both grow continuously on NOG mice, indicating that there is no difference in the in vivo proliferation ability of the two cells, as shown in Figure 32The difference in survival time between NHP iPSC-DKO+FASLG cells and other cells observed on NHPs is due to the speed of rejection by the immune system, indicating that NHP iPSC-DKO+FASLG cells can resist immune rejection and have low immunogenicity.

[0270] The above describes embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of protection of the present application.

Claims

1. A universal cell, characterized in that, The universal cell type, relative to the wild-type cell type, is: 1) The expression of MHC-I and MHC-II human leukocyte antigens is reduced or absent; 2) Overexpression of FASLG, wherein the amino acid sequence of FASLG is shown in SEQ ID NO:1; the cells are human embryonic stem cell line H1 or human induced pluripotent stem cells; In the cells, gene editing tools are used to target transcriptional regulators encoding MHC-I and MHC-II to reduce or eliminate the expression of MHC-I and / or MHC-II genes. The transcriptional regulators are B2M and CIITA.

2. The universal cell according to claim 1, characterized in that, The cells also include genetic modifications targeting the CIITA gene by selectively inactivating rare cleaving endonucleases of the CIITA gene, and genetic modifications targeting the B2M gene by selectively inactivating rare cleaving endonucleases of the B2M gene.

3. The universal cell according to claim 2, characterized in that, The rare cleaving endonuclease is selected from CAS protein, TALE-nuclease, zinc finger nuclease, large nuclease, and homing nuclease.

4. The universal cell according to claim 3, characterized in that, The genetic modifications that target the CIITA or B2M gene via rare cleavage endonucleases include the CAS protein or a polynucleotide encoding the CAS protein, and at least one guide RNA sequence for specifically targeting the CIITA or B2M gene.

5. The universal cell according to claim 4, characterized in that, The B2M and CIITA exon regions were directly knocked out at both ends using the CRISPR / CAS9 system. The target sequences of the guide RNA gRNA for the B2M gene are SEQ ID NO:2 and 3, and the target sequences of the guide RNA gRNA for the CIITA gene are SEQ ID NO:4 and 5.

6. A method for preparing a universal cell according to any one of claims 1-5, characterized in that, It includes the following steps: 1) Knock out B2M, the transcriptional regulator of MHC-I in stem cells; 2) Knock out CIITA, the MHC-II transcriptional regulator of stem cells; 3) Introduce a nucleic acid sequence encoding the FASLG protein into the cell.

7. The preparation method according to claim 6, characterized in that, The knockout described in step 1) or 2) is a genetic modification of the CIITA or B2M gene by selectively inactivating rare cleaving endonucleases that target the CIITA or B2M gene. The rare cleaving endonucleases are selected from CAS proteins, TALE-nucleases, zinc finger nucleases, large nucleases, and homing nucleases.

8. The preparation method according to claim 7, characterized in that, The genetic modifications that target the CIITA or B2M gene via rare cleavage endonucleases include the CAS protein or a polynucleotide encoding the CAS protein, and at least one guide RNA sequence for specifically targeting the CIITA or B2M gene.

9. The preparation method according to claim 8, characterized in that, Steps 1) and 2) use the CRISPR system to directly knock out the exon regions of B2M and CIITA at both ends, respectively. The target sequences of gRNA for B2M gene are SEQ ID NO:2 and 3, and the target sequences of gRNA for CIITA gene are SEQ ID NO:4 and 5.

10. The preparation method according to claim 6, characterized in that, In step 3), an expression vector is used to introduce the nucleic acid sequence encoding the FASLG protein into the cell.

11. The preparation method according to claim 10, characterized in that, The expression vector used in step 3) is a viral vector.

12. The preparation method according to claim 11, characterized in that, The viral vector is a lentivirus.

13. The preparation method according to claim 12, characterized in that, Step 3) involves introducing the nucleic acid sequence encoding the FASLG protein into a selected site on the universal stem cell.

14. The preparation method according to claim 13, characterized in that, The selected site for the universal stem cell is the safe harbor gene site.

15. A method for preparing differentiated universal cells, comprising culturing the universal cells according to any one of claims 1-5 under differentiation conditions to prepare differentiated low-immunogenic cells.

16. The method of claim 15, wherein the differentiation conditions are adapted to differentiate 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.

17. A composition, characterized in that, The composition comprises the universal cells as described in any one of claims 1-5.

18. The composition according to claim 17, characterized in that, The composition further comprises one or more therapeutic agents, said therapeutic agents comprising peptides, cytokines, checkpoint inhibitors, mitogens, growth factors, small RNAs, double-stranded RNAs, monocytes, feeder cells, feeder cell components or their replacement factors, vectors containing one or more of the polynucleotides of interest, and antibodies.

19. Use of the universal cell according to any one of claims 1-5, or the composition according to claim 17 or 18, in the preparation of products for cell therapy.

20. The use of the universal cell according to any one of claims 1-5, or the composition according to claim 17 or 18, in the preparation of products for organ transplantation.

21. Use of the universal cells according to any one of claims 1-5, or the composition according to claim 17 or 18, in constructing a universal PSCs cell bank.

22. The use of the universal cell according to any one of claims 1-5, or the composition according to claim 17 or 18, as a gene drug carrier.

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