Universal engineered cell as well as preparation method and application thereof

Abstract

The invention provides an engineered cell as well as a preparation method and application thereof. Relative to a wild type cell, the engineered cell overexpresses a CD47 molecule; based on the density of the CD47 molecules on the cell surface, the distribution density of the CD47 molecules on the surface of the engineered cell is not less than 2700 molecules / mu m < 2 >. The engineered cell has good ultra-low immunogenicity, can avoid killing of T cells, PBMC cells and NK cells at the same time, remarkably prolongs the in-vivo survival time of transplanted cells, is expected to generate universal cell products, and solves the problem of immunological rejection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell technology, and in particular to a universal engineered cell and a preparation method and application thereof. BACKGROUND

[0002] One of the directions of regenerative medicine research is to replace diseased or lost cells by transplanting new, healthy cells. Human pluripotent stem cells (hPSCs) or mesenchymal stem cells (MSCs) have good proliferative capacity and multilineage differentiation potential, and are expected to provide a reliable cell source for regenerative strategies. Although reprogramming technology can generate autologous iPSCs for patient-specific treatment, this way is costly, time-consuming, labor-intensive, difficult to control the quality, and has high uncertainty of curative effect, and is only suitable for chronic diseases. Allogeneic cell therapy for a large number of patient populations may be more feasible in practice, but at the same time, allogeneic cells will cause strong immune rejection problems, thereby failing to bring the expected therapeutic effect.

[0003] Immune rejection is mainly caused by the mismatch of human leukocyte antigens (HLAs) on the cell surface between allogeneic cells. HLA genes, as the major histocompatibility complex (MHC), provide foreign peptides and self-peptides for T cells. MHC is mainly composed of classical class I molecules and classical class II molecules, among which the classical class I genes (HLA-A, HLA-B and HLA-C) are widely expressed on nucleated cells, while the classical class II genes (HLA-DP, HLA-DQ and HLA-DR) are mainly expressed in T cells, B cells and antigen-presenting cells. In addition, under the stimulation of interferon, MHC class II can be induced to express in other cell types. Each polymorphic HLA gene contains a large number of multiple alleles, so it is difficult to find a cell, organ or tissue donor that matches a specific pair of HLA alleles. Usually, immunosuppressive agents are used for patients in the prior art to solve the problem of immune rejection caused by HLA haplotype mismatch. However, long-term treatment with immunosuppressive drugs usually causes serious side effects.

[0004] CD47 is a ubiquitous membrane protein that can interact with a variety of cell surface receptors to inhibit immune action. It is related to a variety of functions such as neutrophil migration, axon extension and T cell costimulation. In addition, CD47 can interact with the receptor SIRPα on immune cells to inhibit immune response. Therefore, CD47 can provide a "don't eat me" signal to ensure that autologous cells are not recognized for erroneous clearance. The prior art discloses that by knocking out B2M and CIITA genes and using lentivirus to transduce CD47, low immunogenicity induced pluripotent stem cells are constructed, and further differentiated into cardiomyocytes and endothelial cells and transplanted into humanized mice, and the genetically modified cells achieve a higher level of survival. However, the use of lentivirus for transduction has a relatively low infection efficiency, especially in mesenchymal stem cells or induced pluripotent stem cells and their derived cells, it may be difficult to achieve a high level of CD47 expression, and may cause gene insertion mutation with safety risks, and this scheme cannot achieve good and safe ultra-low immunogenicity.

[0005] In view of the above problems, the present application expects to construct a universal type of engineered super low immunogenicity cells (SLIC) without using any immunosuppressants, and to provide a universal cell source for allogeneic cell therapy. SUMMARY

[0006] In order to solve the problem that the engineered cells in the prior art are difficult to achieve excellent ultra-low immunogenicity, the present application provides a universal type of engineered cells and a preparation method thereof.

[0007] In a first aspect of the present application, an engineered cell is provided, wherein the engineered cell overexpresses CD47 molecules relative to wild-type cells;

[0008] In terms of the density of CD47 molecules on the surface of the cells, the distribution density of the CD47 molecules on the surface of the engineered cells is not less than 2700 molecules / μm 2 .

[0009] In a preferred embodiment of the present application, in terms of the density of CD47 molecules on the surface of the cells, the distribution density of the CD47 molecules on the surface of the engineered cells is not less than 2800, or not less than 2900, or not less than 3000 molecules / μm 2 .

[0010] In an embodiment of the present application, the engineered cells have lower immunogenicity or higher immunological tolerance relative to wild-type cells or cells with a surface distribution density of CD47 molecules less than 2700 / μm 2 .

[0011] In one embodiment of the present application, the engineered cell is immune tolerant to multiple immune cells.

[0012] In one embodiment of the present application, the immune cell comprises one or more of, but not limited to, PBMC cell, T cell, NK cell, macrophage, neutrophil, eosinophil, mast cell, dendritic cell. In one preferred embodiment of the present application, the immune cell comprises one or more of PBMC cell, T cell, NK cell. More preferably, the immune cell comprises all of T cell, PBMC cell and NK cell.

[0013] In one embodiment of the present application, the immune cell is autologous or allogeneic to the individual.

[0014] In one embodiment of the present application, the engineered cell has a longer in vivo survival time relative to a wild-type cell or a cell with a surface distribution density of CD47 molecules less than 2700 / μm2. 2

[0015] In one embodiment of the present application, the in vivo survival time is at least 2 days. Preferably, the in vivo survival time is at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 2 weeks, 3 weeks, 4 weeks or more.

[0016] In one embodiment of the present application, the engineered cell comprises an engineered stem cell.

[0017] In one embodiment of the present application, the engineered stem cell comprises an engineered mesenchymal stem cell, an engineered induced pluripotent stem cell and a cell derived therefrom.

[0018] In one embodiment of the present application, the engineered mesenchymal stem cell is derived from a pluripotent stem cell, more preferably, the pluripotent stem cell is selected from an induced pluripotent stem cell (iPSC).

[0019] In one preferred embodiment of the present application, the engineered mesenchymal stem cell is derived from bone marrow, fat, muscle, heart, umbilical cord blood or umbilical cord.

[0020] In one embodiment of the present application, the derived cell comprises CAR-iNK, dopaminergic neural precursor cell, CAR-iMac, cardiomyocyte, endothelial progenitor cell, iNK cell, retinal cell, neural cell, osteoblast, hematopoietic stem cell, blood cell, Β cell, T cell, fibroblast, hair cell, monocyte, macrophage, Treg cell, kidney progenitor cell, lung epithelial cell, endothelial cell, megakaryocyte, smooth muscle cell, skeletal muscle cell, chondrocyte, bone cell, adipocyte, hepatocyte, pancreatic islet cell, keratinocyte, melanocyte, dendritic cell. ​

[0021] In one embodiment of the present application, the engineered cell is an engineered mesenchymal stem cell.

[0022] In one embodiment of the present application, the individual engineered mesenchymal stem cell expresses at least 1.9 million CD47 molecules.

[0023] In one preferred embodiment of the present application, the individual engineered mesenchymal stem cell expresses at least 2 million, 2.1 million, 2.2 million, 2.3 million, 2.4 million, 2.5 million, 2.6 million, 2.7 million, 2.8 million, 2.9 million, 3 million CD47 molecules.

[0024] In one embodiment of the present application, the engineered mesenchymal stem cell has lower immunogenicity or higher immune tolerance compared to wild-type mesenchymal stem cells or mesenchymal stem cells with less than 1.9 million CD47 molecules distributed on the surface of individual cells.

[0025] In one embodiment of the present application, the engineered mesenchymal stem cell has a longer in vivo survival time compared to wild-type mesenchymal stem cells or mesenchymal stem cells with less than 1.9 million CD47 molecules distributed on the surface of individual cells.

[0026] In one embodiment of the present application, the CD47 molecules are selected from modified or unmodified natural CD47 molecules or variants thereof. The natural CD47 molecules include but are not limited to different CD47 subtypes that exist in nature. The variants of CD47 molecules perform the same immune tolerance function as the natural CD47 molecules, including but not limited to different forms of CD47 molecule truncations, fusion proteins that retain the CD47 immune tolerance function, or CD47 mutants that insert, replace, or delete one or more amino acids in the amino acid sequence of the natural CD47 molecules. The modifications of CD47 molecules include but are not limited to glycosylation modification, glutamine cyclization modification, phosphorylation, ubiquitination, other post-translational modifications, etc.

[0027] In one embodiment of the present application, the expression of HLA class I molecules and / or HLA class II molecules in the engineered cell is regulated. In one embodiment of the present application, the regulated expression is no expression or reduced expression.

[0028] In one embodiment of the present application, the genes associated with HLA class I molecules and / or HLA class II molecules in the engineered cell include, but are not limited to, HLA-A, HLA-B, HLA-C, B2M, HLA-DP, HLA-DQ, HLA-DR, TAP1, TAP2, LMP2, LMP7, RFX5, CIITA, RFXANK, RFXAP, NLRC5, HDACs, PSMB, HLA-DM, HLA-DO, HLA-DRA, SUGT1, FoxO1, and the like. Preferably, the genes associated with HLA class I molecules and / or HLA class II molecules in the engineered cell include B2M and CIITA.

[0029] In one embodiment of the present application, the expression of CD54 and / or CD58 in the engineered cell is modulated. In one embodiment of the present application, the modulated expression is no expression or reduced expression.

[0030] In one embodiment of the present application, the nucleic acid encoding the CD47 molecule is site-integrated into one or more gene loci in the genome of the engineered cell.

[0031] In one embodiment of the present application, the gene loci are selected from one or more of AAVS1, CCR5, HTRP, H11, GAPDH, TCR, ROSA26, RUNX1, a gene associated with HLA class I molecules, a gene associated with HLA class II molecules, or an rDNA region; more preferably, the gene loci are selected from one or more of a gene associated with HLA class I molecules, AAVS1, a gene associated with HLA class II molecules, or an rDNA region; further preferably, the gene loci are a plurality of a gene associated with HLA class I molecules, AAVS1, a gene associated with HLA class II molecules, or an rDNA region; more further preferably, the gene associated with HLA class I molecules and / or the gene associated with HLA class II molecules; most preferably, the gene loci are B2M and / or CIITA.

[0032] In one embodiment of the present application, the engineered cell is autologous or allogeneic to the individual.

[0033] In a second aspect of the present application, a method for preparing the engineered cell is provided, comprising introducing the nucleic acid encoding the CD47 molecule into a cell, thereby obtaining the engineered cell.

[0034] In one embodiment of the present application, the nucleic acid encoding the CD47 molecule is site-integrated into one or more gene loci in the genome of the cell, and the engineered cell is obtained by screening the expression amount of CD47 on the surface of a monoclonal cell.

[0035] In an embodiment of the present application, the genetic locus is selected from one or more of AAVS1, CCR5, HTRP, H11, GAPDH, TCR, ROSA26, RUNX1, a gene associated with HLA class I molecule, a gene associated with HLA class II molecule, or a rDNA region; more preferably the genetic locus is selected from one or more of a gene associated with HLA class I molecule, AAVS1, a gene associated with HLA class II molecule, or a rDNA region; further preferably the genetic locus is a plurality of a gene associated with HLA class I molecule, AAVS1, a gene associated with HLA class II molecule, or a rDNA region; more preferably a gene associated with HLA class I molecule and / or a gene associated with HLA class II molecule; most preferably the genetic locus is B2M and / or CIITA.

[0036] In an embodiment of the present application, the one or more genes in the cell are knocked out, including a gene associated with HLA class I molecule, a gene associated with HLA class II molecule, CD54 gene, and CD58 gene.

[0037] In an embodiment of the present application, the gene associated with HLA class I molecule and / or HLA class II molecule includes, but is not limited to, HLA-A, HLA-B, HLA-C, B2M, HLA-DP, HLA-DQ, HLA-DR, TAP1, TAP2, LMP2, LMP7, RFX5, CIITA, RFXANK, RFXAP, NLRC5, HDACs, PSMB, HLA-DM, HLA-DO, HLA-DRA, SUGT1, FoxO1, and the like.

[0038] In an embodiment of the present application, the introduction of the nucleic acid encoding the CD47 molecule and the knockout of the gene are performed simultaneously or stepwise in the cell.

[0039] In an embodiment of the present application, the nucleic acid encoding the CD47 molecule is integrated into the genetic locus associated with HLA class I molecule and / or HLA class II molecule.

[0040] In an embodiment of the present application, the nucleic acid encoding the CD47 molecule is integrated into the B2M locus and / or CIITA locus.

[0041] In an embodiment of the present application, a gene editing tool is used to introduce the nucleic acid or knockout the gene.

[0042] In an embodiment of the present application, the gene editing tool comprises a Cre-lox system, Zinc Finger Nucleases (ZFNs), CRISPR-Cas or Transcription Activator-Like Effector Nucleases (TALENs), preferably CRISPR-Cas or TALENs.

[0043] In an embodiment of the present application, the nucleic acid introduction or gene knockout is performed by a non-viral method.

[0044] In a third aspect of the present application, a composition comprising the engineered cell or the cell obtained by the preparation method is provided.

[0045] In a fourth aspect of the present application, the use of the engineered cell or the composition in the preparation of a medicament for treating a disease is provided.

[0046] In an embodiment of the present application, the disease comprises, but is not limited to, a tumor, an infectious disease, an immune rejection, an autoimmune disease, a genetic disease, a neurological disease, a metabolic disease, a fibrotic disease or tissue regeneration.

[0047] In an embodiment of the present application, the tumor includes, but is not limited to, skin cancer, lung cancer, breast cancer, prostate cancer, gastric cancer, colon cancer, rectal cancer, sarcoma derived from muscle, bone, cartilage, fat or connective tissue, leukemia, lymphoma, myeloma, leiomyosarcoma or osteosarcoma, brain cancer, thyroid cancer, ovarian cancer, pancreatic cancer, kidney cancer, etc. The infectious disease includes, but is not limited to, hepatitis B, tuberculosis, AIDS (HIV / AIDS), influenza (influenza), tuberculosis, rabies, hepatitis B, human papilloma virus infection (associated with cervical cancer), malaria, syphilis, mycoplasma infection, diphtheria, tetanus, scarlet fever, hand-foot-and-mouth disease, acute rheumatic fever, gas gangrene, septicemia, cholecystitis, appendicitis, skin abscess, gingivitis, infectious endocarditis, chronic rhinitis, etc. The autoimmune disease includes, but is not limited to, rheumatoid arthritis, systemic lupus erythematosus (SLE), Sjogren's syndrome (SS), multiple sclerosis, type 1 diabetes, thyroid autoimmune disease, psoriasis (psoriasis), etc. The genetic disease includes, but is not limited to, Down syndrome, cystic fibrosis, hereditary muscular dystrophy, phenylketonuria, sickle cell anemia, Huntington's disease, congenital deafness, etc. The neurological disease includes, but is not limited to, Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis (ALS), etc. The metabolic disease includes, but is not limited to, diabetes, gout, Wilson's disease (copper metabolism disorder), phenylketonuria, congenital hypothyroidism, etc. The fibrosis disease includes cirrhosis, idiopathic pulmonary fibrosis, systemic sclerosis (scleroderma), etc. The tissue regeneration includes, but is not limited to, chronic wound healing, bone regeneration after fracture, stem cell therapy, such as repairing myocardial tissue using stem cells, etc.

[0048] In a fifth aspect of the present application, the use of a CD47 molecule in the preparation of a super-low immunogenic cell is provided, wherein the cell surface overexpresses the CD47 molecule; and the distribution density of the CD47 molecule on the surface of the cell is not less than 2700 molecules / μm 2 .

[0049] Compared with the prior art, the present application has the following beneficial effects:

[0050] In one aspect of the present application, a super-low immunogenic engineered cell is prepared. In terms of the density of CD47 molecules on the surface of the cell, the distribution density of CD47 molecules on the surface of the engineered cell is not less than 2700, 2800, 2900, 3000 molecules / μm 2The engineered cell has good immune tolerance to multiple allogeneic immune cells (including but not limited to PBMC cells, T cells, NK cells, etc.), overcoming the problem that the engineered cell with low-level expression of CD47 molecules in the prior art can only exhibit limited immune suppression ability. In particular, the engineered cell in the present application is a mesenchymal stem cell, which is an important cell bank involved in tissue regeneration in the normal tissue damage repair process of the body. The mesenchymal stem cell has a much stronger proliferation capacity than myocardial cells and endothelial cells, and it still maintains the ability of multi-directional differentiation, and has more advantages in the process of damage repair and transdifferentiation colonization. In the preparation process, after monoclonal selection, cells with a surface distribution density of CD47 molecules of not less than 2700, 2800, 2900, 3000 molecules / μm 2 , or mesenchymal stem cells expressing at least 2 million, 2.1 million, 2.2 million, 2.3 million, 2.4 million, 2.5 million, 2.6 million, 2.7 million, 2.8 million, 2.9 million, 3 million CD47 molecules, realize good immune tolerance to multiple allogeneic immune cells, and can better meet the actual application requirements of allogeneic cell therapy process, provide a new universal cell source, and is expected to greatly reduce the treatment cost of allogeneic cells.

[0051] In the present application, only when the expression amount of CD47 exceeds a certain threshold value, the mesenchymal stem cell can exhibit good ultra-low immunogenicity, and can avoid the killing of T cells, PBMC cells and NK cells at the same time, and significantly prolong the in vivo survival time of the transplanted cells. The ultra-low immunogenicity mesenchymal stem cells prepared in the embodiment of the present application are expected to generate universal cell products, and solve the problem of immune rejection. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 The technical flowchart of the embodiment of the present application is described in the following.

[0053] Figure 2 It is a Sanger sequencing diagram for CIITA knockout clone in Example 1.

[0054] Figure 3 It is a flow cytometry detection diagram of HLA-II expression of CIITA knockout clone in Example 1.

[0055] Figure 4 It is a schematic diagram of CD47 expression frame site integration in Example 2.

[0056] Figure 5 It is a PCR identification diagram of CD47 site integration in Example 2.

[0057] Figure 6Figure for analysis of flow cytometry results of surface markers and HLA-ABC of iMSCs in Example 2.

[0058] Figure 7 Figure for killing of iMSCs by T cells in Example 4.

[0059] Figure 8 Figure for killing of iMSCs by PBMC in Example 4.

[0060] Figure 9 Figure for killing of iMSCs by NK cells in Example 4.

[0061] Figure 10 Figure for monitoring survival of iMSCs by in vivo imaging in Example 5. DETAILED DESCRIPTION

[0062] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively used to explain and describe the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application.

[0063] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods, and the same product source is used in different processes or experimental steps. The methods, steps, etc. not specifically described in the examples are carried out by methods, steps known to those skilled in the art, or according to the instructions of the commercially available products used.

[0064] In the examples of the present application, CD47 molecules are highly expressed on the membrane surface of the engineered cells, and the distribution density of CD47 molecules on the surface of the engineered cells is not less than 2700 molecules / μm 2 By highly expressing CD47 molecules, the ultra-low immunogenicity of the engineered cells is achieved, and when the above engineered cells are co-cultured with allogeneic PBMC cells, T cells, NK cells and other cells, good immune tolerance effect can be achieved.

[0065] In the embodiments of the present application, the expression of HLA-I and HLA-II molecules is further reduced by knocking out the B2M and CIITA genes. The HLA-I molecule is composed of a highly polymorphic heavy chain alpha and beta 2-microglobulin (B2M). The beta 2-microglobulin forms a heterodimer with the HLA-I protein and is essential for the expression of cell surface HLA-I molecules. Knockout of the B2M gene can further limit the immune response of cytotoxic CD8+ T cells by depleting HLA-I molecules. And the deletion of the expression of the HLA-II class transactivator CIITA gene can lead to low expression of HLA-II molecules to inhibit the effect of CD4+ T cells. On this basis, the simultaneous high expression of CD47 molecules further inhibits the immune rejection response, and establishes a super-low immunogenicity strategy to overcome the immune rejection problem in allogeneic cell therapy.

[0066] iPSC, also known as iPSCs, Induced pluripotent stem cells or induced pluripotent stem cells, refers to the same meaning in this paper. It refers to a stem cell that is reprogrammed from a somatic cell, such as a differentiated somatic cell, and has higher potential than the somatic cell. The iPS cell can self-renew and differentiate into mature cells such as smooth muscle cells. The induced pluripotent stem cells used in the specific embodiments of the present application are not strictly limited and can be commercially available products or can be prepared by reprogramming using known methods disclosed in the prior art.

[0067] iMSC, also known as iMSCs, is a mesenchymal stem cell differentiated from an induced pluripotent stem cell.

[0068] In this paper, the CD47 protein, also known as the CD47 molecule, is selected from a modified or unmodified natural CD47 molecule or a variant thereof. Among them, the natural CD47 molecule includes but is not limited to different CD47 subtypes that naturally exist. The variant of the CD47 molecule performs the same immune tolerance function as the natural CD47 molecule, including but not limited to different forms of CD47 molecule truncation, fusion protein retaining CD47 immune tolerance function, or CD47 mutant with one or more amino acids inserted, replaced, or deleted in the amino acid sequence of the natural CD47 molecule.

[0069] The method and timing of knocking out the CIITA gene and B2M gene and knocking in the CD47 cell in some embodiments of the present application are not particularly limited, as long as the CIITA gene and B2M gene are not expressed and the CD47 is safely and effectively expressed, including but not limited to the method of simultaneously or stepwise gene editing.

[0070] The ultra-low immunogenicity of the cells in the present application refers to the ability of the cells to be immune tolerant to multiple allogeneic immune cells, including but not limited to allogeneic PBMC cells, T cells and NK cells, and not to be immune tolerant to a single type of allogeneic immune cells.

[0071] In an embodiment of the present application, the expression of CD47 is preferably carried out by site-specific integration into the genome. The integration mentioned in the embodiments of the present application can be carried out at any safe site in the genome, which can enable safe and effective expression of the CD47 protein. The safe integration gene site in the present application can be one or more of AAVS1, B2M, CCR5, HTRP, H11, GAPDH, TCR, ROSA26, RUNX1, CIITA or rDNA region. The integration has little effect on the normal function of the host cell and can maintain stable transgene expression. Considering the three genomic modification points of CIITA, B2M and CD47, the gene expressing CD47 is further preferably integrated into the B2M site of the genome to achieve the purpose of knocking out B2M and overexpressing CD47, reduce the gene editing operation, reduce the off-target risk, and further improve the safety of the engineered cells.

[0072] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system.

[0073] The term "gene editing" refers to editing (directed modification) of a target gene and its transcription product, achieving addition, deletion, deletion, substitution, insertion, etc. of specific DNA bases, so as to change the sequence, expression amount or function of the target gene or regulatory element.

[0074] The term "stem cell" refers to an undifferentiated cell that can be induced to proliferate. Stem cells can self-maintain, meaning that through each cell division, one daughter cell will also be a stem cell. Stem cells can be obtained from embryonic, fetal, neonatal, juvenile or adult tissues.

[0075] The terms "overexpression", "increased expression" and similar terms refer to the expression of a gene or protein of interest at a higher level than normal compared to a parent or "wild type" cell that does not include the specified genetic modification.

[0076] The gene editing method includes but is not limited to, such as Cre-lox system, Zinc Finger Nucleases (ZFN), CRISPR-Cas or TALEN, preferably CRISPR-Cas or TALEN mediated gene editing.

[0077] The primers involved in the present application are as follows:

[0078] CIITA-F (SEQ ID NO: 4) 5'-TCTTTCCAACACCCTGTGAGGTGA-3' CIITA-R (SEQ ID NO: 5) 5'-TCATCACCTTCCATGTCACACAAC-3' F1 (SEQ ID NO: 6) 5'-TAGAGGGCGCTGGAAGCTCTAA-3' R1 (SEQ ID NO: 7) 5'-GGGAACCACACACGGCACTTAC-3' F2 (SEQ ID NO: 8) 5'-GATCTCCAAGCGCAAGTGTG-3' R2 (SEQ ID NO: 9) 5'-GCAAAGCACATAAAGTCCTTGGCAC-3'

[0079] The present application first synthesizes a CD47 expression vector driven by a broad-spectrum promoter EF1a (the nucleotide sequence of which is shown as SEQ ID NO: 1) by Shengong Bioengineering (Shanghai) Co., Ltd., which is completely matched with the target sequence by Sanger sequencing. CIITA-sgRNA (the nucleotide sequence of which is shown as SEQ ID NO: 2) and B2M-sgRNA (the nucleotide sequence of which is shown as SEQ ID NO: 3) are synthesized by KingsRiver Biotech Co., Ltd. As shown in Figure 1 Figure 1 The technical flowchart of the specific embodiments of the present application is described in the

[0080] Preparation of normal human iPSCs cells with CIITA gene knocked out

[0081] 1. Knocking out CIITA gene of normal human iPSCs using CRISPR / Cas9 technology

[0082] Normal human iPSCs, i.e. hiPSCs (Chinese Academy of Sciences Stem Cell Bank, DYR0100) were selected for nucleic transfection targeting. The CIITA gene was disrupted using CRISPR / Cas9 gene editing tool (the nucleotide sequence of CIITA-sgRNA is shown as SEQ ID NO: 2) to achieve the knockout of HLA-II, thereby inhibiting T helper cell activation and antibody production. Neon transfection kit (Invitrogen, MPK10096) was used for single cell nucleic transfection of iPSCs. The kit was pre-warmed at room temperature for 30 min before nucleic transfection. The specific method is as follows:

[0083] (1) Replace fresh mTeSR Plus medium (STEMCELL, 100-0276) in the SCs culture dish and add 10 μM Y27632 (STEMCELL, 72304).

[0084] (2) Aspirate the culture medium, wash the cells with DPBS (Gibco, C14190500CP) for 2 times, add an appropriate amount of TrypLE Express enzyme (Gibco, 12604021), and digest the cells at 37°C for 3-5 min. When it is observed under a microscope that the cells are rounded and gradually detached, aspirate the TrypLE Express and add 3-5 mL of culture medium.

[0085] (3) Blow up the cells and resuspend them into a single cell suspension, and then centrifuge at 300g at room temperature for 5 min.​

[0086] (4) After the supernatant is discarded, the cells are resuspended by adding DPBS, and the cells are resuspended by adding DPBS at a ratio of 1.8 x 10 6 The number of iPSCs cells per tube is divided into a 15 mL centrifuge tube, and centrifugation is performed again.

[0087] (5) While centrifuging, prepare the nucleic acid transfection solution, add 12 μg of Cas9 protein (Invitrogen, A36498) and 120 pmol of CIITA-sgRNA in a 1.5 mL EP tube, and use Neon Resuspension Buffer R to make up to 20 μL (the Neon Resuspension Buffer R is a component in the Neon transfection kit of Invitrogen), mix well, and stand at room temperature for 5 min.

[0088] (6) Discard the supernatant of the DPBS in the centrifuge tube, resuspend the iPSCs with Neon Resuspension Buffer R, and mix them with the 20 μL of nucleic acid transfection solution in the previous step, and use a Neon suction head to take 100 μL.

[0089] (7) Place the Neon suction head in a pre-added 3 mL Neon Electrolytic Buffer E2 electroporation tube, and perform nucleic acid transfection on the Neon transfection system nucleic acid transfection instrument.

[0090] (8) After nucleic acid transfection is completed, the iPSCs are inoculated into a six-well plate pre-coated with Matrigel (Corning, 354277), and 10 μM Y27632 is added, and after cross shaking, it is placed in a 37°C 5% CO2 saturated humidity incubator and cultured with mTeSR Plus medium.

[0091] (9) After the cells are expanded, single cell inoculation is performed using a Nanocellect WOLF cell sorter, and about 10-12 days iPSCs can grow from a single cell into a large clonal group, which can be subcultured and expanded.

[0092] 2. PCR identification of single cell clones

[0093] (1) Discard the culture medium in the iPSCs cell clone culture dish, wash the cells twice with DPBS. After the DPBS is discarded, the cells are digested at 37°C with TrypLE Express enzyme for 3-5 min, and under a microscope, it is observed that the cells become round and gradually fall off, and the TrypLE Express is discarded.

[0094] (2) Blow the cells down with 1 mL DPBS, and transfer the suspension to a 1.5 mL EP tube, centrifuge at 300g for 5 min at room temperature. After aspirating the DPBS, use the genomic DNA mini extraction kit (Bi Yun Tian, D0063) to extract gDNA.

[0095] (3) Use the extracted iPSCs cell clone gDNA as a template, use primers CIITA-F / CIITA-R for PCR amplification, and perform Sanger sequencing identification.

[0096] Sanger sequencing found that one clone lacked a C base ( Figure 2 ), resulting in a stop codon immediately, which preliminarily indicated that the CIITA gene of the clone had been successfully knocked out.

[0097] 3. Flow cytometry identification of HLA-II expression of CIITA-KO-iMSCs

[0098] (1) Use STEMdiff Mesenchymal Progenitor Kit (STEMCELL, 05240) to differentiate iPSCs into iMSCs according to the method in its instructions.

[0099] (2) Use 100 ng / ml IFN-γ to culture iMSCs for 48h to stimulate the expression of HLA-II molecules. Aspirate the culture medium of iMSCs, wash twice with DPBS, digest the cells with TrypLE Express at room temperature for 1 min, resuspend with an appropriate amount of complete culture medium, then transfer to a 15 mL centrifuge tube and centrifuge at 300g for 5 min.

[0100] (3) After aspirating the supernatant, wash the cells once with DPBS and centrifuge at 300g for 5 min.

[0101] (4) After centrifugation, aspirate the supernatant, take 500,000 cells per tube, add 100 μL of 5% FBS (Gibco, 12664025C) prepared with DPBS to resuspend the cells, then add AF647-HLA-DR, DP, DQ (BD Biosciences, 563591) and mix well, incubate at room temperature for 20 min in the dark.

[0102] (5) After incubation, add 2 volumes of 5% FBS to terminate incubation, centrifuge at 300g for 5 min, discard the supernatant, resuspend the cells with 300 μL of DPBS per tube, and detect by flow cytometry within 1 h.

[0103] Analyze the HLA-DR, HLA-DP, HLA-DQ (HLA-II) flow cytometry data, and the results are as follows Figure 3As shown, WT-iMSCs (WT) that were not genetically edited expressed a certain level of HLA-II molecules, while CIITA-knockout iMSCs (CIITA-KO) did not express HLA-II even under IFN-γ stimulation, thus confirming that knockout of HLA-II was achieved by disrupting the CIITA gene.

[0104] Example 2: Site-specific integration of an expression cassette expressing an exogenous CD47 molecule at the B2M site using CRISPR / Cas9 technology

[0105] In this example, knockout of HLA-I was achieved by disrupting the B2M gene using CRISPR / Cas9 technology, in order to inhibit T cell proliferation and activation. At the same time that the B2M gene was disrupted, an expression cassette expressing an exogenous CD47 molecule was site-specifically integrated into the B2M site by a CD47 expression vector, so that the edited cells overexpressed CD47.

[0106] Based on the cells prepared in Example 1, CIITA-KO-iPSCs were further subjected to B2M knockout and CD47 targeting, using the same experimental methods as above. The nucleofection solution was prepared as follows: 12 μg of Cas9 protein, 120 pmol of B2M-sgRNA, and 6 μg of a CD47 expression vector, and the Neon Resuspension Buffer R was used to make up to 20 μL. After the Cas9 protein cut the B2M gene, the FHA and BHA homologous arms on the CD47 expression vector allowed the CD47 expression cassette to be site-specifically integrated into the B2M site by homologous recombination (as shown in Figure 4 ). Primers F1 / R1 (PCR product 1.1 kb in size) spanning the upstream homologous arm FHA, and primers F2 / R2 (PCR product 1.2 kb in size) spanning the downstream homologous arm BHA were designed. After obtaining monoclonal cells, positive monoclonal cells were identified by PCR, and five CD47 site-specific integration monoclonal cells were obtained (as shown in Figure 5 ), which were sequentially named M2, M3, M4, M5, and M6, respectively. iPSCs cells that had knockout of the CIITA gene and the B2M gene, but had not targeted CD47, were also screened and used as a control group for subsequent differentiation detection, and were named M1. The above monoclonal cells were differentiated into iMSCs, and the resulting iMSC cells were still named using the above numbering.

[0107] 2. Identification of iMSC surface markers and detection of HLA-ABC expression

[0108] The single clone cells of the site-specific integration iPSCs were respectively directionally differentiated into iMSCs, and each iMSC surface marker was identified by flow cytometry, and the experimental method was the same as above. The iMSC surface marker antibodies used were PE-CD105 (Biolegend, 800504), FITC-CD73 (Biolegend, 400114), FITA-CD90 (BD Biosciences, 555595), BV421-CD11b (BD Biosciences, 562632), BV421-CD19 (BD Biosciences, 562440), BV421-CD34 (BD Biosciences, 562577), BV421-CD45 (BD Biosciences, 563879), BV421-HLA-DR (BD Biosciences, 562804), and PE-HLA-ABC (BD Biosciences, 555553) was used to detect the expression of HLA-I molecules.

[0109] Flow cytometry was performed on each iMSC surface marker, and the results of iMSC cell surface marker detection were all positive for CD73, CD90, and CD105 expression, and negative for CD11b, CD19, CD34, CD34, CD45, and HLA-DR expression, which met the identification standard of normal iMSCs Figure 6 ). In addition, except for M0 (WT-iMSC cells without gene editing), the rest of the iMSC cell lines did not express HLA-ABC, proving that the knockout of HLA-I molecules had been achieved by disrupting the B2M gene.

[0110] Example 3 Detection of iMSCs cell surface CD47 molecule expression level

[0111] 1. Flow cytometry quantitative detection of CD47 expression level

[0112] PE quantitative microspheres were used for CD47 quantitative detection, and according to the instructions for using PE quantitative microspheres (BD Biosciences, 340495), the CD47 expression level of iMSCs was detected by flow cytometry, and the flow cytometry operation steps were the same as above. The antibody used was PE-CD47 (BD Biosciences, 556046), and the quantitative data results are shown in Table 1.

[0113] Table 1 CD47 density of different iMSC cell lines

[0114]

[0115]

[0116] 2. iMSC diameter measurement and CD47 density calculation

[0117] iMSCs in suspension were measured for diameter D using a countstar cell counter, and the surface area formula for a sphere S = πD 2 Cell surface area was calculated. Flow cytometry data for CD47 were divided by the corresponding cell surface area to obtain the expression level of CD47 per unit area, i.e. CD47 density (as shown in Table 1). M0 is WT-iMSC, M1 is HLA-I / II double knockout iMSC. M2-M6 are HLA-I / II double knockout iMSCs with site-specific integration of CD47 overexpression, in which M2 and M3 are high expression strains with CD47 expression level exceeding 2 million molecules per cell, and M4, M5 and M6 are low expression strains with CD47 expression level less than 2 million molecules per cell. M0 and M1 have very low levels of endogenous CD47 expression.

[0118] Example 4. Detection of low immunogenicity of cells by cell co-culture killing experiment

[0119] In the embodiments of the present application, various immune cells are used for co-culture killing experiments, such as T cells, PBMC cells and NK cells, which are closer to the actual application environment in allogeneic cell therapy.

[0120] 1. T cell killing detection

[0121] The co-culture medium used in the cell co-culture killing experiment is RPMI 1640 added with 10% FBS and 100 IU / mL IL-2. The killing experiment is performed on an xCELLigence RTCA DP cell analyzer. Before co-culturing T cells, iMSCs are treated with 100 ng / mL IFN-γ for 48 h, and 100 ng / mL IFN-γ is also added to the co-culture medium. The specific method is as follows:

[0122] (1) Prepare iMSC cell suspension

[0123] 1) In a sterile condition in a clean bench, aspirate the old culture solution in the culture dish.

[0124] 2) Wash 1-2 times with DPBS solution, add an appropriate amount of TrypLE Express enzyme, incubate in a 37°C incubator for 1 min, and observe the digestion of cells under an inverted microscope. If the cytoplasm retracts and the cell gap increases, stop the digestion.

[0125] 3) Aspirate the digestion solution, add culture medium to repeatedly blow and stick the cells to form a cell suspension.

[0126] 4) Transfer the cell suspension into 15 mL centrifuge tube, centrifuge at 300g for 5 min, remove supernatant, add fresh medium, and blow the cells evenly. After counting the concentration of cell suspension using countstar, prepare the required cell concentration of 1.5 x 10 5 cells / mL.

[0127] (2) E-Plate 16 preparation

[0128] 1) Add 50 μl medium into the wells of E-Plate 16.

[0129] 2) Place E-Plate 16 on xCELLigence RTCA DP instrument.

[0130] 3) The RTCA system automatically scans (“Scan Plate”) to check if the contact is good (Connection OK is displayed on the “Message” page).

[0131] 4) Start detecting the baseline (Background) to determine if the selected wells are in good contact, and the Cell Index of all wells should be lower than 0.063.

[0132] 5) Take out E-Plate 16, add 100 μl of iMSCs cell suspension mixed evenly into the wells, so that the number of cells in each well is 1.5 x 10 4 .

[0133] 6) Place E-Plate 16 in a clean bench at room temperature for 30 min.

[0134] 7) Place E-Plate 16 on xCELLigence RTCA DP instrument in the incubator.

[0135] 8) Start Step 2, detect cell index every 10 min (detect cell proliferation curve).

[0136] (3) Add immune cells

[0137] 1) Prepare the appropriate concentration of effector cells (T cells). The effector-to-target ratio of T cells is 5:1.

[0138] 2) Terminate Step 2, take out E-Plate 16, and place it in a clean bench.

[0139] 3) Add 50 μl of effector cell (T cell) suspension to the experimental group.

[0140] 4) Place E-Plate 16 on xCELLigence RTCA DP instrument.

[0141] 5) Start Step 3, observe the effect of effector cells (T cells) on the growth of target cells (iMSCs cells).

[0142] The killing of cells by co-culturing iMSCs with T cells was detected, and the experimental results showed that the killing of T cells on M1-M6 was significantly weaker than that on M0 (WT-iMSC) ( Figure 7 ), indicating that HLA-I / II double knockout can effectively reduce the killing of T cells. At the same time, M1 cells showed that iMSCs with very low endogenous expression of CD47 could not resist the killing of T cells for a long time, and showed a gradual small downward trend with the extension of time.

[0143] 2. PBMC killing detection

[0144] iMSCs were co-cultured with PBMCs to detect cell killing, and the experimental method was the same as above. The effector-target ratio of PBMC co-culture was 1:1. PBMC is a single-nucleated cell in peripheral blood, which contains a variety of different immune cells, and can reflect the overall killing effect of peripheral immune cells. The experimental results showed that PBMC had strong killing effect on M0 and M1, and the survival of CD47 high expression strains (M2 and M3) was the best ( Figure 8 ). Therefore, when the expression of CD47 molecules on the surface of iMSCs cells is more than 2 million, iMSCs cells can continue to resist the killing of PBMC, and have low immunogenicity. When the expression of CD47 molecules is less than 2 million, iMSCs cells are gradually killed by PBMC, and do not have low immunogenicity. This also proves that iMSCs cells with low expression of CD47 molecules (insufficient CD47 distribution density on the surface of iMSCs cell membrane) cannot provide sufficient immune tolerance when facing the killing of PBMC.

[0145] 3. NK cell killing detection

[0146] iMSCs were co-cultured with NK cells to detect cell killing, and the experimental method was the same as above. The effector-target ratio of NK cell co-culture was 1:5. As Figure 9 , the experimental results showed that NK cells had the strongest killing effect on M1 (HLA-I / II double knockout); the killing effect on CD47 low expression strains iMSCs cells (M4, M5 and M6) was also strong; and the survival of CD47 high expression strains (M2 and M3) and M0 (WT-iMSC) was similar, and the survival was good. This indicates that in iMSCs, the knockout of HLA molecules can lead to the activation of NK cells and produce killing, and low-level overexpression of CD47 cannot effectively rescue this killing, while high-level overexpression of CD47 (more than 2 million molecules per cell) can effectively avoid the activation of NK cells caused by HLA molecule knockout, so that iMSCs obtain low immunogenicity.

[0147] In this embodiment, by co-culturing T cells, PBMCs, and NK cells with iMSC cell strains of different genotypes and different CD47 expression levels in vitro, the experimental results show that iMSCs with high-level CD47 expression (more than 2 million CD47 molecules per cell) can effectively avoid the activation of NK cells caused by HLA molecule knockout, and can simultaneously escape the killing of T cells, PBMCs, and NK cells. Low-level CD47 expression is not enough to save the killing caused by NK cells. iMSCs with HLA-I / II double knockout and high-level CD47 molecule expression are a kind of ultra-low immunogenicity cells that can escape immune killing.

[0148] Example 5 Monitoring cell survival by transplanting iMSCs into humanized mice

[0149] After M0, M2, and M4 iMSCs were transduced with luciferase using lentivirus, they were transplanted into HSC-NCG-hIL15 humanized model mice (provided by Jiangsu Jizu Yekang Biotechnology Co., Ltd.). Each mouse was transplanted with 1.5 x 10 6 The cells were resuspended with 140 μL DPBS and mixed with 70 μL Matrigel glue, and then injected into the subcutaneous position of the hind limbs of the mice.

[0150] Live imaging was performed, and the experimental results are shown in Figure 10 At D3 (third day) and D6 (sixth day), M2 and M4 with HLA-I / II double knockout and CD47 overexpression showed stronger cell signals than M0 (wild-type iMSC); at D11 (eleventh day), only the M2 group (HLA-I / II double knockout and CD47 high-expression strain) showed cell signal survival. The experimental results show that iMSCs with HLA-I / II double knockout and CD47 high expression can significantly prolong cell survival time, and are a kind of ultra-low immunogenicity cells that can escape immune killing, while iMSCs with HLA-I / II double knockout and low CD47 expression (less than 2 million molecules per cell) cannot achieve long-term cell survival in mice.

[0151] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. An engineered cell, characterized in that, The engineered cells overexpress CD47 molecules relative to wild-type cells; The distribution density of the CD47 molecules on the surface of the engineered cells is not less than 2700 molecules per pm in terms of the density of the CD47 molecules on the surface of the cells 2 .

2. The engineered cell of claim 1, wherein, In terms of the density of CD47 molecules on the surface of the engineered cells, the surface distribution density of the CD47 molecules on the surface of the engineered cells is not less than 2800, or not less than 2900, or not less than 3000 molecules per pm 2 .

3. The engineered cell of claim 1, wherein the engineered cell is a T cell. less than 2700 per pm relative to wild-type cells or CD47 molecules surface distribution density 2 The engineered cells have lower immunogenicity or higher immunological tolerance.

4. The engineered cell of claim 1, wherein the engineered cell is a T cell. The engineered cells are immune-tolerant to a plurality of immune cells.

5. The engineered cell of claim 4, wherein the cell is a T cell. The immune cells include, but are not limited to, one or several of the following: PBMC cells, T cells, NK cells, macrophages, neutrophils, eosinophils, mast cells, dendritic cells.

6. The engineered cell of claim 5, wherein, The immune cells include one or several of the following: PBMC cells, T cells, NK cells.

7. The engineered cell of claim 6, wherein the cell is a T cell. The immune cells include all of the following: T cells, PBMC cells, and NK cells.

8. The engineered cell of claim 4, wherein, The immune cells are autologous or allogeneic cells to the individual.

9. The engineered cell of claim 1, wherein, The engineered cells include engineered stem cells.

10. The engineered cell of claim 9, wherein the cell is a T cell. The engineered stem cells include engineered mesenchymal stem cells, engineered induced pluripotent stem cells, and their derivative cells.

11. The engineered cell of claim 10, wherein the cell is a T cell. The engineered mesenchymal stem cells are derived from pluripotent stem cells.

12. The engineered cell of claim 11, wherein, The pluripotent stem cells are selected from induced pluripotent stem cells (iPSCs).

13. The engineered cell of claim 10, wherein the cell is a T cell. The engineered mesenchymal stem cells are derived from bone marrow, fat, muscle, heart, umbilical cord blood, or umbilical cord.

14. The engineered cell of claim 10, wherein the cell is a T cell. The derivative cells include CAR-iNKs, dopaminergic neural precursor cells, CAR-iMac, cardiomyocytes, endothelial progenitor cells, iNK cells, retinal cells, neural cells, osteoblasts, hematopoietic stem cells, blood cells, B cells, T cells, fibroblasts, hair cells, monocytes, macrophages, Treg cells, kidney progenitor cells, lung epithelial cells, endothelial cells, megakaryocytes, smooth muscle cells, skeletal muscle cells, chondrocytes, bone cells, adipocytes, hepatocytes, pancreatic islet cells, keratinocytes, melanocytes, dendritic cells.

15. The engineered cell of claim 10, wherein the cell is a T cell. The engineered cells are engineered mesenchymal stem cells, and each of the engineered mesenchymal stem cells expresses at least 1.9 million CD47 molecules.

16. The engineered cell of claim 15, wherein the cell is a T cell. Each of the engineered mesenchymal stem cells expresses at least 2 million, 2.1 million, 2.2 million, 2.3 million, 2.4 million, 2.5 million, 2.6 million, 2.7 million, 2.8 million, 2.9 million, or 3 million CD47 molecules.

17. The engineered cell of claim 15, wherein the cell is a T cell. The engineered mesenchymal stem cells have lower immunogenicity or higher immune tolerance relative to wild-type mesenchymal stem cells or mesenchymal stem cells with less than 1.9 million CD47 molecules distributed on the surface of each cell.

18. The engineered cell of claim 1, wherein, The CD47 molecules are selected from modified or unmodified natural CD47 molecules or variants thereof.

19. The engineered cell of claim 18, wherein, The natural CD47 molecules include, but are not limited to, different CD47 subtypes that naturally exist.

20. The engineered cell of claim 18, wherein the cell is a T cell. The variants of CD47 molecules perform the same immune-tolerant functions as natural CD47 molecules, including but not limited to different forms of CD47 molecule truncations, fusion proteins that retain CD47 immune-tolerant functions, or CD47 mutants with one or more amino acids inserted, replaced, or deleted in the amino acid sequence of natural CD47 molecules.

21. The engineered cell of claim 18, wherein the nucleic acid molecule is integrated into the genome of the engineered cell. The modifications of CD47 molecules include, but are not limited to, glycosylation modifications, glutamine cyclization modifications, phosphorylation, ubiquitination, and other post-translational modifications.

22. The engineered cell of claim 1, wherein the cell is a T cell. The expression of HLA class I molecules and / or HLA class II molecules in the engineered cells is modulated, which is no expression or reduced expression.

23. An engineered cell as described in claim 22, characterized in that, The gene associated with HLA-class I molecule and / or HLA-class II molecule in the engineered cell includes but is not limited to HLA-A, HLA-B, HLA-C, B2M, HLA-DP, HLA-DQ, HLA-DR, TAP1, TAP2, LMP2, LMP7, RFX5, CIITA, RFXANK, RFXAP, NLRC5, HDACs, PSMB, HLA-DM, HLA-DO, HLA-DRA, SUGT1, FoxO1.

24. The engineered cell of claim 1, wherein the cell is a T cell. 25 The expression of CD54 and / or CD58 in the engineered cell is modulated, which is no expression or reduced expression.

25. The engineered cell of claim 1, wherein the cell is a T cell. 25 The nucleic acid encoding CD47 molecule is site-integrated into one or more gene sites in the genome of the engineered cell.

26. The engineered cell of claim 25, wherein the cell is a T cell. The gene site is selected from one or more of AAVS1, CCR5, HTRP, H11, GAPDH, TCR, ROSA26, RUNX1, HLA-class I molecule associated gene, HLA-class II molecule associated gene or rDNA region.

27. The engineered cell of claim 26, wherein the cell is a T cell. The gene site is selected from one or more of HLA-class I molecule associated gene, AAVS1, HLA-class II molecule associated gene or rDNA region.

28. An engineered cell as described in claim 27, characterized in that, The gene site is multiple of HLA-class I molecule associated gene, AAVS1, HLA-class II molecule associated gene or rDNA region.

29. An engineered cell as described in claim 28, characterized in that, The gene site is HLA-class I molecule associated gene and / or HLA-class II molecule associated gene.

30. The engineered cell of claim 29, wherein the nucleic acid molecule is integrated into the genome of the engineered cell. 30 The gene site is B2M and / or CIITA.

31. The engineered cell of claim 1, wherein the cell is a T cell. 30 The engineered cell is autologous or allogeneic to the individual.

32. A method of making an engineered cell of any one of claims 1-31, wherein, The nucleic acid encoding the CD47 molecule is introduced into the cell to obtain the engineered cell.

33. The method for preparing engineered cells as described in claim 32, characterized in that, The nucleic acid encoding the CD47 molecule is site-integrated into one or more gene sites in the genome of the cell, and the engineered cell is obtained by screening the expression amount of CD47 on the surface of monoclonal cells.

34. The method for preparing engineered cells as described in claim 33, characterized in that, The gene site is selected from one or more of AAVS1, CCR5, HTRP, H11, GAPDH, TCR, ROSA26, RUNX1, HLA-class I molecule associated gene, HLA-class II molecule associated gene or rDNA region.

35. The method for preparing engineered cells as described in claim 34, characterized in that, The gene site is selected from one or more of HLA-class I molecule associated gene, AAVS1, HLA-class II molecule associated gene or rDNA region.

36. The method for preparing engineered cells as described in claim 35, characterized in that, The gene site is multiple of HLA-class I molecule associated gene, AAVS1, HLA-class II molecule associated gene or rDNA region.

37. The method for preparing engineered cells as described in claim 36, characterized in that, The gene site is HLA-class I molecule associated gene and / or HLA-class II molecule associated gene.

38. The method for preparing engineered cells as described in claim 37, characterized in that, The gene site is B2M and / or CIITA.

39. The method for preparing engineered cells as described in claim 32, characterized in that, The knockout of one or more of the following genes in the cell is also included, including HLA-class I molecule associated gene, HLA-class II molecule associated gene, CD54 gene, CD58 gene.

40. The method of making an engineered cell of claim 39, wherein, The introduction of the nucleic acid encoding the CD47 molecule and the knockout of the gene in the cell are carried out simultaneously or step by step.

41. The method of making an engineered cell of claim 40, wherein, integrating a nucleic acid encoding the CD47 molecule into a gene locus associated with HLA class I molecule and / or HLA class II molecule.

42. The method of making an engineered cell of claim 41, wherein, integrating a nucleic acid encoding the CD47 molecule into a B2M locus and / or CIITA locus.

43. The method for preparing engineered cells as described in claim 32, characterized in that, using a gene editing tool to introduce the nucleic acid or knock out the gene.

44. The method for preparing engineered cells as described in claim 43, characterized in that, the gene editing tool includes Cre-lox system, Zinc Finger Nucleases (ZFNs), CRISPR-Cas or Transcription Activator-Like Effector Nucleases (TALENs), preferably CRISPR-Cas or TALENs.

45. The method for preparing engineered cells as described in claim 43, characterized in that, using a non-viral method to introduce the nucleic acid or knock out the gene.

46. A composition comprising, a cell obtained by any of the preparation methods of claims 32-45.

47. Use of the engineered cell of any of claims 1-31 or the composition of claim 46 in the preparation of a medicament for treating a disease. the disease includes but is not limited to tumor, infectious disease, immune rejection, autoimmune disease, genetic disease, neurological disease, metabolic disease.

48. Use of a CD47 molecule in the preparation of a cell with ultra-low immunogenicity or high immunological tolerance, characterized in that, the cell surface overexpresses a CD47 molecule; and the CD47 molecule is distributed on the cell surface at a density of no less than 2700 molecules per pm2 2 .

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