A low immunogenic cell expressing cd300ld and a method of preparing the same

Abstract

The present application provides a kind of low immunogenic cell expressing CD300LD and a preparation method thereof.The low immunogenic pluripotent cell can significantly reduce or escape the recognition and attack of the immune system, especially the attack of T cells, natural killer cells, macrophages and the like, while not changing the stem cell update and differentiation characteristics.

Description

[0001] This application claims priority to Chinese patent application 2023111948810 with the filing date of September 15, 2023. This application incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application belongs to the field of genetic engineering and stem cell technology, and specifically relates to a low immunogenic cell expressing CD300LD and a preparation method thereof. BACKGROUND

[0003] Stem cells are a class of cells with self-renewal ability and differentiation ability to specific functional cells. According to the degree of stem cell characteristics, stem cells are mainly divided into totipotent stem cells, pluripotent stem cells and adult stem cells. Induced pluripotent stem cells (iPSCs) have the potential for unlimited proliferation, self-renewal and differentiation into various types of cells, and have important application prospects in the treatment of cancer, neurological, cardiovascular and other diseases. However, immune incompatibility and immune rejection of transplanted cells hinder the clinical application of transplanted allogeneic functional cells for treatment.

[0004] Human major histocompatibility complex (MHC), also known as human leukocyte antigen (HLA), is the main cause of immune incompatibility. MHC is composed of a series of genes and 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-I class molecules will stimulate the activation of CD8+ T cells and be eliminated. CD4+ helper T cells recognize "non-self" cells of MHC-II genes, thereby causing immune rejection, and III class molecules do not participate in immune activity.

[0005] In recent years, it has been reported that by knocking out B2M, CIITA and other genes, the expression of MHC-I and MHC-II cell surface or itself genes can be deleted, thereby making the cells immune tolerant or escaping T cell / B cell specific immune response, and producing immune compatible low immunogenic pluripotent stem cells.

[0006] CD300LD, also known as CD300D, is a type I membrane protein with a single extracellular IgV domain. The short cytoplasmic tail lacks any known signal motif, but there is a negatively charged residue (glutamic acid) within the transmembrane domain. It can enable transmembrane signal receptor activity and viral receptor activity, and is involved in immune system processes. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application provides a new method for obtaining low immunogenicity by modifying cells, which first adopts a strategy of screening a large number of molecules related to each mechanism of maternal-fetal tolerance and tumor escape, and finally identifies a representative new gene CD300LD through multiple rounds of functional detection. The gene can significantly reduce or escape the recognition and attack of the immune system, especially the attack of natural killer cells, T cells, macrophages and the like. The present application proposes a feasible strategy for realizing the immunological exemption of cells by transforming new genes in an unpredictable field.

[0008] 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 overexpresses the new gene CD300LD identified by the present application in the DKO cell using a lentiviral vector, so that the human pluripotent stem cell obtained can further escape the killing of NK cells on the basis of escaping the attack of T cells. 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.

[0009] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0010] In one aspect, the present application provides a low immunogenic pluripotent stem cell, comprising: reduced endogenous major histocompatibility class I antigen (MHC-I) function compared to a parental pluripotent stem cell; reduced endogenous major histocompatibility class II antigen (MHC-II) function compared to the parental pluripotent stem cell; and reduced sensitivity to killing by NK cells and T cells compared to the parental pluripotent stem cell, wherein the reduced sensitivity to killing by NK cells and T cells is caused by increased expression of CD300LD protein.

[0011] In some embodiments, the MHC-I function is reduced by reducing the activity of an MHC class I protein or an MHC-I transcriptional regulator.

[0012] In one embodiment, the MHC class I protein comprises an HLA-A protein, an HLA-B protein, or an HLA-C protein.

[0013] In one embodiment, the MHC-I transcriptional regulator comprises a B2M protein, a TAP1 protein, a TAP2 protein, a TAP-related glycoprotein, or an NLRC5 protein.

[0014] In one embodiment, the MHC-I function is reduced by reducing the activity of a B2M protein.

[0015] In an embodiment, the B2M protein is a human B2M protein comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence having 90% identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0016] In an embodiment, the MHC-II function is reduced by reducing the activity of an MHC- class II protein or an MHC-II transcriptional regulator.

[0017] In an embodiment, the MHC-class II protein comprises an HLA-DR protein, an HLA-DQ protein, or an HLA-DP protein.

[0018] In an embodiment, the MHC-II transcriptional regulator comprises a CIITA2 protein, an RFXANK protein, an RFX5 protein, or an RFXAP protein.

[0019] In an embodiment, the MHC-II function is reduced by reducing the activity of a CIITA protein.

[0020] In an embodiment, the CIITA protein is a human CIITA protein comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence having 90% identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0021] In some embodiments, the CD300LD protein is a human CD300LD protein comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence having 90% identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0022] In some embodiments, the CD300LD protein is a human CD300LD protein comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence having 90% identity to the amino acid sequence set forth in SEQ ID NO: 4.

[0023] In an embodiment, the hypoimmunogenic pluripotent stem cell comprises one or more alterations that reduce endogenous B2M protein activity, one or more alterations that reduce endogenous CIITA protein activity, and one or more alterations that result in increased CD300LD protein expression in the hypoimmunogenic pluripotent stem cell.

[0024] In an embodiment, the hypoimmunogenic pluripotent stem cell further comprises one or more alterations that increase expression of a polypeptide selected from the group consisting of: DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9.

[0025] In an embodiment, the hypoimmunogenic pluripotent stem cell comprises: one or more alterations that inactivate both alleles of an endogenous B2M gene; one or more alterations that inactivate both alleles of an endogenous CIITA gene;

[0026] and one or more alterations that cause increased expression of a CD300LD gene in the hypoimmunogenic pluripotent stem cell.

[0027] In another aspect, the present application also provides a method of producing the above-mentioned hypoimmunogenic pluripotent stem cell of the present application, the method comprising: reducing endogenous major histocompatibility class I antigen (MHC-I) function in the pluripotent stem cell; reducing endogenous major histocompatibility class II antigen (MHC-II) function in the pluripotent stem cell; and increasing expression of a protein that reduces the susceptibility of the pluripotent stem cell to killing by NK cells and T cells, wherein the protein is a CD300LD protein.

[0028] In an embodiment, the method comprises: reducing activity of a B2M protein in the pluripotent stem cell; reducing activity of a CIITA protein in the pluripotent stem cell; and increasing expression of a CD300LD protein in the pluripotent stem cell.

[0029] In an embodiment, the method comprises: eliminating activity of both alleles of a B2M gene in the pluripotent stem cell; eliminating activity of both alleles of a CIITA gene in the pluripotent stem cell; and increasing expression of a CD300LD gene in the pluripotent stem cell.

[0030] In an embodiment, the method further comprises: increasing expression of at least one gene selected from the group consisting of: DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9 in the pluripotent stem cell.

[0031] In an embodiment, the activity of the B2M protein in the pluripotent stem cell and / or the activity of the CIITA protein in the pluripotent stem cell is reduced by a technique selected from the group consisting of:

[0032] introducing a gene expression modifying molecule, clustered regularly interspaced short palindromic repeats (CRISPR) technology, transcription activator-like effector nucleases (TALEN) technology, zinc finger nuclease (ZFN) technology or homologous recombination technology; preferably, the gene expression modifying molecule comprises an siRNA, an shRNA, a microRNA, an antisense RNA, an antisense oligonucleotide ASO or an anti-miRNA oligonucleotide AMO.

[0033] In one embodiment, the activity of the B2M protein in the pluripotent stem cell is reduced by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 gene editing technology.

[0034] In one embodiment, the activity of the CIITA protein in the pluripotent stem cell is reduced by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 gene editing technology.

[0035] In one embodiment, the activity of the B2M protein and the CIITA protein in the pluripotent stem cell is reduced by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 gene editing technology.

[0036] In one embodiment, the expression of the CD300LD protein is increased by modification of an endogenous locus.

[0037] In one embodiment, the expression of the CD300LD protein is increased by expression of a transgene.

[0038] In a preferred embodiment, the expression of the CD300LD protein is increased by introducing at least one copy of the CD300LD gene under the control of a promoter into the pluripotent stem cell by a lentiviral vector, wherein the CD300LD gene is constructed into the lentiviral vector by synthesizing a nucleic acid sequence encoding the CD300LD protein.

[0039] In one embodiment, the nucleic acid sequence encoding the CD300LD protein comprises a nucleic acid sequence as set forth in SEQ ID NO: 25 or a nucleic acid sequence having at least 80% identity to the nucleic acid sequence as set forth in SEQ ID NO: 25.

[0040] In one embodiment, the nucleic acid sequence encoding the CD300LD protein comprises a nucleic acid sequence as set forth in SEQ ID NO: 26 or a nucleic acid sequence having at least 80% identity to the nucleic acid sequence as set forth in SEQ ID NO: 26.

[0041] In another aspect, the present application also provides the use of the low immunogenic pluripotent stem cell of the present application or the low immunogenic pluripotent stem cell prepared by the method of the present application in the preparation of a medicament for preventing or treating a disease in need of cell transplantation.

[0042] In one embodiment, the disease comprises acute leukemia, chronic leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, small cell lung cancer, breast cancer, testicular cancer, neuroblastoma, ovarian cancer, melanoma, aplastic anemia, congenital immunodeficiency, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, type I diabetes, Parkinson's disease, Alzheimer's disease, spinal cord injury, retinal degenerative disease, stroke, Huntington's disease, amyotrophic lateral sclerosis, atherosclerosis, hypertension, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, myocarditis, myocardial infarction, heart failure, aortic aneurysm, peripheral arterial disease, type II diabetes, sepsis, hypoglycemia, hyperlipidemia, or osteoporosis.

[0043] In another aspect, the present application provides a composition comprising the hypoimmunogenic cell of the first aspect. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The knock-out strategy of B2M gene and the results of B2M gene knock-out verification by PCR of B2M and CIITA double knock-out cell line (DKO) are shown.

[0045] Figure 2 The knock-out strategy of CIITA gene and the results of CIITA gene knock-out verification by PCR of B2M and CIITA double knock-out cell line (DKO) are shown.

[0046] Figure 3 The results of qPCR detecting the expression of B2M and CIITA at RNA level of B2M / CIITA double allelic knock-out clone H1 cell DKO are shown.

[0047] Figure 4 The results of Western blot detecting the B2M protein level of B2M / CIITA double allelic knock-out clone H1 cell DKO are shown. Figure 5A The results of FACS detection using IFN-γ stimulation of WT and DKO cells to detect HLA class I / II molecules on the surface of H1 cells are shown.

[0048] Figure 5B The results of FACS detection using IFN-γ stimulation of WT and DKO cells to detect HLA class I / II molecules on the surface of iPSC cells are shown.

[0049] Figure 6 The results of karyotype detection of B2M / CIITA double allelic knock-out clone H1 cell DKO are shown.

[0050] Figure 7A-Figure 7FResults of the detection of the expression of sternness genes in DKO cells.

[0051] Figure 7A Results of the detection of the protein levels of sternness genes POU5F1 and NANOG in H1 cells (WT cells) and H1 -derived DKO cells (DKO cells) by immunofluorescence;

[0052] Figure 7B Results of the detection of the expression of sternness genes POU5F1, NANOG and SOX2 at the RNA level in H1 cells (WT cells) and H1 -derived DKO cells (DKO cells) by RT-qPCR;

[0053] Figure 7C Results of the detection of the expression of surface sternness genes SSEA-4 and Tra1-81 in H1 cells (WT cells) and H1 -derived DKO cells (DKO cells) by flow cytometry;

[0054] Figure 7D Results of the detection of the expression of surface sternness genes SSEA-4, TRA-1-60, TRA-1-81, OCT-4, SOX2 in iPSC-WT and iPSC-DKO cells by flow cytometry, Figure 7E Results of the detection of the protein levels of sternness genes TRA-1-60, TRA-1-81, OCT-4, SOX2 and NANOG in iPSC-WT cells by immunofluorescence;

[0055] Figure 7F Results of the detection of the protein levels of sternness genes TRA-1-60, TRA-1-81, OCT-4, SOX2 and NANOG in iPSC-DKO cells by immunofluorescence.

[0056] Figure 8A and Figure 8B Results of the detection of the immune escape function of WT and DKO cells by RTCA.

[0057] Figure 8A Results of the detection of the killing rate of NK cells and T cells against H1 cells WT and H1 cells-derived DKO cells by RTCA;

[0058] Figure 8B Results of the detection of the killing rate of NK cells and T cells against iPSC-WT and iPSC-DKO cells by RTCA, i.e. the detection of the immune escape function of iPSC-WT and iPSC-DKO cells against NK cells.

[0059] Figure 9 Schematic representation of the structure of the lentiviral vector pGC-EF1 a vector.

[0060] Figure 10A and Figure 10B Results showing verification of CD47 overexpression in H1 cell DKO+CD47 cells. Figure 10A Results of detection of CD47 expression levels in H1 cell DKO+CD47 cell lines by FACS; Figure 10B Results of detection of CD47 expression levels in H1 cell DKO+CD47 cell lines by qPCR.

[0061] Figure 11 Results showing detection of immune escape function of WT cells and derived DKO+CD47 cells of H1 cells by RTCA.

[0062] Figure 12 Results showing detection of protein levels of stemness genes OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81 in constructed iPSC-DKO+CD300LD cells by immunofluorescence.

[0063] Figure 13 Results showing detection of expression levels of surface stemness genes SSEA-4, TRA-1-60, TRA-1-81 and OCT4 in constructed iPSC-DKO+CD300LD cells by flow cytometry.

[0064] Figure 14 Results showing detection of triderm differentiation ability of constructed iPSC-DKO+CD300LD cells by immunofluorescence.

[0065] Figure 15 Results showing NK cell killing experiment of iPSC-DKO+CD300LD cells detected by RTCA, wherein B is a multiple killing statistical chart, N=6.

[0066] Figure 16 Results showing PBMC killing experiment of iPSC-DKO+CD300LD cells detected by RTCA, wherein B is a multiple killing statistical chart, N=4.

[0067] Figure 17 Results showing macrophage killing experiment of iPSC-DKO+CD300LD cells detected by RTCA, wherein B is a multiple killing statistical chart, N=4. DETAILED DESCRIPTION

[0068] The present application is further illustrated by the following examples, which are not intended to limit the application to the examples described. The experimental methods in the following examples, for which specific conditions are not indicated, are selected according to conventional methods and conditions, or according to the instructions of the commercial products.

[0069] General Definitions and Terminology

[0070] All patents, patent applications, scientific publications, manufacturer's specifications and handbooks, etc., cited herein are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication.

[0071] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. Also, all patents, patent applications, scientific publications, manufacturer's specifications and handbooks, etc., cited herein are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication.

[0072] As used herein, the recitations "comprising", "containing", "including", and "having" are open-ended, and mean including but not limited to the listed elements, steps or components. The recitation "consisting of means excluding any element, step or component not specified. The recitation "consisting essentially of means limited to the specified elements, steps or components, plus an optional presence of other elements, steps or components that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. It is understood that the recitations "consisting essentially of and "consisting of encompass the meaning of the recitation "comprising".

[0073] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The recitation "one" or "at least one" encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0074] The recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0075] Unless specifically stated otherwise, the numerical values or ranges recited herein are approximations. Although the numerical values and ranges recited herein are approximations, the numerical values are provided to give an approximate understanding of the ranges encompassed. The numerical values and ranges recited herein are intended to be approximations.

[0076] Unless otherwise indicated, designations such as 1), 2),..., i), ii),..., a), b),... in the steps of the methods described herein are merely used as examples of differentiation and do not imply that the steps of the methods described are performed in such order.

[0077] The term "pluripotent cell" refers to a cell that is capable of self-renewal and proliferation while maintaining an undifferentiated state and can be induced to differentiate into specialized cell types under appropriate conditions.

[0078] As used herein, the term "pluripotent stem cell" has the potential to differentiate into any of the three germ layers: endoderm (e.g., stomach attachment, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue). The term "pluripotent stem cell" as used herein also includes "induced pluripotent stem cell" or "iPSC," a pluripotent stem cell derived from a non-pluripotent cell. Exemplary human pluripotent stem cell lines include the Hl human pluripotent stem cell line, the H9 human pluripotent stem cell line, and the iPSC human pluripotent stem cell line. Additional exemplary pluripotent stem cell lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection (as described in Cowan CA, et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med. 2004 Mar 25; 350(13): 1353-6.).

[0079] As used herein, the term "totipotent" refers to the ability of a cell to form a complete organism. For example, in mammals, only the zygote and the first cleavage blastomere are totipotent. In an embodiment, the pluripotent stem cells described herein are not totipotent and do not form a complete organism.

[0080] As used herein, the term "hypoinmunogenic cell" refers to a cell that is engineered using gene editing techniques to eliminate immune rejection, achieving hypoinmunogenicity.

[0081] The cells can be from, for example, a human or a non-human mammal. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cows, and non-human primates. In some embodiments, the cells are from an adult human or non-human mammal. In some embodiments, the cells are from a neonatal human, adult human, or non-human mammal.

[0082] As used herein, the term "immune rejection" or "immunological incompatibility" refers to the attack of a foreign cell, tissue or organ by the immune cells of the recipient itself after transplantation into the recipient, thereby failing to ensure its normal physiological function. The human major histocompatibility complex (MHC), also known as human leukocyte antigen (HLA), is the main cause of "immune rejection" or "immunological incompatibility".

[0083] As used herein, the term "subject" or "patient" refers to any animal, such as a domestic animal, a zoo animal, or a human. The "subject" or "patient" can be a mammal, such as a dog, a cat, a bird, a livestock, or a human. Specific examples of "subjects" and "patients" include, but are not limited to, individuals (particularly humans) having a disease or disorder related to the liver, heart, lung, kidney, pancreas, brain, neural tissue, blood, bone, bone marrow, and the like.

[0084] A "hypoinmunogenic pluripotent stem cell" herein refers to a pluripotent stem cell that retains its pluripotent stem cell characteristics and produces a reduced immune rejection response when transplanted into an allogeneic host. In preferred embodiments, the hypoinmunogenic pluripotent stem cell does not produce an immune response. Thus, "hypoinmunogenic" refers to a significantly reduced or eliminated immune response as compared to the immune response of the parental ("WT") stem cell prior to immunomodification. For example, such a hypoinmunogenic cell can be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or greater than 99% less likely to produce an immune rejection relative to a wild-type cell that has not been immunomodified.

[0085] The term "major histocompatibility complex (MHC)" relates to a complex of genes that occurs in all vertebrates. MHC proteins or molecules play a function in the signaling between lymphocytes and antigen-presenting cells in the normal immune response. The human MHC, also known as HLA for human leukocyte antigen, is located on chromosome 6 and includes MHC-I and MHC-II.

[0086] The term "MHC-I" or "MHC class I" relates to major histocompatibility complex class I proteins or genes. Within the human MHC-I region, there are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, CDla, CDlb, and CDlc subregions. MHC class I proteins are present on the surface of almost all cells, including most tumor cells. MHC-I proteins are loaded with antigens, which are usually derived from endogenous proteins or pathogens present within the cell, and then presented to cytotoxic T lymphocytes (CTLs, also known as CD8+ T cells). T cell receptors are able to recognize and bind to peptides complexed with MHC class I molecules. Each cytotoxic T lymphocyte expresses one unique T cell receptor, which is able to bind to a specific MHC / peptide complex. MHC class I molecules primarily mediate the presentation process of endogenous antigens.

[0087] The term "MHC-II" or "MHC class II" relates to major histocompatibility complex class II proteins or genes. MHC II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR. MHC class II proteins are primarily expressed on antigen-presenting cells such as B cells, monocyte macrophages, dendritic cells, etc. MHC class II molecules primarily mediate the presentation process of exogenous antigens, which present exogenous antigenic polypeptide molecules to Th cells (T helper cells), i.e., stimulate CD4+ T cells.

[0088] The term "MHC / peptide complex" relates to a non-covalent complex of the binding domain of a MHC class I or MHC class II molecule and a MHC class I or MHC class II binding peptide.

[0089] "Knockout" herein refers to a process of rendering a particular gene inactive in the host cell in which it resides, resulting in no production of the protein of interest or an inactive form. As understood by those skilled in the art and further described below, this can be accomplished in a variety of different ways, including removal of nucleic acid sequences from the gene, or interruption of the sequence with other sequences, altering the reading frame, or altering the regulatory elements of the nucleic acid. For example, all or part of the coding region of the gene of interest can be removed or replaced with a "nonsense" sequence, all or part of the regulatory sequences (e.g., promoters) can be removed or replaced, translation initiation sequences can be removed or replaced, etc.

[0090] In this paper, the terms “reduction” and “reduction” are generally used to indicate a reduction that is statistically significant. However, to avoid ambiguity, “reduction” or “reduction” includes a reduction of at least 10% compared to a reference level, such as a reduction of at least about 20% or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% reduction (i.e., a level that does not exist compared to the reference sample), or any reduction between 10% and 100%.

[0091] In this article, "knock-in" or "overexpression" refers to the process of adding genetic function to a host cell. This results in an increase in the level of the encoded protein. As those skilled in the art will understand, this can be achieved in several ways, including adding one or more additional copies of a gene to the host cell or altering the regulatory components of an endogenous gene to increase protein expression. This can be achieved by modifying a promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences.

[0092] In this document, the term “increase” is generally used to indicate an increase that is statistically significant; to avoid any ambiguity, the term “increase” means an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% increase or any increase between 10 and 100%, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times or 10 times compared to a reference level, or any increase between 2 and 10 times or greater than 10 times.

[0093] β-2 microglobulin, or β2M, or B2M protein, is a component of MHC-I molecules. B2M protein is expressed in all nucleated cells (except erythrocytes), can non-covalently bind to the α chain of MHC-I molecules, attach to the cell membrane, and can also be released into various tissue fluids.

[0094] CD47 protein, or integrin-associated protein (IAP), is an important self-signal that can inhibit macrophage phagocytosis and induce immune escape by binding to the N-terminus of SIRPα, a ligand signaling regulatory protein on immune cells.

[0095] The MHC-II transactivator protein (CIITA) is a key molecule that regulates MHC-II expression. The body mainly regulates the expression level of the MHC-II gene by controlling the expression of CIITA.

[0096] CD300LD is a tumor immunosuppressive receptor specific to polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). PMN-MDSCs are a type of neutrophils that are pathologically induced and widely present in various tumors. They can suppress the function of effector cells such as T cells and NK cells, promote tumor development, invasion and metastasis, and play a key role in tumor immune regulation.

[0097] As used herein, the term “homogeneous” refers to the genetic similarity or identity between a host organism and a cell transplant, wherein the organism is immunocompatible; for example, it does not produce an immune response.

[0098] As used in this article, the term "allogeneic" refers to the genetic differences between the host organism and the transplanted cell, which generate an immune response.

[0099] As used in this article, the term "B2M- / -" refers to diploid cells having an inactivated B2M gene on both chromosomes.

[0100] As used in this article, the term "CIITA- / -" refers to diploid cells having the inactivated CIITA gene on both chromosomes.

[0101] As used herein, the term "peptide" refers to a polymer containing two or more amino acids covalently linked by peptide bonds. A "protein" may contain one or more polypeptides, wherein the polypeptides interact with each other covalently or non-covalently. Unless otherwise stated, "peptide" and "protein" are used interchangeably.

[0102] In the context of cells, "wild-type" refers to cells found in nature. However, in the context of pluripotent stem cells, as used herein, it also refers to pluripotent stem cells that have not undergone gene-editing procedures to achieve low immunogenicity, such as parental pluripotent stem cells (WT) as described herein. Parental pluripotent stem cells of the H1 cell line are also referred to as WT in this disclosure, and parental pluripotent stem cells of the iPSC cell line are also referred to as iPSC-WT.

[0103] As used herein, the term "% identity" for sequences refers to the percentage of identical nucleotides or amino acids in the best alignment between sequences to be compared. Differences between two sequences can be distributed across local regions (segments) or the entire length of the sequences being compared. Identity between two sequences is typically determined after the best alignment of a segment or "comparison window." Best alignment can be performed manually or with the aid of algorithms known in the art, including but not limited to the local homology algorithms described in Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search methods described in Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percentage similarity between two sequences can be determined using the BLASTN or BLASTP algorithms publicly available on the National Center for Biotechnology Information (NCBI) website.

[0104] The percentage similarity is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100. In some embodiments, a degree of similarity is given for at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the region. In some embodiments, a degree of similarity is given for the entire length of the reference sequence. Sequence similarity can be determined using tools known in the art, preferably using optimal sequence alignment, such as Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, or Gap Extend 0.5.

[0105] In this article, "nucleotide" includes deoxyribonucleotides and ribonucleotides and their derivatives. As used herein, "ribonucleotide" is the building block of ribonucleic acid (RNA), consisting of one base, one pentose sugar, and one phosphate molecule; it refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotide," on the other hand, is the building block of deoxyribonucleic acid (DNA), also consisting of one base, one pentose sugar, and one phosphate molecule; it refers to a nucleotide where the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen, and it is a major chemical component of chromosomes. Nucleotides are usually identified by a single letter representing the bases in them: "A(a)" refers to deoxyadenosine or adenosine containing adenine, "C(c)" refers to deoxycytidine or cytidine containing cytosine, "G(g)" refers to deoxyguanosine or guanosine containing guanine, "U(u)" refers to uridine containing uracil, and "T(t)" refers to deoxythymidine containing thymine.

[0106] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to polymers of deoxyribonucleotides (DNA) or polymers of ribonucleotides (RNA). The terms "polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the sequence of nucleotides in a polynucleotide. Those skilled in the art will understand that the DNA coding strand (sense strand) and its encoded RNA can be considered to have the same nucleotide sequence, with the deoxythymidine nucleotide in the DNA coding strand sequence corresponding to the uridine nucleotide in its encoded RNA sequence.

[0107] As used herein, the term “expression” includes the transcription and / or translation of nucleotide sequences. Therefore, expression can involve the production of transcripts and / or polypeptides. The term “transcription” refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcription). The term “in vitro transcription” refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in a suitable cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). 9In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). Vectors that can be used to produce transcripts are also called “transcription vectors,” which contain the regulatory sequences required for transcription. The term “transcription” encompasses “in vitro transcription.”

[0108] As used in this article, "encoding" refers to the inherent characteristics of a specific nucleotide sequence in a polynucleotide. For example, genes, cDNA, or mRNA can serve as templates to synthesize polymers and macromolecules in other biological processes, as long as a definite nucleotide sequence or a definite amino acid sequence is available. Therefore, a gene encoding a protein means that the gene's mRNA produces a protein in a cell or other biological system through transcription and translation.

[0109] Unless otherwise stated, all methods described herein may be performed in any suitable order.

[0110] pluripotent stem cells

[0111] In one aspect, the present invention provides a low-immunogenic pluripotent stem cell, said low-immunogenic pluripotent stem cell comprising:

[0112] Reduced endogenous major histocompatibility class I (MHC-I) function compared to parental pluripotent stem cells;

[0113] Reduced function of endogenous major histocompatibility class II (MHC-II) antigens compared to parental pluripotent stem cells; and

[0114] Reduced sensitivity to NK and T cell killing compared to parental pluripotent stem cells.

[0115] In this article, parental pluripotent stem cells refer to parental pluripotent stem cells (also referred to as “WT” or “iPSC-WT”) that have not undergone gene editing procedures to achieve low immunogenicity before immunomodulation.

[0116] In a particularly preferred embodiment, the reduced sensitivity to NK cell and T cell killing is caused by increased expression of the CD300LD protein.

[0117] As those skilled in the art will understand, functional reduction can be achieved in a variety of ways, including removing nucleic acid sequences from a gene, interrupting the sequence with other sequences, or altering the regulatory components of the nucleic acid. For example, all or part of the coding region of the target gene can be removed or replaced with a "nonsense" sequence, frameshift mutations can be performed, regulatory sequences such as all or part of a promoter can be removed or replaced, or translation initiation sequences can be deleted or replaced, etc.

[0118] As those skilled in the art will understand, the reduction in MHC I (HLA I when cells are derived from human cells) function in pluripotent stem cells can be measured using techniques known in the art and described below; for example, using FACS techniques with labeled antibodies that bind to HLA complexes; for example, using commercially available HLA-A, HLA-B, and HLA-C antibodies that bind to human major histocompatibility HLA classes I. The reduction in MHC II (HLA II when cells are derived from human cells) function in pluripotent stem cells can be measured using techniques known in the art and described below; for example, using FACS techniques with labeled antibodies that bind to HLA complexes; for example, using commercially available HLA-DQ, HLA-DR, and HLA-DP antibodies that bind to human major histocompatibility HLA classes II.

[0119] In some implementations, the MHC-I function is reduced by decreasing the activity of MHC-I class proteins.

[0120] In one embodiment, the MHC-I class of proteins includes human leukocyte antigen-A (HLA-A) protein, human leukocyte antigen-B (HLA-B) protein, or human leukocyte antigen-C (HLA-C) protein.

[0121] In some implementations, the MHC-I function is reduced by decreasing the activity of MHC-I transcriptional regulators.

[0122] In some preferred embodiments, the transcriptional regulator of MHC-I may be selected from one or more of the following: β2 microglobulin (B2M), antigen processing-associated transporter 1 (TAP1), antigen processing-associated transporter 2 (TAP2), antigen processing-associated transporter (TAP)-associated glycoprotein (Tapasin), or NOD-like receptor family caspase recruitment domain 5 (NLRC5).

[0123] In one embodiment, the MHC-I function is reduced by decreasing the activity of the HLA-A protein.

[0124] In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the HLA-A protein.

[0125] In one embodiment, the MHC-I function is reduced by decreasing the activity of HLA-B proteins.

[0126] In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the HLA-B protein.

[0127] In one embodiment, the MHC-I function is reduced by decreasing the activity of the HLA-C protein.

[0128] In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the HLA-C protein.

[0129] In one embodiment, the MHC-I function is reduced by decreasing the activity of the TAP1 protein.

[0130] In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the TAP1 protein.

[0131] In one embodiment, the MHC-I function is reduced by decreasing the activity of the TAP2 protein.

[0132] In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the TAP2 protein.

[0133] In one embodiment, the MHC-I function is reduced by decreasing the activity of the Tapasin protein.

[0134] In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the Tapasin protein.

[0135] In one embodiment, the MHC-I function is reduced by decreasing the activity of the NLRC5 protein.

[0136] In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the NLRC5 protein.

[0137] In a preferred embodiment, the MHC-I function is reduced by decreasing the activity of the B2M protein.

[0138] In one embodiment, the B2M protein is a human B2M protein comprising the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1.

[0139] In one embodiment, the MHC-I function is reduced by knocking out the gene encoding the B2M protein.

[0140] In some embodiments, the MHC-II function is reduced by decreasing the activity of MHC-II proteins.

[0141] In one embodiment, the MHC-II proteins include human leukocyte antigen-DR (HLA-DR) protein, human leukocyte antigen-DQ (HLA-DQ) protein, or human leukocyte antigen-DP (HLA-DP) protein.

[0142] In some embodiments, MHC-II function is reduced by decreasing the activity of MHC-II transcriptional regulators. In some preferred embodiments, the MHC-II transcriptional regulators may be selected from one or more of the following: MHC-II transactivator protein (CIITA), regulatory factor X-associated ankylosing protein (RFXANK), regulatory factor X5 (RFX5), and regulatory factor X-associated protein (RFXAP).

[0143] In one embodiment, the MHC-II function is reduced by decreasing the activity of the HLA-DR protein.

[0144] In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the HLA-DR protein.

[0145] In one embodiment, the MHC-II function is reduced by decreasing the activity of the HLA-DQ protein.

[0146] In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the HLA-DQ protein.

[0147] In one embodiment, the MHC-II function is reduced by decreasing the activity of the HLA-DP protein.

[0148] In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the HLA-DP protein.

[0149] In one embodiment, the MHC-II function is reduced by decreasing the activity of the RFXANK protein.

[0150] In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the RFXANK protein.

[0151] In one embodiment, the MHC-II function is reduced by decreasing the activity of the RFX5 protein.

[0152] In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the RFX5 protein.

[0153] In one embodiment, the MHC-II function is reduced by decreasing the activity of the RFXAP protein.

[0154] In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the RFXAP protein.

[0155] In a preferred embodiment, the MHC-II function is reduced by decreasing the activity of the CIITA protein.

[0156] In one embodiment, the CIITA protein is a human CIITA protein comprising the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:2.

[0157] In one embodiment, the MHC-II function is reduced by knocking out the gene encoding the CIITA protein.

[0158] In a preferred embodiment, gene knockout is performed using CRISPR technology. In some cases, CRISPR technology is used to introduce small deletions / insertions into the coding region of a gene, resulting in the non-functional protein, often as a result of frameshift mutations that lead to the generation of stop codons, resulting in truncated, non-functional proteins.

[0159] Successful reductions in MHC-I (HLA-I when the cells are derived from human cells) and MHC-II (HLA-II when the cells are derived from human cells) function in pluripotent stem cells can be measured using techniques known in the art, such as Western blotting with protein antibodies, FACS, RT-PCR, qPCR, etc.

[0160] In some implementations, the reduced sensitivity to NK and T cell killing is caused by increased expression of the CD300LD protein in pluripotent stem cells. This is accomplished in several ways, including, as those skilled in the art, by using "knock-in" or transgenic techniques. In some cases, the increased CD300LD expression is caused by one or more CD300LD transgenes.

[0161] Therefore, in some embodiments, one or more copies of the CD300LD gene are added to pluripotent stem cells under the control of an inducible or constitutive promoter. In some embodiments, lentiviral constructs are used, as described herein or known in the art. As is known in the art, the CD300LD gene can be integrated into the genome of a host cell under the control of a suitable promoter.

[0162] In one embodiment, the increased CD300LD protein expression is caused by the CD300LD transgene.

[0163] In one embodiment, the CD300LD protein is a human CD300LD protein comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:3.

[0164] In one embodiment, the CD300LD protein is a human CD300LD protein comprising the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4.

[0165] The presence of sufficient CD300LD protein expression can be determined using known techniques, such as those described in the examples, for instance, using Western blotting, ELISA, or FACS. Generally, in this context, "sufficient" means increased expression of the CD300LD protein on the surface of pluripotent stem cells, which silences NK and T cell killing.

[0166] In another aspect, the present invention also provides a low-immunogenic pluripotent stem cell, comprising:

[0167] One or more alterations that reduce the activity of endogenous B2M proteins;

[0168] One or more alterations that reduce the activity of endogenous CIITA proteins; and

[0169] One or more alterations that induce increased expression of CD300LD protein in the said low-immunogenic pluripotent stem cells.

[0170] In one embodiment, the low-immunogenicity pluripotent stem cells comprise:

[0171] One or more alterations that inactivate two alleles of the endogenous B2M gene;

[0172] One or more alterations that inactivate two alleles of the endogenous CIITA gene; and

[0173] One or more alterations that induce increased expression of the CD300LD gene in the said low-immunogenic pluripotent stem cells.

[0174] In one embodiment, the low immunogenic pluripotent stem cells further comprise one or more alterations that increase the expression levels of genes selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9.

[0175] As used herein, the term “alteration” or “genetic alteration” refers to changes that cause changes in cells, such as the pluripotent stem cells described herein, which can be achieved, for example, by modifying the genome or introducing new gene fragments. In this context, modifying the genome refers to modifying nucleic acid sequences within cells or under cell-free conditions to produce modified pluripotent cells and pluripotent stem cells. Exemplary techniques for “alteration” or “genetic alteration” include, but are not limited to, homologous recombination, knock-in, ZFN (zinc finger nucleases), TALEN (transcription activator-like effector nucleases), CRISPR (clustered regularly spaced short palindromic repeats) / Cas9, and other site-specific nuclease techniques. These techniques enable double-strand DNA breaks at desired gene loci. These controlled double-strand breaks promote homologous recombination at specific gene loci. The process focuses on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to the sequences and induce double-strand breaks in the nucleic acid molecules. Double-strand breaks are repaired by error-prone non-homologous end joining (NHEJ) or by homologous recombination (HR). Exemplary techniques for “altering” or “genetically altering” also include the introduction of gene expression modifying molecules, including but not limited to siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotides (ASO), or anti-miRNA oligonucleotides (AMO).

[0176] Those skilled in the art will understand that many different techniques can be used to modify the pluripotent cells and pluripotent stem cells of the present invention to make them less immunogenic.

[0177] Typically, these technologies can be used alone or in combination. For example, CRISPR technology can be used to reduce the expression of active B2M and / or CIITA proteins in engineered cells, and viral technologies (e.g., lentiviruses) can be used to knock in the CD300LD gene. Furthermore, those skilled in the art will understand that these genes can be manipulated using different technologies in different sequences. In some embodiments, one or more alterations included in the low immunogenic pluripotent stem cells of the present invention reduce the function of endogenous major histocompatibility class I (MHC-I) antigens. In some embodiments, one or more alterations included in the low immunogenic pluripotent stem cells of the present invention reduce the function of endogenous major histocompatibility class II (MHC-II) antigens.

[0178] In some embodiments, one or more modifications to the low-immunogenic pluripotent stem cells of the present invention can reduce sensitivity to NK cell and T cell killing.

[0179] In some embodiments, one or more alterations included in the low immunogenic pluripotent stem cells of the present invention can reduce the activity of endogenous B2M protein. In some embodiments, one or more alterations included in the low immunogenic pluripotent stem cells of the present invention can reduce the activity of endogenous CIITA protein. In some embodiments, one or more alterations included in the low immunogenic pluripotent stem cells of the present invention can increase CD300LD protein expression.

[0180] In some embodiments, one or more alterations comprising the low immunogenic pluripotent stem cells of the present invention can inactivate two alleles of the endogenous B2M gene. In some embodiments, one or more alterations comprising the low immunogenic pluripotent stem cells of the present invention can inactivate two alleles of the endogenous CIITA gene. In some embodiments, one or more alterations comprising the low immunogenic pluripotent stem cells of the present invention can increase CD300LD gene expression.

[0181] In some embodiments, one or more alterations comprising the low immunogenic pluripotent stem cells of the present invention can suppress the expression of endogenous B2M protein. In some embodiments, one or more alterations comprising the low immunogenic pluripotent stem cells of the present invention can suppress the expression of endogenous CIITA protein.

[0182] In some embodiments, one or more alterations comprising the low immunogenic pluripotent stem cells of the present invention can interfere with the expression of endogenous B2M protein. In some embodiments, one or more alterations comprising the low immunogenic pluripotent stem cells of the present invention can interfere with the expression of endogenous CIITA protein.

[0183] In some embodiments, one or more alterations included in the low immunogenic pluripotent stem cells of the present invention can reduce the expression of endogenous B2M protein. In some embodiments, one or more alterations included in the low immunogenic pluripotent stem cells of the present invention can reduce the expression of endogenous CIITA protein.

[0184] In some embodiments, one or more alterations in the low immunogenic pluripotent stem cells of the present invention can knock out the endogenous B2M protein. In some embodiments, one or more alterations in the low immunogenic pluripotent stem cells of the present invention can knock out the endogenous CIITA protein.

[0185] In one embodiment, the pluripotent stem cells are altered using clustered regularly spaced short palindromic repeats / Cas (“CRISPR”) technology known in the art to reduce the activity of endogenous B2M proteins.

[0186] In one embodiment, the pluripotent stem cells are altered using clustered regularly spaced short palindromic repeats / Cas (“CRISPR”) technology known in the art to reduce the activity of endogenous CIITA protein.

[0187] In one embodiment, the pluripotent stem cells are altered using clustered regularly spaced short palindromic repeats / Cas (“CRISPR”) technology known in the art to inactivate two alleles of the endogenous B2M gene.

[0188] In one embodiment, the pluripotent stem cells are altered using clustered regularly spaced short palindromic repeats / Cas (“CRISPR”) technology known in the art, to inactivate two alleles of the endogenous CIITA gene.

[0189] Assays for determining whether a gene has been inactivated are known and described herein. In one embodiment, the assay involves detecting a protein blot of cell lysate using an antibody against either the B2M or CIITA protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) confirms the presence of inactivation alterations.

[0190] In one embodiment, viral technologies known in the art can be used to induce increased expression of the CD300LD gene in the said low-immunogenic pluripotent stem cells. The viral technologies include, but are not limited to, the use of retroviral vectors, lentiviral vectors, adenoviral vectors, and Sendai viral vectors. In some embodiments, a nucleic acid sequence encoding the CD300LD protein is introduced into a selected site in the cells; the selected site is a safe harbor gene site such as AAVS1 or CCR5. As used herein, a "safe harbor gene site" refers to a site that can be used for safe gene knock-in and ensures the normal and stable expression of the introduced gene.

[0191] In a preferred embodiment, a lentiviral vector is used to induce increased expression of the CD300LD gene in the hypoimmunogenic pluripotent stem cells.

[0192] In one embodiment, the low-immunogenic pluripotent stem cells are human pluripotent stem cells.

[0193] In one embodiment, the B2M protein is a human B2M protein comprising the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1.

[0194] In one embodiment, the CIITA protein is a human CIITA protein comprising the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:2.

[0195] In one embodiment, the CD300LD protein is a human CD300LD protein comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:3.

[0196] In one embodiment, the CD300LD protein is a human CD300LD protein comprising the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4.

[0197] In one embodiment, the low-immunogenic stem cells comprise:

[0198] Reduced endogenous major histocompatibility class I (MHC-I) function compared to parental pluripotent stem cells;

[0199] Compared with parental pluripotent stem cells, it has reduced endogenous major histocompatibility class II (MHC-II) function; and reduced sensitivity to NK cell and T cell killing compared with parental pluripotent stem cells.

[0200] In one embodiment, the T-cell response induced by the low-immunogenic pluripotent stem cells is lower than that induced by parental pluripotent stem cells, which do not contain the alterations that reduce the activity of B2M and CIITA proteins or the alterations that cause increased expression of CD300LD protein. In one embodiment, the T-cell response is measured by real-time label-free dynamic cell analysis (RTCA) to determine the cytotoxicity of T cells against the low-immunogenic pluripotent stem cells or parental pluripotent stem cells.

[0201] In one embodiment, the natural killer (NK) cell response induced by the low-immunogenic pluripotent stem cells is lower than that induced by NK cell responses from B2M / CIITA biallelic knockout cloned DKO cells, which contain the alterations that reduce the activity of B2M and CIITA proteins but do not contain the alterations that cause increased expression of CD300LD protein. In one embodiment, the NK cell response is measured by determining the IFN-γ levels of NK cells incubated in vitro with the low-immunogenic pluripotent stem cells or DKO cells. In one embodiment, the NK cell response is measured by determining the cytotoxicity of NK cells against the low-immunogenic pluripotent stem cells or DKO cells using real-time label-free dynamic cell analysis (RTCA).

[0202] Method for generating low-immunogenic pluripotent stem cells of the present invention

[0203] The present invention also provides a method for generating the low immunogenic pluripotent stem cells of the present invention, the method comprising: reducing the function of endogenous major histocompatibility class I (MHC-I) antigens in the pluripotent stem cells; reducing the function of endogenous major histocompatibility class II (MHC-II) antigens in the pluripotent stem cells; and increasing the expression of a protein that reduces the sensitivity of the pluripotent stem cells to NK cell and T cell killing, wherein the protein is CD300LD protein.

[0204] In some embodiments, the method includes: reducing the activity of B2M protein in the pluripotent stem cells; reducing the activity of CIITA protein in the pluripotent stem cells; and increasing the expression of CD300LD protein in the pluripotent stem cells.

[0205] In one embodiment, the method includes: eliminating the activity of two alleles of the B2M gene in the pluripotent stem cells; eliminating the activity of two alleles of the CIITA gene in the pluripotent stem cells; and increasing the expression of the CD300LD gene in the pluripotent stem cells.

[0206] In some embodiments, the activity of the B2M protein in the pluripotent stem cells can be reduced using the "alteration" or "genetic alteration" techniques described above. In some embodiments, the activity of the CIITA protein in the pluripotent stem cells can be reduced using the "alteration" or "genetic alteration" techniques described above. These techniques include, for example, the introduction of gene expression modification molecules, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, TALEN (Transcription Activator-like Effector Nuclease) technology, ZFN (Zinc Finger Nuclease) technology, or homologous recombination technology. In a preferred embodiment, the gene expression modification molecule comprises siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotides (ASO), or anti-miRNA oligonucleotides (AMO).

[0207] In some embodiments, the CRISPR / Cas system includes a Cas protein or a nucleic acid sequence encoding a Cas protein and at least one or two ribonucleic acids (e.g., gRNAs) capable of guiding the Cas protein to and hybridizing with a target motif of a target polynucleotide sequence. In some embodiments, the CRISPR / Cas system includes a Cas protein or a nucleic acid sequence encoding a Cas protein and a single ribonucleic acid or at least one ribonucleic acid (e.g., gRNA) pair capable of guiding the Cas protein to and hybridizing with a target motif of a target polynucleotide sequence.

[0208] In some embodiments, the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprise conserved amino acid substitutions. In some cases, substitution and / or modification can prevent or reduce protein hydrolytic degradation and / or prolong the half-life of the polypeptide in the cell. In some embodiments, the Cas protein may comprise peptide bond substitutions (e.g., urea, thiourea, carbamate, sulfonylurea, etc.). In some embodiments, the Cas protein may comprise naturally occurring amino acids. In some embodiments, the Cas protein may comprise optional amino acids (e.g., D-amino acids, β-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein may comprise modifications to include portions (e.g., PEGylation, glycosylation, esterification, acetylation, end-capping, etc.).

[0209] In some embodiments, the Cas protein comprises a core Cas protein. Exemplary Cas core proteins include, but are not limited to, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, and Cas9. In some embodiments, the Cas protein comprises an E. coli subtype Cas protein (also known as CASS2). Exemplary E. coli subtype Cas proteins include, but are not limited to, Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, the Cas protein comprises a Ypest subtype Cas protein (also known as CASS3). Exemplary Ypest subtype Cas proteins include, but are not limited to, Csy1, Csy2, Csy3, and Csy4. In some embodiments, the Cas protein comprises an Nmeni subtype Cas protein (also known as CASS4). Exemplary Nmeni subtype Cas proteins include, but are not limited to, Csn1 and Csn2. In some embodiments, the Cas protein comprises a Dvulg subtype Cas protein (also known as CASS1). Exemplary Dvulg subtype Cas proteins include, but are not limited to, Csd1, Csd2, and Cas5d. In some embodiments, the Cas protein comprises a Tneap isotype Cas protein (also known as CASS7). Exemplary Tneap isotype Cas proteins include, but are not limited to, Cst1, Cst2, and Cas5t. In some embodiments, the Cas protein comprises a Hmari isotype Cas protein. Exemplary Hmari isotype Cas proteins include, but are not limited to, Csh1, Csh2, and Cas5h. In some embodiments, the Cas protein comprises an Apern isotype Cas protein (also known as CASS5). Exemplary Apern isotype Cas proteins include, but are not limited to, Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, the Cas protein comprises an Mtube isotype Cas protein (also known as CASS6). Exemplary Mtube isotype Cas proteins include, but are not limited to, Csm1, Csm2, Csm3, Csm435, and Csm5. In some embodiments, the Cas protein comprises a RAMP type Cas protein. Exemplary RAMP type Cas16 proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.

[0210] In some embodiments, the Cas protein is the Cas9 protein of *Streptococcus pyogenes* or a functional portion thereof. In some embodiments, the Cas protein is the Cas9 protein of *Streptococcus aureus* or a functional portion thereof. In some embodiments, the Cas protein is the Cas9 protein of *Streptococcus thermophilus* or a functional portion thereof. In some embodiments, the Cas protein is the Cas9 protein of *Neisseria meningitides* or a functional portion thereof. In some embodiments, the Cas protein is the Cas9 protein of *Treponema denticola* or a functional portion thereof. In some embodiments, the Cas protein is the Cas9 protein or a functional portion thereof from any bacterial species. The Cas9 protein is a member of the type II CRISPR system, which typically includes a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas protein. The Cas9 protein (also known as the CRISPR-associated endonuclease Cas9 / Csn1) is a polypeptide containing 12 amino acids.

[0211] In one embodiment, the activity of the B2M protein in the pluripotent stem cells is reduced by CRISPR / Cas9 gene editing technology.

[0212] In one embodiment, the activity of two alleles of the B2M gene in the pluripotent stem cells is eliminated by CRISPR / Cas9 gene editing technology.

[0213] In one embodiment, the activity of the CIITA protein in the pluripotent stem cells is reduced by CRISPR / Cas9 gene editing technology.

[0214] In one embodiment, the activity of two alleles of the CIITA gene in the pluripotent stem cells is eliminated by CRISPR / Cas9 gene editing technology.

[0215] In one embodiment, the expression of CD300LD protein is increased by modifying an endogenous locus. In some embodiments, the endogenous locus is modified using techniques described above for “alteration” or “genetic alteration.” These techniques include, for example, gene knock-in, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Transcription Activator-like Effector Nuclease), ZFN (Zinc Finger Nuclease), or homologous recombination.

[0216] In one embodiment, the expression of the CD300LD protein is increased by transgenic expression. Transgenic expression techniques known in the art can be used to increase the expression of the CD300LD protein, including but not limited to viral techniques, Piggybac transposon techniques, and Sleeping Beauty transposon techniques.

[0217] In this document, known recombination techniques can be used to generate expression constructs as described herein. In some embodiments, a nucleic acid sequence encoding a target protein may be operatively linked to one or more regulatory nucleotide sequences in the expression construct. Regulatory nucleotide sequences are generally suitable for host cells and subjects to be treated. Various types of suitable expression vectors and suitable regulatory sequences are known in the art for use in a variety of host cells. Typically, one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. The expression constructs used herein may use constitutive or inducible promoters known in the art. Promoters may be naturally occurring promoters or heterozygous promoters combining elements of more than one promoter. The expression construct may be present in a cell on an episome (e.g., a plasmid), or the expression construct may be inserted into a chromosome. In one specific embodiment, the expression vector includes a selection marker gene to allow selection of transformed host cells. Some embodiments include an expression vector containing a nucleotide sequence encoding a target protein operatively linked to at least one regulatory sequence. Regulatory sequences used herein include promoters, enhancers, and other expression control elements. In some embodiments, the expression vector is designed to select the host cell to be transformed, the target protein to be expressed, the copy number of the vector, the ability to control that copy number, or the expression of any other protein encoded by the vector, such as an antibiotic marker. In some embodiments, the promoter is the EF1a promoter.

[0218] Viral technologies can be used to induce increased expression of the CD300LD gene in the said low-immunogenic pluripotent stem cells. These viral technologies include, but are not limited to, the use of retroviral vectors, lentiviral vectors, adenoviral vectors, and Sendai virus vectors.

[0219] In a preferred embodiment, a nucleic acid sequence encoding the CD300LD protein is synthesized and constructed into a lentiviral vector, and then at least one copy of the promoter-controlled CD300LD gene is introduced into the pluripotent stem cells via the lentiviral vector to increase the expression of the CD300LD protein.

[0220] In one embodiment, a nucleic acid sequence encoding the CD300LD protein is introduced into a selected site in the pluripotent stem cell genome. In a preferred embodiment, the selected site is a safe harbor gene site such as AAVS1 or CCR5. As used herein, a "safe harbor gene site" refers to a site that can be used for safe gene knock-in and ensures the normal and stable expression of the transferred gene.

[0221] In one embodiment, the CD300LD protein is the human CD300LD protein.

[0222] In one embodiment, the nucleic acid sequence encoding the CD300LD protein comprises a nucleic acid sequence as shown in SEQ ID NO:25 or a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleic acid sequence as shown in SEQ ID NO:25.

[0223] In one embodiment, the nucleic acid sequence encoding the CD300LD protein comprises a nucleic acid sequence as shown in SEQ ID NO:26 or a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleic acid sequence as shown in SEQ ID NO:26.

[0224] Prevention or treatment

[0225] The present invention further provides the use of the low immunogenic pluripotent stem cells of the present invention or the low immunogenic pluripotent stem cells prepared by the method of the present invention in the preparation of medicaments for the prevention or treatment of diseases requiring cell transplantation.

[0226] The low-immunogenic pluripotent stem cells of the present invention, or those prepared by the methods of the present invention, can be induced to differentiate into different cell types, which can be used for different preventive or therapeutic purposes to prevent or treat different diseases. As those skilled in the art will understand, the differentiation method depends on the desired cell type using known techniques. For example, cells can be differentiated in suspension and then formulated into a gel matrix form, such as matrix gelatin, gelatin, or fibrin / thrombin, to promote cell survival. Differentiation can usually be determined by assessing the presence of cell-specific markers, as is known in the art. For example, cells can be differentiated into 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, or epithelial cells under certain differentiation conditions.

[0227] In some embodiments, the disease is cancer, which includes solid tumors and hematologic malignancies. In some embodiments, the solid tumors include small cell lung cancer, breast cancer, testicular cancer, neuroblastoma, ovarian cancer, or melanoma. In some embodiments, the hematologic malignancies include acute leukemia, chronic leukemia, lymphoma, myelodysplastic syndrome, or multiple myeloma.

[0228] In one implementation, the disease includes aplastic anemia.

[0229] In one implementation, the disease includes congenital immunodeficiency diseases.

[0230] In some implementations, the disease is an autoimmune disease, including systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, or type I diabetes.

[0231] In some implementations, the disease is a neurodegenerative disease, including Parkinson's disease, Alzheimer's disease, spinal cord injury, retinal degeneration, stroke, Huntington's disease, or amyotrophic lateral sclerosis.

[0232] In some implementations, the disease is a cardiovascular disease, including atherosclerosis, hypertension, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, myocardial infarction, heart failure, aortic aneurysm, or peripheral artery disease.

[0233] In some implementations, the disease is a metabolic-related disease, including type II diabetes, scurvy, hypoglycemia, hyperlipidemia, or osteoporosis.

[0234] Differentiation of pluripotent stem cells

[0235] This invention provides pluripotent stem cells that can differentiate into different cell types for subsequent transplantation into recipient subjects. As those skilled in the art will understand, differentiated, low-immunogenic pluripotent cell derivatives can be transplanted using techniques known in the art, depending on the cell type and the end use of these cells.

[0236] 1. Cardiac cells are derived from pluripotent stem cells.

[0237] This invention provides pluripotent stem cells that can differentiate into different types of heart cells for subsequent transplantation or implantation into a subject (e.g., a recipient).

[0238] As those skilled in the art will understand, the differentiation method depends on the desired cell type using known techniques. Exemplary cardiac cell types include, but are not limited to, cardiomyocytes, nodular cardiomyocytes, conduction cardiomyocytes, working cardiomyocytes, cardiomyocyte precursor cells, cardiac stem cells, atrial cardiac stem cells, ventricular cardiac stem cells, epicardial cells, hematopoietic cells, vascular endothelial cells, endocardial endothelial cells, cardiac valve interstitial cells, cardiac pacemaker marker cells, etc.

[0239] In some implementations, cardiomyocyte precursors comprise cells capable of producing progeny including mature (terminal) cardiomyocytes (without dedifferentiation or reprogramming). Cardiomyocyte precursor cells can typically be identified using one or more markers selected from the GATA-4, Nkx2.5, and MEF-2 transcription factor families.

[0240] In some implementations, the cardiomyocytes are low-immunogenic cardiomyocytes.

[0241] In some implementations, the cardiomyocytes described herein are administered to recipient subjects to treat a select group of heart conditions, including: pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, idiopathic cardiomyopathy, other cardiomyopathy, myocardial ischemia-reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, and rheumatoid arthritis. Wet heart disease, arteritis, cardiovascular disease, myocardial infarction, myocardial ischemia, congestive heart failure, myocardial infarction, myocardial ischemia, heart injury, myocardial ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, coronary artery disease, conduction system dysfunction, coronary artery dysfunction, pulmonary hypertension, arrhythmia, muscle dysfunction, muscle mass abnormalities, muscle degeneration, myocarditis, infectious myocarditis, drug- or toxin-induced muscle abnormalities, allergic myocarditis, and autoimmune endocarditis.

[0242] In some embodiments, a method for generating a population of low-immunogenic cardiomyocytes from a population of low-immunogenic pluripotent (HIP) stem cells via in vitro differentiation includes: (a) culturing the HIP cell population in a medium containing a GSK inhibitor; (b) culturing the HIP cell population in a medium containing a WNT antagonist to generate a precardiomyocyte population; and (c) culturing the precardiomyocyte population in a medium containing insulin to generate a low-immunogenic cardiomyocyte population. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration range of the GSK inhibitor is from about 2 mM to about 10 mM. In some embodiments, the WNT antagonist is IWR1, a derivative thereof, or a variant thereof. In some cases, the concentration range of the WNT antagonist is from about 2 mM to about 10 mM.

[0243] Other useful methods for differentiating induced pluripotent stem cells or pluripotent stem cells into cardiomyocytes are described, for example, in US2017 / 0152485; US2017 / 0058263; US2017 / 0002325; US2016 / 0362661; US2016 / 0068814; US9,062,289; US7,897,389; and US7,452,718. Other methods for generating cardiomyocytes from induced pluripotent stem cells or pluripotent stem cells are described, for example, in Xu et al., Stem Cells and Development, 2006, 15(5):631-9; Burridge et al., Cell Stem Cell, 2012, 10:16-28; and Chen et al., Stem Cell Res, 2015, 15(2):365-375.

[0244] In various implementation schemes, low-immunogenic cardiomyocytes can be cultured in a medium containing BMP pathway inhibitors, WNT signaling activators, WNT signaling inhibitors, WNT agonists, WNT antagonists, Src inhibitors, EGFR inhibitors, PCK activators, cytokines, growth factors, cardiomyocytes, and other compounds.

[0245] WNT signaling activators include, but are not limited to, CHIR99021. PCK activators include, but are not limited to, PMA. WNT signaling inhibitors include, but are not limited to, compounds selected from KY02111, SO3031 (KY01-I), SO2031 (KY02-I), SO3042 (KY03-I), and XAV939. Src inhibitors include, but are not limited to, A419259. The EGFR inhibitors include, but are not limited to, AG1478.

[0246] Non-limiting examples of reagents for generating cardiomyocytes from iPSCs include activin A, BMP-4, Wnt3a, VEGF, soluble curly protein, cyclosporine A, angiotensin II, phenylephrine, ascorbic acid, dimethyl sulfoxide, 5-aza-2'-deoxycytidine, etc.

[0247] 2. Nerve cells differentiated from pluripotent stem cells

[0248] This invention provides pluripotent stem cells capable of differentiating into various neural cell types for subsequent transplantation or implantation into recipient subjects. As those skilled in the art will understand, the differentiation method depends on the desired cell type using known techniques. Exemplary neural cell types include, but are not limited to, brain endothelial cells, neurons, glial cells, etc.

[0249] In some implementations, nerve cells are administered to subjects to treat Parkinson's disease, Huntington's disease, multiple sclerosis, other neurodegenerative diseases or conditions, attention deficit hyperactivity disorder (ADHD), Tourette syndrome (TS), schizophrenia, psychosis, depression, and other neuropsychiatric disorders. In some implementations, the nerve cells described herein are administered to subjects to treat or improve stroke.

[0250] In some embodiments, neurons and glial cells are administered to subjects with amyotrophic lateral sclerosis (ALS). In some embodiments, cerebral endothelial cells are administered to alleviate the symptoms or effects of cerebral hemorrhage. In some embodiments, dopaminergic neurons are administered to patients with Parkinson's disease. In some embodiments, norepinephrine neurons and GABAergic interneurons are administered to patients who have experienced seizures. In some embodiments, motor neurons, interneurons, Schwann cells, oligodendrocytes, and microglia are administered to patients who have experienced spinal cord injury.

[0251] In some embodiments, brain endothelial cells (ECs), their precursors, and progenitors are differentiated from surface pluripotent stem cells (e.g., induced pluripotent stem cells) by culturing cells in a medium containing one or more factors that promote the production of brain endothelial cells or nerve cells. In some cases, the medium contains one or more of the following: CHIR-99021, VEGF, basic FGF, and Y-27632. In some embodiments, the medium includes supplements designed to promote the survival and function of nerve cells. In some embodiments, brain endothelial cells (ECs), their precursor cells, and progenitors are differentiated from surface pluripotent stem cells by culturing cells in unconditioned or conditioned medium. In some cases, the medium contains factors or small molecules that promote or facilitate differentiation. In some embodiments, the medium contains one or more factors or small molecules selected from VEGR, FGF, SDF-1, CHIR-99021, Y-27632, SB431542, and any combination thereof. In some embodiments, the surface for differentiation contains one or more extracellular matrix proteins. In some cases, as is known in the art, differentiation is typically determined by assessing the presence of cell-specific markers. In some implementations, brain endothelial cells express or secrete factors selected from CD31, VE cadherin, and combinations thereof.

[0252] In some embodiments, neurons, their precursors, and progenitor cells differentiate into pluripotent stem cells by culturing cells in a culture medium containing one or more factors. The one or more factors are selected from the group consisting of GDNF, BDNF, GM-CSF, B27, basic FGF, basic EGF, NGF, CNTF, SMAD inhibitors, Wnt antagonists, SHH signaling activators, and any combination thereof. In some embodiments, the SMAD inhibitor is selected from SB431542, LDN-193189, NogginPD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, lerdebmumab, metebmumab, GC-I008, AP-12009, AP-11014, LY 550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSoxALK inhibitor), SD-208, SMI6, NPC-30345, K26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, dorsomorphin dihydrochloride and its derivatives.

[0253] In some embodiments, the Wnt antagonist is selected from the group consisting of XAV939, DKK1, DKK-2, DKK-3, Dkk-4, SFRP-1, SFRP-2, SFRP-5, SFRP-3, SFRP-4, WIF-1, Soggy, IWP-2, IWR1, ICG-001, KY0211, Wnt-059, LGK974, IWP-L6, and their derivatives. In some embodiments, the SHH signaling activator is selected from the group consisting of smoothed agonists (SAG), SAG analogs, SHH, C25-SHH, C24-SHH, puromorphamine, Hg-Ag, and their derivatives.

[0254] In some embodiments, the stem cells described herein differentiate into dopaminergic neurons, including dopaminergic progenitor cells. The stem cells are cultured in a differentiation medium containing supplements or additives to induce neuronal differentiation. In some embodiments, cells are cultured in the presence of supplements or additives to induce basal plate cells. In some embodiments, the supplements or additives include the BMP inhibitor LDN193189, the ALK-5 inhibitor A83-01, the smoothed agonist puromorphamine, FGF8, the GSK3 inhibitor CHIR99021, glial cell line-derived neurotrophic factors, GDNF, ascorbic acid, the brain-derived neurotrophic factor BDNF, dibutyryl adenosine monophosphate (dbcAMP), the ROCK inhibitor Y-27632, etc.

[0255] In some embodiments, a method for generating a population of low-immunogenic dopaminergic neurons from a population of low-immunogenic induced pluripotent stem cells (HIP cells) through in vitro differentiation includes (a) culturing the HIP cell population in a first culture medium containing one or more factors selected from the group consisting of sound hedgehog factor (SHH), BDNF, EGF, bFGF, FGF8, WNT1, retinoic acid, a GSK3 inhibitor, an ALK inhibitor, and a ROCK inhibitor to generate a population of immature dopaminergic neurons; and (b) culturing the immature dopaminergic neuron population in a second culture medium different from the first culture medium to generate the dopaminergic neuron population. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration range of the GSK inhibitor is from about 2 mM to about 10 mM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some cases, the concentration range of the ALK inhibitor is from about 1 mM to about 10 mM. In some embodiments, the first and / or second culture media are free of animal serum.

[0256] Methods for differentiating pluripotent stem cells have been described, for example, in Kikuchi et al., Nature, 2017, 548, 592-596; Kriks et al., Nature, 2011, 547-551; Doi et al., Stem Cell Reports, 2014, 2, 337-50; Perrier et al., Proc Natl Acad Sci USA, 2004, 101, 12543-12548; Chambers et al., Nat Biotechnol, 2009, 27, 275-280; Kirkeby et al., Cell Reports, 2012, 1, 703-714. Useful descriptions of stem cell-derived neurons and their preparation methods can be found, for example, Kirkeby et al., Cell Rep, 2012, 1:703-714; Kriks et al., Nature, 2011, 480:547-551; Wang et al., Stem Cell Reports, 2018, 11(1):171-182; Lorenz Studer, Progress in Brain Research, Chapter 8 - Strategies for Introducing Stem Cell-Derived Dopamine Neurons into the Clinical - NYSTEM Trials, 2017, Vol. 230, p. 15. 191-212; Liu et al., Nat Protoc, 2013, 8:1670-1679; Upadhya et al., Curr Protoc StemCell Biol, 38, 2D.7.1-2D.7.47; the contents of U.S. Patent Applications 20160115448 and 8,252,586; 8,273,570; 9,487,752 and 10,093,897 are incorporated herein by reference in their entirety.

[0257] In some implementations, glial cells are generated by differentiating pluripotent stem cells into therapeutically effective glial cells, including microglia, astrocytes, oligodendrocytes, ependymal cells and Schwann cells, glial precursors, and glial progenitors. Differentiation of low-immunogenic pluripotent stem cells yields low-immunogenic neural cells, such as low-immunogenic glial cells.

[0258] In some embodiments, glial cells, precursors, and progenitor cells are generated by culturing pluripotent stem cells in a medium containing one or more reagents selected from retinoic acid, IL-34, M-CSF, FLT3 ligand, GM-CSF, CCL2, TGFβ inhibitors, BMP signaling inhibitors, SHH signaling activators, FGF, platelet-derived growth factor PDGF, PDGFR-α, HGF, IGF-1, hog(SHH), dorsomorphin, noggin, and any combination thereof. In some cases, the BMP signaling inhibitor is LDN193189, SB431542, or a combination thereof.

[0259] In some implementations, glial cells are identified as expressing known glial cell biomarkers. Useful methods for generating glial cells, their precursor cells, and progenitor cells from stem cells can be found, for example, in US7,579,188; US7,595,194; US8,263,402; US8,206,699; US8,252,586; US9,193,951; US9,862,925; US8,227,247; US9,709,553; US2018 / 0187148; US2017 / 0198255; US2017 / 0183627.

[0260] US2017 / 0182097; US2017 / 253856; US2018 / 0236004; WO2017 / 172976; and WO2018 / 093681.

[0261] In some implementations, pluripotent stem cells are differentiated by exposing or exposing them to specific factors known to produce specific cell lineages, so as to target their differentiation to specific, desired lineages and / or cell types.

[0262] In some embodiments, terminally differentiated cells exhibit specific phenotypic properties or characteristics. In some embodiments, the stem cells described herein differentiate into neuroectoderm, neurons, neuroendocrine, dopaminergic, cholinergic, serotonergic (5-HT), glutamatergic, GABAergic, adrenergic, noradrenergic, sympathetic neurons, parasympathetic neurons, sympathetic peripheral neurons, or glial cell populations. In some cases, glial cell populations include microglia or macroglia (central nervous system cells: astrocytes, oligodendrocytes, ependymal cells, and radial glial cells; and peripheral nervous system cells: Schwann cells and satellite cells), or precursors and progenitors of any of the aforementioned cells.

[0263] Protocols for generating different types of nerve cells are described in PCT application WO2010144696, US patents 9,057,053; 9,376,664; and 10,233,422. Additional descriptions of methods for differentiating low-immunogenic pluripotent cells can be found, for example, in Deuse et al., Nature Biotechnology, 2019, 37, 252-258 and Han et al., Proc Natl Acad Sci USA, 2019, 116(21), 10441-10446. Methods for determining the efficacy of neural cell transplantation in animal models of neurological diseases or conditions are described in the following references: For spinal cord injury - Curtis et al., Cell Stem Cell, 2018, 22, 941-950; For Parkinson's disease - Kikuchi et al., Nature, 2017, 548: 592-596; For ALS - Izrael et al., Stem Cell Research, 2018, 9(1): 152 and Izrael et al., Intech Open, DOI: 10.5772 / intechopen.72862; For epilepsy - Upadhya et al., PNAS, 2019, 116(l): 287-296. The efficacy of neural cell transplantation for spinal cord injury can be evaluated in rat models of acute spinal cord injury, such as McDonald et al., Nat. Med., 1999, 5: 1410 and Kim et al., Nature, 2002, 418: 50. For example, a successful transplant may show the presence of transplanted cells at the lesion site 2–5 weeks later, differentiating into astrocytes, oligodendrocytes, and / or neurons, and migrating from the lesion end along the spinal cord, resulting in improved gait, coordination, and weight-bearing. Depending on the type of nerve cells and the specific animal model to be treated, nerve cells can be administered in a manner that allows them to be transplanted to the intended tissue site and to reconstruct or regenerate areas of functional deficiency. For example, depending on the disease being treated, nerve cells can be directly transplanted into the parenchyma or intrathecal site of the central nervous system. In some embodiments, any of the nerve cells described herein, including endothelial cells, neurons, dopaminergic neurons, ependymal cells, astrocytes, microglia, oligodendrocytes, and Schwann cells, are injected into the patient via intravenous, intraspinal, intraventricular, intrathecal, intraarterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, intraperitoneal, intraocular, retrobulbar, or combinations thereof. In some embodiments, cells are injected or deposited in the form of a bolus or continuous infusion. In some implementations, nerve cells are administered by injection into the brain, near the brain, or a combination thereof. For example, injection can be performed by drilling a hole in the subject's skull.Appropriate sites for delivering nerve cells to the brain include, but are not limited to, the ventricles, lateral ventricles, cerebellar cisterns, putamen, basal ganglia, hippocampal cortex, striatum, caudate region of the brain, and combinations thereof.

[0264] Additional descriptions of the neural cells including dopaminergic neurons used in this invention can be found in WO2020 / 018615, the disclosure of which is incorporated herein by reference in its entirety.

[0265] 3. Endothelial cells differentiated from pluripotent stem cells

[0266] This invention provides pluripotent stem cells that can differentiate into various endothelial cell types for subsequent transplantation or implantation into a subject (e.g., a recipient). As those skilled in the art will understand, the differentiation method depends on the desired cell type using known techniques. Exemplary endothelial cell types include, but are not limited to, capillary endothelial cells, vascular endothelial cells, aortic endothelial cells, arterial endothelial cells, venous endothelial cells, renal endothelial cells, brain endothelial cells, liver endothelial cells, etc.

[0267] In some implementations, the endothelial cells described herein are administered to recipient subjects to treat vascular conditions selected from the group consisting of: vascular injury, cardiovascular disease, vascular disease, peripheral vascular disease, ischemic disease, myocardial infarction, congestive heart failure, peripheral vascular occlusive disease, hypertension, ischemic tissue injury, reperfusion injury, limb ischemia, stroke, neuropathy (e.g., peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver failure, kidney failure, etc.), diabetes, rheumatoid arthritis, osteoporosis, cerebrovascular disease, hypertension, angina pectoris and myocardial infarction due to coronary artery disease, renovascular hypertension, renal failure due to renal artery stenosis, lower extremity claudication, and other vascular conditions or diseases.

[0268] In some implementations, low-immunogenic pluripotent cells differentiate into endothelial colony-forming cells (ECFCs) to form new blood vessels to address peripheral artery disease. Techniques for differentiating endothelial cells are known. See, for example, Prasain et al., doi:10.1038 / nbt.3048, the entire contents of which are incorporated herein by reference, and specifically for methods and reagents for generating endothelial cells from human pluripotent stem cells, and also for transplantation techniques. Differentiation can be assessed as is known in the art, typically by assessing the presence of endothelial cell-related or specific markers or by functional measurements.

[0269] In some embodiments, a method for generating a population of low-immunogenic endothelial cells from a population of low-immunogenic pluripotent cells by in vitro differentiation includes: (a) culturing the HIP cell population in a first medium containing a GSK inhibitor; (b) culturing the HIP cell population in a second medium containing VEGF and bFGF to generate a population of preendothelial cells; and (c) culturing the preendothelial cell population in a third medium containing a ROCK inhibitor and an ALK inhibitor to generate a population of low-immunogenic endothelial cells.

[0270] In some embodiments, the GSK inhibitor is CHIR-99021, its derivatives, or variants thereof. In some cases, the concentration range of the GSK inhibitor is from about 1 mM to about 10 mM. In some embodiments, the ROCK inhibitor is Y-27632, its derivatives, or variants thereof. In some cases, the concentration range of the ROCK inhibitor is from about 1 pM to about 20 pM. In some embodiments, the ALK inhibitor is SB-431542, its derivatives, or variants thereof. In some cases, the concentration range of the ALK inhibitor is from about 0.5 pM to about 10 pM.

[0271] In some embodiments, the first culture medium contains 2 pM to about 10 pM of CHIR-99021. In some embodiments, the second culture medium contains 50 ng / ml VEGF and 10 ng / ml bFGF. In other embodiments, the second culture medium also contains Y-27632 and SB-431542. In various embodiments, the third culture medium contains 10 pM Y-27632 and 1 pM SB-431542. In some embodiments, the third culture medium also includes VEGF and bFGF. In certain cases, the first and / or second culture media do not contain insulin.

[0272] In some embodiments, the low-immunogenic endothelial cell population is separated from non-endothelial cells. In some embodiments, the separated low-immunogenic endothelial cell population is amplified prior to administration. In some embodiments, the separated low-immunogenic endothelial cell population is amplified and cryopreserved prior to administration.

[0273] Additional description of the endothelial cells used in this invention can be found in WO2020 / 018615, the disclosure of which is incorporated herein by reference in its entirety.

[0274] 4. Thyroid cells are derived from pluripotent stem cells.

[0275] In some implementations, pluripotent stem cells can differentiate into thyroid progenitor cells and thyroid follicular organoids, which can secrete thyroid hormones to resolve autoimmune thyroiditis. Techniques for differentiating thyroid cells are known in the art. See, for example, Kurmann et al., Cell Stem Cell, 5 November 2015; 17(5):527-42, the entire contents of which are incorporated herein by reference, and particularly for methods and reagents for generating thyroid cells from human pluripotent stem cells and for transplantation techniques. Differentiation can be assessed as is known in the art, typically by assessing the presence of thyroid cell-related or specific markers or by functional measurements.

[0276] 5. Pluripotent stem cells differentiate into hepatocytes

[0277] In some implementations, pluripotent stem cells can differentiate into hepatocytes to address hepatocyte dysfunction or cirrhosis. Various techniques are available for differentiating HIP cells into hepatocytes; see, for example, Pettinato et al., doi:10.1038 / spre32888, Snykers et al., Methods Mol Biol 698:305-314 (2011), Si-Tayeb et al., Hepatology 51:297-305 (2010), and Asgari et al., Stem Cell Rev(:493-504 (2013), all of which are incorporated herein by reference in their entirety and specifically for methods and reagents used for differentiation. As is known in the art, differentiation is typically assessed by evaluating the presence of hepatocyte-related and / or specific markers, including but not limited to albumin, alpha-fetoprotein, and fibrinogen. Differentiation can also be functionally measured, such as ammonia metabolism, LDL storage and uptake, ICG uptake and release, and glycogen storage.

[0278] 6. Pancreatic islet cells differentiated from pluripotent stem cells

[0279] This invention provides pluripotent stem cells capable of differentiating into various islet cell types for subsequent transplantation or implantation into a subject (e.g., a recipient). As those skilled in the art will understand, the differentiation method depends on the desired cell type using known techniques. Exemplary islet cell types include, but are not limited to, islet progenitor cells, immature islet cells, mature islet cells, etc. In some embodiments, the pancreatic cells described herein are administered to a subject to treat diabetes.

[0280] In some embodiments, islet cells are derived from the low-immunogenicity pluripotent cells described herein. Useful methods for differentiating pluripotent stem cells into islet cells are described, for example, in US9,683,215; US9,157,062; and US8,927,280.

[0281] In some implementations, low-immunogenic pluripotent cells are differentiated into β-like cells or pancreatic islet organoids for transplantation to address type 1 diabetes mellitus (T1DM). Cellular systems are a promising approach to address T1DM; see Ellis et al., NatRev Gastroenterol Hepatol. Oct 2017; 14(10):612-628, incorporated herein by reference. Furthermore, Pagbuca et al. (Cell, 2014, 159(2):428-39) reported the successful differentiation of B cells from hiPSCs, the contents of which are incorporated herein by reference in their entirety, particularly the methods and reagents outlined therein for the large-scale production of functional human B cells from human pluripotent stem cells. In addition, Vegas et al. produced human B cells from human pluripotent stem cells and then encapsulated them to avoid host immune rejection; Vegas et al., NatMed, 2016, 22(3):306-11, the entire contents of which are incorporated herein by reference, particularly the methods and reagents outlined therein for the large-scale production of functional human B cells from human pluripotent stem cells.

[0282] In some embodiments, a method for generating a population of low-immunogenic islet cells from a population of low-immunogenic pluripotent cells via in vitro differentiation includes: (a) culturing the HIP cell population in a first medium containing one or more factors selected from insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sound hedgehog (SHH) factors, vascular endothelial growth factor (VEGF), transforming growth factor-b (TORb) superfamily, bone morphogenetic protein-2 (BMP2), bone morphogenetic protein-7 (BMP7), a GSK inhibitor, an ALK inhibitor, a BMP type 1 receptor inhibitor, and retinoic acid to generate an immature islet cell population; and (b) culturing the immature islet cell population in a second medium different from the first medium to generate a low-immunogenic islet cell population. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration range of the GSK inhibitor is from about 2 mM to about 10 mM. In some embodiments, the ALK inhibitor is SB-431542, its derivatives, or variants thereof. In some cases, the concentration range of the ALK inhibitor is from about 1 pM to about 10 pM. In some embodiments, the first and / or second culture media are free of animal serum.

[0283] In some embodiments, the low-immunogenic islet cell population is separated from non-islet cells. In some embodiments, the separated low-immunogenic islet cell population is amplified prior to administration. In some embodiments, the isolated low-immunogenic islet cell population is amplified and cryopreserved prior to administration.

[0284] As is known in the art, differentiation is typically assessed by evaluating the presence of β-cell-related or specific markers, including but not limited to insulin. Differentiation can also be measured functionally, for example, by measuring glucose metabolism, generally see Muraro et al., Cell Syst. 2016 Oct 26; 3(4):385-394.e3, the entire contents of which are hereby incorporated by reference, and particularly for the biomarkers outlined therein. Once β-cells are generated, they can be transplanted (either as a cell suspension or in the gel matrix discussed herein) into the portal vein / liver, omentum, gastrointestinal mucosa, bone marrow, muscle, or subcutaneous sac.

[0285] Additional description of pancreatic islet cells including dopaminergic neurons used in this invention can be found in WO2020 / 018615, the disclosure of which is incorporated herein by reference in its entirety.

[0286] 7. Retinal pigment epithelial (RPE) cells differentiated from pluripotent stem cells

[0287] This invention provides low-immunogenic pluripotent cells that can differentiate into various RPE cell types for subsequent transplantation or implantation into a subject (e.g., a recipient). As those skilled in the art will understand, the differentiation method depends on the desired cell type using known techniques. Exemplary RPE cell types include, but are not limited to, retinal pigment epithelial (RPE) cells, RPE progenitor cells, immature RPE cells, mature RPE cells, functional RPE cells, etc.

[0288] Useful methods for differentiating pluripotent stem cells into RPE cells are described, for example, in US9,458,428 and US9,850,463, which are incorporated herein by reference in their entirety, including the specification. Other methods for generating RPE cells from human induced pluripotent stem cells can be found, for example, Lamba et al., PNAS, 2006, 103(34):12769-12774; Melough et al., Stem Cells, 2012, 30(4):673-686; Idelson et al., Cell Stem Cells, 2009, 5(4):396-408; Rowland et al., Journal of Cellular Physiology, 2012, 227(2):457-466; Buchholz et al., Stem Cells Trans Med, 2013, 2(5):384-393; da Cruz et al., Nat Biotech, 2018, 36:328-337.

[0289] In some implementations, the RPE cells described herein are administered to subjects to treat an ocular condition selected from wet macular degeneration, dry macular degeneration, juvenile macular degeneration (e.g., Staggart's disease, Best's disease, and juvenile retinoschisis), Leber's congenital tarsal membrane disease, retinitis pigmentosa, retinal detachment, age-related macular degeneration (AMD), early AMD, intermediate AMD, late AMD, and non-neovascular age-related macular degeneration.

[0290] Human pluripotent stem cells have been differentiated into RPE cells using techniques outlined in Kamao et al., Stem Cell Reports 2014:2:205-18, which is incorporated herein by reference in its entirety, particularly the methods and reagents used for differentiation techniques and reagents outlined therein; see also Mandai et al., N Engl J Med, 2017, 376:1038-1046, the entire contents of which are incorporated herein by reference for understanding techniques used to generate RPE cell sheets and transplant them into patients. Differentiation can be assessed as is known in the art, typically by assessing the presence of RPE-related and / or specific markers or by functional measurements. See, for example, Kamao et al., Stem Cell Reports, 2014, 2(2):205-18, the entire contents of which are incorporated herein by reference, particularly for the markers outlined in the first paragraph of the results section.

[0291] In some embodiments, a method for generating a population of low-immunogenic retinal pigment epithelial (RPE) cells from a low-immunogenic pluripotent cell population via in vitro differentiation includes: (a) culturing the low-immunogenic pluripotent cell population in a first culture medium containing a subset of activin A, bFGF, BMP4 / 7, DKK1, IGF1, headprotein, BMP inhibitors, ALK inhibitors, ROCK inhibitors, and VEGFR inhibitors to generate a pre-RPE cell population; and (b) culturing the pre-RPE cell population in a second culture medium different from the first culture medium to generate a low-immunogenic RPE cell population. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some cases, the concentration range of the ALK inhibitor is from about 2 mM to about 10 mM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some cases, the concentration range of the ROCK inhibitor is from about 1 pM to about 10 pM. In some embodiments, the first and / or second culture media are free of animal serum.

[0292] Differentiation can be determined as is known in the art, typically by assessing the presence of RPE-related and / or specific markers or by functional measurements. See, for example, Kamao et al., Stem Cell Reports, 2014, 2(2):205-18, the contents of which are incorporated herein by reference in their entirety and specifically for the results section.

[0293] Additional description of the RPE cells used in this invention can be found in WO2020 / 018615, the disclosure of which is incorporated herein by reference in its entirety.

[0294] 8. NK cells differentiated from pluripotent stem cells

[0295] This invention provides low-immunogenic pluripotent cells that can differentiate into natural killer (NK) cells. In some embodiments, a method for generating a natural killer (NK) cell population from a low-immunogenic pluripotent cell population by in vitro differentiation includes: (a) culturing the stem cell population in a first medium containing a ROCK inhibitor under conditions sufficient for aggregate formation; (b) culturing the aggregates in a second medium containing BMP-4; (c) culturing the aggregates in a third medium containing BMP-4, FGF2, a WNT pathway activator, and activin A; and (d) culturing the cells in a third medium containing FGF2, VEGF, TPO, SCF, IL-3, FLT3L, and WNT. (e) The cell population was cultured in a fourth medium containing C-59 and activin / node inhibitors to form a cell population containing hematopoietic stem cells and progenitor cells (HSPCs); (f) The cell population was cultured in a fifth medium containing FGF2, VEGF, TPO, SCF, IL-3, and FLT3L; (g) The cell population was cultured in a sixth medium containing IL-3, IL-7, FLT3L, IL-15, and SCF for a period of time sufficient to generate NK cells.

[0296] In some embodiments, the first culture medium contains 10 μM of ROCK inhibitor. In some embodiments, the second culture medium contains 30 ng / mL BMP-4. In some embodiments, the second culture medium contains 30 ng / mL BMP-4 and 10 μM ROCK inhibitor. In some embodiments, the third culture medium comprises 30 ng / mL BMP-4, 100 ng / mL FGF2, 6 μM CHIR-99021, and 2.5-5 ng / mL Activin A. In some embodiments, the third culture medium comprises 30 ng / mL BMP-4, 100 ng / mL FGF2, 7 μM CHIR-99021, and 2.5-5 ng / mL Activin A. In some embodiments, the fourth and fifth culture media comprise 20 ng / mL FGF, 20 ng / mL VEGF, 20 ng / mL TPO, 100 ng / mL SCF, 40 ng / mL IL-3, and 10-20 ng / mL FLT3L. In some embodiments, the fourth culture medium further comprises 2 μM WNT C-59 and 5 μM SB-431542. In some embodiments, the fourth culture medium further comprises 5 μM SB-431542. In some embodiments, the fourth culture medium does not include WNT C-59. In some embodiments, the sixth and seventh culture media comprise 20 ng / mL IL-7, 10-20 ng / mL FLT3L, 10-20 ng / mL IL-15, and 20 ng / mL SCF. In some embodiments, the sixth culture medium comprises 5 ng / mL IL-3. In some embodiments, the eighth culture medium comprises IL-7, FLT3L, IL-15, SCF, and nicotinamide.

[0297] Additional description of the NK cells used in this invention can be found in US20220169700A1, the disclosure of which is incorporated herein by reference in its entirety.

[0298] For therapeutic applications, cells prepared according to the disclosed methods are generally available as pharmaceutical compositions containing isotonic excipients and prepared under sufficiently sterile conditions for human administration. For general principles of pharmaceutical formulation of cell compositions, see “Cell Therapy: Stem Cell Transplantation, Gene Therapy and Cell Immunotherapy,” edited by Morstyn & Sheridan, Cambridge University Press, 1996; and “Hematopoietic Stem Cell Therapy,” EDBall, J. Lister & P. ​​Law, Churchill Livingstone, 2000. Cells can be packaged in devices or containers suitable for dispensing or clinical use.

[0299] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0300] The reagents and raw materials used in this invention are all commercially available.

[0301] The positive and progressive effects of the present invention are as follows: the pluripotent stem cells of the present invention and the low immunogenicity pluripotent stem cells prepared by the method of the present invention can exhibit excellent effects, such as, but not limited to: (1) having good self-renewal and differentiation capabilities; (2) being able to escape T cell killing; (3) being able to escape NK cell killing; (4) being able to escape PBMC cell killing; (5) being able to escape MAC cell killing; and / or (6) the differentiated cells can also escape NK cell, T cell or MAC cell killing; thus showing excellent application potential.

[0302] The invention is further described with reference to the following embodiments. It should be understood that these embodiments are merely examples and do not constitute a limitation of the invention. All materials and instruments described below are commercially available or prepared according to methods known in the art. All experiments described below were performed according to the manufacturer's instructions or according to methods and procedures known in the art.

[0303] Experimental methods not specifically described in the following examples are generally performed under standard conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0304] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0305] Example 1. Construction of a B2M and CIITA double knockout cell line (DKO)

[0306] 1.1 Construction of a B2M and CIITA double knockout cell line (DKO)

[0307] The following examples use human induced pluripotent stem cells (iPSCs) for constructing the target cell line. The iPSCs used were selected according to the manufacturer's instructions and constructed using CTS. TM CytoTune TM -iPS 2.1 Sendai Virus Reprogramming Kit (CTS) TM CytoTune TMThe iPS2.1 Sendai Reprogramming Kit (catalog number: A34546) is prepared from human peripheral blood cells. Alternatively, human pluripotent stem cell lines H1 (Wicell, WA01) or H9 (Wicell, WA09) can be used to construct the target cell line. The cell culture and gene knockout reagents used are shown in Table 1.

[0308] Table 1. Cell Culture and Gene Knockout Reagents

[0309]

[0310] By knocking out β-2-microglobulin (B2M) in the endoplasmic reticulum using CRISPR / CAS9, the cell surface MHC-I cannot form functional molecules, thus allowing the cell to escape allogeneic CD8. + T cell killing; escape CD4+ T cell killing is achieved by knocking out CIITA, a positive regulator of MHC-II gene transcription, thereby reducing the expression of MHC-II molecules. In this disclosure, the double knockout pluripotent stem cells (DKO) of the H1 cell line are also referred to as DKO cells or H1 cell-derived DKO cells, and the double knockout pluripotent stem cells (DKO) of the iPSC cell line are referred to as iPSC-DKO cells or iPSC cell-derived DKO cells.

[0311] B2M CRISPR / CAS9 gene knockout strategy, including the gRNA sequence used and identification primers, etc. Figure 1 As shown in Table 2, B2M-gRNA1 and B2M-gRNA2 (EasyEdit sgRNA, GenScript) were used to directly knock out both ends of the B2M exon region. Then, the knockout of the genome sequence was verified using two pairs of PCR primers, B2M-F1 / R1 and B2M-F2 / R2, respectively. Figure 1 A represents the B2M gene knockout strategy in the B2M and CIITA double knockout cell line (DKO). Figure 1 The result of B in the figure is the result of PCR verification of the B2M gene knockout in the H1 cell line. Figure 1 C represents the result of PCR verification of B2M gene knockout in iPSC cell lines.

[0312] In addition, CIITA's CRISPR / CAS9 gene knockout strategy, the gRNA sequences used, and the identification primers, such as... Figure 2 As shown in Table 2, CIITA-gRNA1 and CIITA-gRNA2 (EasyEdit sgRNA, GenScript) were used to directly knock out both ends of the CIITA exon region. Then, two pairs of PCR primers, CIITA-F1 / R1 and CIITA-F2 / R2, were used to verify the genome sequence knockout. Figure 2A represents the CIITA gene knockout strategy in the B2M and CIITA double knockout cell line (DKO). Figure 2 B represents the result of PCR verification of CIITA gene knockout in the H1 cell line. Figure 2 C represents the result of PCR verification of the CIITA gene knockout in the iPSC cell line.

[0313] Table 2. gRNA sequences and identification primers

[0314]

[0315]

[0316] The specific steps are as follows:

[0317] 1) Human induced pluripotent stem cells were cultured to 80% confluence in mTeSR1 medium supplemented with Y-27632 in Matrigel-coated 6-well plates as usual. After digestion with TrypLE, DMEM / F12 was added for neutralization, and the cells were counted. 2 × 10⁻⁶ cells were aspirated. 6 Cells were placed in EP tubes, centrifuged, and the supernatant was discarded.

[0318] 2) Based on the 100 μL electroporation system of the Neon transfection system (ThermoFisher), add 15 μg of TrueCut. TM The ribonucleoprotein complex (RNP) system was composed of Cas9Protein and 3 μg gRNA (B2M-gRNA1+B2M-gRNA2+CIITA-gRNA1+CIITA-gRNA2), and after mixing, it was placed at room temperature for 20 min.

[0319] 3) Resuspend the cells in 100 μL of RNP electroporation system and perform electroporation using the Neon transfection system at 1200 V, 30 ms, and 1 pause. Quickly add pre-warmed culture medium to the electroporated cells and then seed them evenly into 6-well plates coated with Matrigel.

[0320] 4) Change the mTeSR1 medium daily with fresh medium. Once single cells have grown, pick a single clone and place it in a 48-well plate. After the clone has amplified, collect genomic samples for PCR detection of gene editing. The PCR results are as follows: Figure 1 and Figure 2 As shown. PCR-positive clones were sent to the company for further Sanger sequencing verification.

[0321] 5) Amplification, culture, cryopreservation and identification of the positive B2M / CIITA biallelic knockout clone DKO.

[0322] 1.2 Detection of RNA expression levels of B2M and CIITA in DKO

[0323] Total RNA was extracted from H1 cells and derived DKO cells using the FastPure Cell / Tissue Total RNA Isolation Kit V2 (Novazia, RC112-01), and then the RNA was reverse-engineered into cDNA using the HiScript III RT SuperMix for qPCR (Novazia, R323-01) according to the manufacturer's instructions.

[0324] The expression of B2M and CIITA at the RNA level in DKO cells derived from B2M / CIITA biallelic knockout clone H1 cells was detected using qPCR. The primers used are shown below.

[0325] B2M-F(SEQ ID NO:19):AAGATGAGTATGCCTGCCGT

[0326] B2M-R(SEQ ID NO:20):ATGCGGCATCTTCAAACCTC

[0327] CIITA-F(SEQ ID NO:21):CCTGGAGCTTCTTAACAGCGA

[0328] CIITA-R(SEQ ID NO:22):TGTGTCGGGTTCTGAGTAGAG

[0329] Using a Roche 480II instrument, the reaction system is as follows:

[0330] Pre-incubation, 95℃, 30s.

[0331] Amplification, 95℃ for 10s, 60℃ for 30s, 40 cycles.

[0332] Melting curve and Cooling are the default programs.

[0333] qPCR results as follows Figure 3 As shown, compared with untreated wild-type human pluripotent stem cells, the expression of B2M and CIITA at the RNA level in the B2M / CIITA biallelic knockout clone DKO was almost non-existent, confirming the successful knockout of B2M and CIITA.

[0334] 1.3 Detection of B2M protein expression levels in DKO

[0335] Western blotting was used to detect the expression level of B2M protein in DKO cells derived from B2M / CIITA biallelic knockout clone H1 cells (B2M antibody catalog number ab75853; internal control antibody GAPDH catalog number ab181602, both purchased from Abcam).

[0336] Western blot results as follows Figure 4 As shown, the B2M protein in H1 cell-derived DKO was significantly reduced compared to wild-type human pluripotent stem cells, confirming the successful knockout of the B2M protein.

[0337] 1.4 Detection of HLA class I / II molecules in DKO

[0338] HLA type I / II molecules were detected on the surface of WT and DKO cells using IFN-γ (PeproTech, Cat#300-02) stimulation. The specific detection method is as follows:

[0339] Cells were plated, and on the second day, when changing the medium, medium containing IFN-γ was added to the cells. After 48 hours of incubation, the cells were digested and the expression of HLA-I / II was detected using flow cytometry (Agilent Technologies, NovoCyte).

[0340] Flow cytometry results as follows Figure 5A and Figure 5B As shown, where Figure 5A The results are for H1 cells and DKO cells. Figure 5B The results for iPSC cells and iPSC-DKO cells are shown. All figures show HLA-ABC on the left, detecting HLA-I molecules; and HLA-DR, DQ, and DP on the right, detecting HLA-II molecules. T cells serve as positive controls. It was observed that DKO cells (DKO cells and iPSC-DKO cells) derived from the B2M / CIITA biallelic knockout clone H1 and iPSC cell lines did not express HLA-I / II molecules in response to IFN-γ stimulation, demonstrating reduced HLA-I and HLA-II function in DKO cells and iPSC-DKO cells.

[0341] 1.5DKO karyotype detection

[0342] Karyotype analysis was performed on the obtained B2M / CIITA biallelic knockout H1 cell positive clones (DKO), and the specific detection method is as follows:

[0343] Chromosome specimens fixed on glass slides were treated with trypsin and then stained with Giemsa stain. Based on characteristics such as chromosome length, centromere position, long and short arm ratio, and presence or absence of satellites, chromosomes in metaphase were analyzed for chromosome number and morphology to determine whether their karyotype was consistent with the normal karyotype.

[0344] Karyotype test results as follows Figure 6 As shown, the karyotype of DKO cells derived from H1 cells was normal, with no significant changes compared to the normal karyotype.

[0345] Example 2. Stem cell line and immune function.

[0346] This embodiment further examines whether the stemness and immune function of pluripotent stem cells change after knocking out the B2M / CIITA biallelic gene.

[0347] 2.1 Expression of stemness genes in DKO cells

[0348] The protein levels of stem gene mutations in WT cells (WT cells and iPSC-WT cells) and DKO cells (DKO cells and iPSC-DKO cells) of iPSC and H1 cell lines were detected by immunofluorescence. The expression of stem gene mutations at the RNA level in H1 cells (WT cells) and DKO cells (DKO cells) was detected by RT-qPCR. The expression of corresponding proteins of stem gene mutations on the surface of H1 cells (WT cells), iPSC-WT cells, and DKO cells (DKO cells and iPSC-DKO cells) was detected by flow cytometry. The specific detection methods are as follows:

[0349] Immunofluorescence assay: WT or DKO cells were seeded in 12-well plates. After the cells reached a density of 60-80%, the culture medium was aspirated, 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 other stem cell proteins such as NANOG. After washing off the primary antibody, the cells were incubated at room temperature with a fluorescently labeled secondary antibody, and then photographed using a fluorescence microscope (Nikon Ts2R-FL).

[0350] RT-qPCR detection:

[0351] Using a Roche 480II instrument, the reaction system is as follows:

[0352] Pre-incubation, 95℃, 30s.

[0353] Amplification, 95℃ for 10s, 60℃ for 30s, 40 cycles.

[0354] Melting curve and Cooling are the default programs.

[0355] Mesenchymal stem cells (MSCs) served as a negative control for stem gene expression.

[0356] Flow cytometry:

[0357] After cell collection, the antibodies were incubated in EP tubes at 4°C in the dark for 30 minutes. Then, the appropriate fluorescence acquisition channel was selected based on the antibody information, and fluorescence was detected by fluorescence detection. All antibodies were from BD Biosciences.

[0358] Figure 7A The results are for detecting the protein levels of stem genes POU5F1 and NANOG in H1 cells (WT cells) and H1-derived DKO cells (DKO cells) by immunofluorescence. Figure 7B The results of RT-qPCR detection of the expression of stem genes POU5F1, NANOG and SOX2 at the RNA level in H1 cells (WT cells) and H1-derived DKO cells (DKO cells); Figure 7C To detect the expression of stemness genes SSEA-4 and Tra1-81 on the surface of H1 cells (WT cells) and H1-derived DKO cells (DKO cells) by flow cytometry, Figure 7D To detect the expression of stemness genes SSEA-4, TRA-1-60, TRA-1-81, OCT-4, and SOX2 on the surface of iPSC-WT and iPSC-DKO cells by flow cytometry, Figure 7E To obtain the results of immunofluorescence detection of the protein levels of stem genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in iPSC-WT cells, Figure 7F The results are based on the detection of protein levels of stem genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in iPSC-DKO cells by immunofluorescence.

[0359] Immunofluorescence detection results as follows Figure 7A As shown, both H1 cell-derived WT cells and H1 cell-derived DKO cells expressed the stemness genes POU5F1 and NANOG at the protein level, and the expression of POU5F1 and NANOG in H1 cell-derived DKO cells was not significantly different from that in WT cells. The RT-qPCR results are as follows. Figure 7B As shown, H1 cell-derived WT cells and H1 cell-derived DKO cells expressed stem genes POU5F1, NANOG, and SOX2 at the RNA level, and the expression of these genes in H1 cell-derived DKO cells was not significantly different from that in H1 cell-derived WT cells. Flow cytometry results are as follows. Figure 7CAs shown, both H1 cells derived from WT and H1 cells derived from DKO highly expressed the stemness gene corresponding proteins SSEA-4 (WT 100% and DKO 99.98%) and Tra1-81 (WT 96.75% and DKO 99.13%) on their surface. Figure 7D To detect the expression of stemness genes SSEA-4, TRA-1-60, TRA-1-81, OCT-4, and SOX2 on the surface of iPSC-WT and iPSC-DKO cells by flow cytometry, the results showed that both iPSC-WT cells and iPSC-derived DKO cells highly expressed the corresponding proteins of the stemness genes on their surface. Figure 7E To detect the protein levels of stemness genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in iPSC-WT cells using immunofluorescence, the corresponding proteins of stemness genes were highly expressed on the surface of iPSC-WT cells. Figure 7F To obtain the results of immunofluorescence detection of the protein levels of stem genes TRA-1-60, TRA-1-81, OCT-4, SOX2 and NANOG in iPSC-DKO cells, the corresponding proteins of stem genes were highly expressed on the surface of iPSC-derived DKO cells.

[0360] 2.2 Differentiation capacity of DKO cells

[0361] This embodiment examines the differentiation capacity of DKO cells. The specific detection method is as follows: 100 μL of a solution containing 5 × 10⁻⁶ DKO cells was subcutaneously injected into immunodeficient mice (SCIDBeige, Vitallix). 5 DKO cell suspension, when the teratoma volume is greater than 1.5 cm 3 The sections were then removed and subjected to paraffin sectioning and hematoxylin-eosin staining.

[0362] Staining results showed that DKO cells derived from H1 cells with B2M / CIITA biallelic knockout could form teratomas in vivo and differentiate into cells from the inner, middle, and outer germ layers. DKO cells derived from H1 cells had normal tri-germ layer differentiation capacity.

[0363] 2.3 Immune function of DKO cells

[0364] The cytotoxicity assays of T cells and NK cells were performed using the xCELLigence RTCA Instrument to detect changes in the immune function of H1 cells, iPSC cells, and derived DKO cells. The reagents used in the cytotoxicity assays are shown in Table 3.

[0365] The specific detection method is as follows: Equal amounts of WT and DKO cell lines were resuspended in Essential 8 medium containing human IL-2 and seeded into 96-well E-plates coated with matrix gel. Activated T cells (XC11228, purchased from SAILYBIO) or NK cells (XC11013, purchased from SAILYBIO) were added for cytotoxicity testing. Before use, T cells underwent flow cytometry analysis for CD3, CD4, and CD8, and NK cells underwent flow cytometry analysis for CD16 and CD56 to ensure the function of the T and NK cells used. RTCA detection data were analyzed using xCELLigence software to calculate the cytotoxicity rate and escape function.

[0366] Table 3. Reagents used in lethality tests

[0367] Name Cat No. Size Manufacturer PE anti-human CD4 980804 500 μl / tube Biolegend APC anti-human CD8 980904 500 μl / tube Biolegend FITC anti-human CD3 300440 500 times Biolegend APC anti-human CD16 301012 100 times Biolegend PE anti-human CD56 (NCAM) 318306 100 times Biolegend human IL-2 202-1L-050 / CF 50 μg R&D Y-27632 2HCl S1049 5 mg Selleck Matrigel 354277 5ml Gibco Essential 8 TM culture medium A1517001 500ml Gibco E-Plate VIEW 96 PET 300601030 6 pieces / box Agilent

[0368] RTCA results are as follows Figure 8A and Figure 8B As shown, WT cells can evade NK cell killing due to HLA-I expression, but are killed by T cells. DKO cells can evade T cell killing, while being more sensitive to NK cell killing. Figure 8A The three images above show the results of RTCA detection of the killing rate of NK cells against H1 cells WT and H1 cell-derived DKO cells, that is, the detection results of the immune escape function of H1 cells WT and H1 cell-derived DKO cells against NK cells. Figure 8A The three images in the second row show the results of RTCA detection of the killing rate of T cells against H1 cells (WT cells) and H1 cell-derived DKO cells, i.e., the detection results of the immune escape function of WT cells and H1 cell-derived DKO cells against T cells. Figure 8B The three images above show the results of RTCA detection of the killing rate of NK cells against iPSC-WT and iPSC-DKO cells, that is, the detection results of the immune escape function of iPSC-WT and iPSC-DKO cells against NK cells; Figure 8B The three images in the second row show the results of RTCA detection of the killing rate of T cells against iPSC-WT and iPSC-DKO cells, that is, the detection results of the immune escape function of iPSC-WT and iPSC-DKO cells against T cells.

[0369] Example 3. Construction and Immune Function Verification of DKO+CD47 Cell Line

[0370] 3.1 Construction of H1 cell line DKO+CD47

[0371] In this embodiment, CD47 (NM_198793) was overexpressed in DKO cells obtained in Example 1 using a lentiviral vector. The amino acid sequence of CD47 is shown in SEQ ID NO:5.

[0372] The nucleic acid sequence encoding the CD47 protein (SEQ ID NO:6) was constructed in a lentiviral vector (pGC-EF1a) initiated by EF1a and carrying a puromycin selection marker. The structure of the pGC-EF1a vector is as follows. Figure 9 As shown. The specific operation method is as follows:

[0373] The lentiviral vector was digested with BamHI / NheI, and the nucleic acid sequence encoding the CD47 protein (SEQ ID NO:6) was ligated into the lentiviral vector. After successful ligation, the correctness of the inserted sequence was verified by Sanger sequencing, and the virus was packaged. The lentiviral vector was transfected into the DKO cells constructed in Example 1. The medium was changed after 24 hours, and after 48 hours, the medium was changed to puromycin-containing medium for selection.

[0374] The constructed stable cell line DKO+CD47 was analyzed by flow cytometry (CD47 antibody purchased from FACS: Biolegend, catalog number: 323108) and qPCR. The qPCR primers were CD47-F: AGAAGGTGAAACGATCATCGAGC (SEQ ID NO:27); CD47-R: CTCATCCATACCACCGGATCT (SEQ ID NO:28).

[0375] Test results as follows Figure 10A and 10B As shown, the expression level of CD47 in the constructed DKO+CD47 cell line was significantly higher than that in WT cells. The validated DKO+CD47 cell line was then expanded and its functionality was subsequently assessed.

[0376] 3.2 Verification of Immune Function of DKO+CD47

[0377] RTCA was used to detect whether the DKO+CD47 overexpressing cell line could successfully evade NK cell killing while escaping T cell killing. For specific detection methods, please refer to Example 2.3.

[0378] RTCA detection such as Figure 11 As shown, NK cells can effectively kill DKO cells, while WT and DKO+CD47 overexpressing cells can escape NK cell killing.

[0379] Example 4. Construction of DKO+CD300LD cell line and detection of stemness and differentiation capacity

[0380] 4.1 Construction and overexpression detection of iPSC-DKO+CD300LD cell line

[0381] The nucleic acid sequence encoding the CD300LD protein (the amino acid sequence of the CD300LD protein is shown in SEQ ID NO:3 or 4) was directly synthesized.

[0382] CD300LD amino acid sequence:

[0383] >NCBI Reference Sequence:NP_001108624.1

[0384] CMRF35-like molecule 5precursor[Homo sapiens]

[0385] MWLSPALLLLILPGYSIAAKITGPTTVNGSEQGSLTVQCAYGSGWETYLKWRCQGADWNYCNILVKTNGSEQEVKKNRVSIRDNQKNHVFTVTMENLKRDDADSYWCGTERPGIDLGVKVQVTINPGTQTAVSEWTTTTASLAFTAAATQKTSSPLTRSPLKSTHFLFLFLLELPLLLSMLGTVLWVNRPQRRS(SEQ ID NO:3)

[0386] >NCBI Reference Sequence:XP_047291002.1

[0387] CMRF35-like molecule 5isoform X1[Homo sapiens]

[0388] MNLRFPGYSIAAKITGPTTVNGSEQGSLTVQCAYGSGWETYLKWRCQGADWNYCNILVKTNG SEQEVKKNRVSIRDNQKNHVFTVTMENLKRDDADSYWCGTERPGIDLGVKVQVTINPGTQTAVSE WTTTTASLAFTAAATQKTSSPLTRSPLKSTHFLFLFLLELPLLLSMLGTVLWVNRPQRRS(SEQ ID NO:4)

[0389] As described in Example 3.1, the nucleic acid sequence encoding the CD300LD protein was constructed in a lentiviral vector (pGC-EF1a) initiated by EF1a and carrying a puromycin selection marker. The structure of the pGC-EF1a vector is as follows. Figure 9As shown. The specific operation is as follows: The vector was digested with BamHI / NheI enzymes, and the nucleic acid sequence of the synthesized CD300LD was ligated into the lentiviral vector. After successful ligation, Sanger sequencing was used to verify the correctness of the inserted sequence and virus packaging was performed. The lentiviral vector was transfected into the iPSC-DKO cells constructed in Example 1. The medium was changed after 24 hours, and after 48 hours, the medium was changed to puromycin-containing medium for selection.

[0390] Overexpression was detected in the constructed iPSC-DKO+CD300LD cell line. qRT-PCR was used to detect mRNA overexpression levels, with iPSC-WT cells serving as a negative control. The primers used are shown in Table 4. FACS was used to detect protein overexpression levels, with iPSC-WT cells serving as a negative control and isotype representing the negative control for the detection signal.

[0391] Table 4. Primers for qPCR detection of CD300LD

[0392] Primer sequence Specific sequence CD300LD F1 (SEQ ID NO: 23) TCCCAGGTTACTCCATTGCC CD300LD R1 (SEQ ID NO: 24) GCCTGAGCCATAAGCACACT

[0393] The test results showed that the constructed iPSC-DKO+CD300LD cell line had high expression of CD300LD.

[0394] 4.2 Expression of stemness genes in iPSC-DKO+CD300LD cell lines

[0395] The expression of stemness genes in the DKO+CD300LD cell line was detected by immunofluorescence and flow cytometry, confirming the stemness of the constructed cells. Specific detection methods are described in Example 2.1. The results of the immunofluorescence detection of stemness genes are shown below. Figure 12 As shown, the flow cytometry results for the stem gene are as follows: Figure 13 As shown in the figure. Immunofluorescence assay results showed that the constructed iPSC-DKO+CD300LD cells expressed stemness genes OCT4, NANOG, SOX2, TRA-1-60, and TRA-1-81 at the protein level. Flow cytometry results showed that iPSC-DKO+CD300LD cells highly expressed stemness genes SSEA-4, TRA-1-60, TRA-1-81, and OCT4 on their surface.

[0396] 4.3 Trigerm layer differentiation capacity of iPSC-DKO+CD300LD cell line

[0397] Trigerm layer differentiation capacity was assessed using DKO+CD300LD cells. To generate mesoderm, endoderm, and ectoderm cells, dissociated iPSC-DKO+CD300LD single cells were resuspended in three different germ layer media supplemented with Y27632. A suitable amount of cells were then attached to wells of a matrix gel-coated plate with cell spreaders. After 24 hours, the medium was replaced with preheated differentiation medium, and the medium was changed daily until day seven to obtain mesoderm, endoderm, and ectoderm cells. The expression of trigerm layer marker proteins was detected by immunofluorescence to assess the trigerm layer differentiation capacity of the iPSC-DKO+CD300LD cell line. The results of the trigerm layer marker protein expression detection are shown below. Figure 14 As shown. Immunofluorescence assay results showed that iPSC-DKO+CD300LD cells expressed ectoderm marker proteins: PAX6 and GAD1; mesodermal marker proteins: Brachyury and NCAM; and endoderm marker proteins: SOX17 and FOXA2 at the protein level. Example 5: Immune Escape Function of iPSC-DKO+CD300LD Cell Line

[0398] To verify the escape function of iPSC-DKO+CD300LD cells against different immune cells, NK cells, PBNK (a mixture of T cells and NK cells), and macrophages were used in the experiment. The escape function of iPSC-DKO+CD300LD cells against different immune cells was detected by RTCA. Specific detection methods are described in Example 2.3.

[0399] 5.1 iPSC-DKO+CD300LD cells resist NK cell killing

[0400] NK cell killing assays were performed on the XCelligence platform (ACEA BioSciences). Various iPSC cell types (iPSC-WT cells, iPSC-DKO cells, iPSC-DKO+CD300LD cells) were resuspended in 100 μl of cell-specific culture medium and seeded onto 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 standardized and analyzed using RTCA software (ACEA). The killing rate results are shown below. Figure 15 As shown, Figure 15 A and Figure 15 Figure B shows the results of an experiment on the killing effect of NK cells on iPSC-DKO+CD300LD cells, as detected by RTCA. Figure 15Figure B represents the multiple kill statistics, with N=6 for the DKO cell line, N=5 for the DKO+CD300LD cell line, and N=7 for the WT cell line. The results show that iPSC DKO+CD300LD cells can significantly evade NK cell killing.

[0401] 5.2 iPSC-DKO+CD300LD cells resist PBMC cell killing

[0402] NK cell activating factors were added to PBMCs beforehand to increase the proportion of NK cells and the killing ability of T cells. Activated mixed lymphocytes (PBMCs) were used as effector cells in RTCA experiments to comprehensively evaluate the immune escape ability of iPSC-DKO+CD300LD cells (method reference PMID:33309274). Specifically, PBMC cell killing assays were performed on the XCelligence platform (ACEA BioSciences). Various types of iPSC cells (iPSC-WT cells, iPSC-DKO cells, iPSC-DKO+CD300LD cells) were resuspended in 100 μl of cell-specific culture medium and seeded onto 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, PBMC cells were added at a 2:1 E:T ratio. Data were standardized and analyzed using RTCA software (ACEA). The killing rate results are shown below. Figure 16 As shown, Figure 16 A and Figure 16 Figure B shows the results of an RTCA-based assay of PBMCs' cytotoxicity against iPSC-DKO+CD300LD cells, in which... Figure 16 B represents the statistical plot of multiple kills, with N=4. The results show that iPSC-DKO+CD300LD cells can significantly evade PBMC killing.

[0403] 5.3 iPSC-DKO+CD300LD cells resist macrophage killing

[0404] MAC cell killing assays were performed on the XCelligence platform (ACEA BioSciences). Various iPSC cell types (iPSC-WT cells, iPSC-DKO cells, iPSC-DKO+CD300LD cells) were resuspended in 100 μl of cell-specific culture medium and seeded onto 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, MAC cells were added at a 2:1 E:T ratio. Data were standardized and analyzed using RTCA software (ACEA). The killing rate results are shown below.Figure 17 As shown, Figure 17 A and Figure 17 Figure B shows the results of an experiment on the killing effect of macrophages on iPSC-DKO+CD300LD cells, as detected by RTCA. Figure 17 B represents the statistical plot of multiple kills, with N=4. The results show that iPSC-DKO+CD300LD cells can significantly evade PBMC killing.

[0405] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

[0406] sequence list

[0407]

[0408]

[0409]

Claims

1. A low-immunogenic pluripotent stem cell, comprising: Reduced endogenous major histocompatibility class I (MHC-I) function compared to parental pluripotent stem cells; Reduced endogenous major histocompatibility class II (MHC-II) function compared to parental pluripotent stem cells; and Reduced sensitivity to NK cell killing compared to parental pluripotent stem cells, wherein the reduced sensitivity to NK cell killing is caused by increased expression of CD300LD protein. The MHC-I function is reduced by decreasing the activity of B2M proteins, through one or more alterations that reduce the activity of endogenous B2M proteins. The MHC-II function is reduced by decreasing the activity of CIITA protein, through one or more alterations that reduce the activity of endogenous CIITA protein; and One or more alterations that induce increased expression of CD300LD protein in the hypoimmunogenic pluripotent stem cells, the amino acid sequence of which is shown in SEQ ID NO:

3.

2. The low-immunogenicity pluripotent stem cells as described in claim 1, comprising: One or more alterations that inactivate two alleles of the endogenous B2M gene; One or more alterations that inactivate two alleles of the endogenous CIITA gene; and One or more alterations that induce increased expression of the CD300LD gene in the said low-immunogenic pluripotent stem cells.

3. The low immunogenic pluripotent stem cells according to claim 1, wherein the B2M protein is a human B2M protein, and the amino acid sequence of the human B2M protein is shown in SEQ ID NO:

1.

4. The low immunogenic pluripotent stem cells according to claim 1, wherein the CIITA protein is human CIITA protein, and the amino acid sequence of the human CIITA protein is shown in SEQ ID NO:

2.

5. A method for generating low-immunogenic pluripotent stem cells according to any one of claims 1-4, the method comprising: Reduce the activity of B2M protein in the pluripotent stem cells; Reduce the activity of CIITA protein in the pluripotent stem cells; and Increase the expression of CD300LD protein in the pluripotent stem cells.

6. The method of claim 5, wherein the method comprises: Eliminate the activity of two alleles of the B2M gene in the pluripotent stem cells; Eliminate the activity of two alleles of the CIITA gene in the pluripotent stem cells; and Increase the expression of the CD300LD gene in the pluripotent stem cells.

7. The method of claim 5, wherein the activity of B2M protein in the pluripotent stem cells and / or the activity of CIITA protein in the pluripotent stem cells are reduced by techniques selected from: Introduce gene expression modification molecules, clustered regular-interval short palindromic repeats (CRISPR) technology, transcription activator-like effector nucleases (TALEN) technology, zinc finger nucleases (ZFN) technology, or homologous recombination technology.

8. The method of claim 7, wherein the gene expression modification molecule comprises siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotide ASO, or antimiRNA oligonucleotide AMO.

9. The method of claim 8, wherein the activity of B2M protein in the pluripotent stem cells is reduced by CRISPR / Cas9 gene editing technology; and / or the activity of CIITA protein in the pluripotent stem cells is reduced by CRISPR / Cas9 gene editing technology.

10. The method of claim 5, wherein the expression of CD300LD protein is increased by modifying the endogenous locus.

11. The method of claim 5, wherein the expression of CD300LD protein is increased by the expression of the transgene.

12. The method of claim 11, wherein a nucleic acid sequence encoding the CD300LD protein is synthesized and constructed into a lentiviral vector, and at least one copy of the promoter-controlled CD300LD gene is introduced into the pluripotent stem cells via the lentiviral vector to increase the expression of the CD300LD protein.

13. The method of claim 12, wherein the nucleic acid sequence encoding the CD300LD protein is shown in SEQ ID NO:25.

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