Low-immunogenicity cell for expressing CD300LD and preparation method of low-immunogenicity cell

Abstract

The invention provides a low-immunogenicity cell for expressing CD300LD and a preparation method of the low-immunogenicity cell. The low-immunogenicity pluripotent cell can significantly reduce or escape the recognition and attack of an immune system, especially the attack of T cells, natural killer cells, macrophages and the like, without changing the updating and differentiation characteristics of stem cells.

Description

[0001] This application claims the priority of Chinese patent application No. 2023111948810 filed on September 15, 2023. This application cites the entire text of the above Chinese patent application. Technical Field

[0002] The present invention 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 Art

[0003] Stem cells are a type of cell that has the ability to self-renew and differentiate into specific functional somatic cells. Based on the degree of differences in 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 to proliferate indefinitely, self-renew and differentiate into various types of cells, and have important application prospects in the treatment of cancer, neurological, cardiovascular and other diseases. However, key issues such as immune incompatibility and immune rejection of transplanted cells have hindered the clinical application of transplanting allogeneic functional cells for treatment.

[0004] The 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. Transplanted cells expressing "non-self" MHC-I class molecules will stimulate the activation of CD8+T cells and be eliminated. CD4+ helper T cells recognize the MHC-II genes of "non-self" cells, thereby performing immune rejection, while class III molecules do not participate in immune activities.

[0005] In recent years, it has been reported that by knocking out genes such as B2M and CIITA, the expression of MHC-I and MHC-II cell surface or their own genes can be deleted, thereby making the cells immune tolerant or escaping T cell / B cell-specific immune responses, 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-type domain. The short cytoplasmic tail lacks any known signaling motifs, but there is a negatively charged residue (glutamic acid) within the transmembrane domain. It can enable transmembrane signaling receptor activity and viral receptor activity, and participate in immune system processes. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a new method for transforming cells to obtain low immunogenicity. For the first time, a strategy of mass screening of various mechanism molecules involved in maternal-fetal tolerance and tumor escape was adopted. Through multiple rounds of functional testing, a representative new gene CD300LD was finally identified. This gene can significantly reduce or escape the recognition and attack of the immune system, especially the attack of natural killer cells, T cells, macrophages, etc. The present invention proposes a feasible strategy to achieve the immune immunity of cells by developing new genes through unpredictable field transformation.

[0008] The present invention successfully constructs B2M / CIITA double allele knockout positive clone DKO cells by knocking out β-2-microglobulin (B2M) in the endoplasmic reticulum of human pluripotent stem cells and knocking out CIITA, a positive regulator of MHC-II gene transcription; then uses a lentiviral vector to overexpress the new gene CD300LD identified by the present invention in DKO cells, and the human pluripotent stem cells obtained thereby can further escape the killing of NK cells on the basis of escaping T cell attack. At the same time, these low-immunogenic pluripotent stem cells retain their key biological functions of pluripotent stem cells, such as their stemness and differentiation ability.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] In one aspect, the present invention 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 a parental pluripotent stem cell; and reduced sensitivity to NK cell and T cell killing compared to a parental pluripotent stem cell, wherein the reduced sensitivity to NK cell and T cell killing 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-I class 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-associated glycoprotein or a NLRC5 protein.

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

[0015] In one embodiment, the B2M protein is a human B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence that has 90% identity with the amino acid sequence shown in SEQ ID NO:1.

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

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

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

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

[0020] In one embodiment, the CIITA protein is a human CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence that has 90% identity with the amino acid sequence shown in SEQ ID NO: 2.

[0021] In some embodiments, the CD300LD protein is a human CD300LD protein comprising the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence that is 90% identical to the amino acid sequence shown in SEQ ID NO:3.

[0022] In some embodiments, 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 90% identity to the amino acid sequence shown in SEQ ID NO:4.

[0023] In one embodiment, the low immunogenic pluripotent stem cells comprise: one or more changes that reduce the activity of endogenous B2M protein; one or more changes that reduce the activity of endogenous CIITA protein; and one or more changes that cause increased expression of CD300LD protein in the low immunogenic pluripotent stem cells.

[0024] In one embodiment, the low immunogenic pluripotent stem cells further comprise one or more changes that increase the expression of the following polypeptides, wherein the polypeptides whose expression is increased are selected from: DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8 and SerpinB9.

[0025] In one embodiment, the low immunogenic pluripotent stem cells comprise: one or more alterations that inactivate both alleles of the endogenous B2M gene; one or more alterations that inactivate both alleles of the endogenous CIITA gene;

[0026] and one or more alterations that result in increased CD300LD gene expression in said low immunogenic pluripotent stem cells.

[0027] On the other hand, the present invention also provides a method for producing the above-mentioned low immunogenic pluripotent stem cells of the present invention, the method comprising: reducing the function of endogenous major histocompatibility class I antigen (MHC-I) in the pluripotent stem cells; reducing the function of endogenous major histocompatibility class II antigen (MHC-II) 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.

[0028] In one embodiment, the method comprises: 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.

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

[0030] In one embodiment, the method further comprises increasing the 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 cells.

[0031] In one embodiment, the activity of the B2M protein in the pluripotent stem cells is reduced and / or the activity of the CIITA protein in the pluripotent stem cells is reduced by a technique selected from the following:

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

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

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

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

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

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

[0038] In a preferred embodiment, a nucleic acid sequence encoding CD300LD protein is synthesized and constructed into a lentiviral vector, and then at least one copy of the CD300LD gene under the control of a promoter is introduced into the pluripotent stem cells via the lentiviral vector to increase the expression of CD300LD protein.

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

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

[0041] In another aspect, the present invention also provides 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 preparing a medicament for preventing or treating a disease requiring 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 degeneration, stroke, Huntington's disease, amyotrophic lateral sclerosis, atherosclerosis, hypertension, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, myocardial infarction, heart failure, aortic aneurysm, peripheral arterial disease, type II diabetes, scurvy, hypoglycemia, hyperlipidemia or osteoporosis.

[0043] In another aspect, the present invention provides a composition comprising the low immunogenic cells according to the first aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The diagram shows the knockout strategy of the B2M gene in the B2M and CIITA double knockout cell lines (DKO) and the results of B2M gene knockout verification by PCR.

[0045] Figure 2 The diagram shows the knockout strategy of the CIITA gene in B2M and CIITA double knockout cell lines (DKO) and the results of the CIITA gene knockout verification by PCR.

[0046] Figure 3 The results of qPCR detection of B2M and CIITA expression at the RNA level in B2M / CIITA biallelic knockout clone H1 cells DKO are shown.

[0047] Figure 4 The figure shows the results of Western blot detection of B2M protein levels in B2M / CIITA biallelic knockout clone H1 DKO cells. Figure 5A The figure shows the results of FACS detection of HLA class I / II molecules on the surface of H1 cells by stimulating WT and DKO cells with IFN-γ.

[0048] Figure 5B The figure shows the results of FACS detection of HLA class I / II molecules on the surface of iPSC cells by stimulating WT and DKO cells with IFN-γ.

[0049] Figure 6 The karyotype detection results of the B2M / CIITA biallelic knockout clone H1 cell DKO are shown.

[0050] Figure 7A-7FThe results of detecting the expression of stemness genes in DKO cells are shown.

[0051] Fig. 7A The results are the results of detecting the protein levels of stemness genes POU5F1 and NANOG in H1 cells (WT cells) and H1-derived DKO cells (DKO cells) by immunofluorescence;

[0052] Figure 7B The results are the results of detecting the expression of stemness 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 The results are the results of detecting the expression of surface stemness genes SSEA-4 and Tra1-81 in H1 cells (WT cells) and H1-derived DKO cells (DKO cells) by flow cytometry;

[0054] Fig.7D The results of flow cytometry detection of the expression of stemness genes SSEA-4, TRA-1-60, TRA-1-81, OCT-4, and SOX2 on the cell surface of iPSC-WT and iPSC-DKO. Fig. 7E The results of immunofluorescence detection of the protein levels of stemness genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in iPSC-WT cells;

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

[0056] Fig. 8A and Figure 8B The results of detecting the immune escape function of WT and DKO cells by RTCA are shown.

[0057] Fig. 8A The results of detecting the killing rate of H1 WT cells and H1 cell-derived DKO cells by NK cells and T cells by RTCA are shown;

[0058] Figure 8B The graph shows the results of detecting the killing rates of NK cells and T cells on iPSC-WT and iPSC-DKO cells by RTCA, that is, the detection results of the immune escape function of iPSC-WT and iPSC-DKO cells on NK cells.

[0059] Fig. 9 A schematic diagram showing the structure of the lentiviral vector pGC-EF1a vector is shown.

[0060] Fig. 10A and Fig. 10B Shown are the results confirming the overexpression of CD47 in H1 cell DKO+CD47 cells. Fig. 10A The results are as follows: CD47 expression level in H1 cell DKO+CD47 cell line detected by FACS; Fig. 10B The results are shown in Figure 2, which show the expression level of CD47 in the H1 cell DKO+CD47 cell line detected by qPCR.

[0061] Fig.11 The results of RTCA detection of the immune escape function of WT H1 cells and derived DKO+CD47 cells are shown.

[0062] Fig.12 The graph shows the results of immunofluorescence detection of the protein levels of stemness genes OCT4, NANOG, SOX2, TRA-1-60, and TRA-1-81 in constructed iPSC-DKO+CD300LD cells.

[0063] Fig.13 The graph shows the results of detecting the expression levels of stemness genes SSEA-4, TRA-1-60, TRA-1-81, and OCT4 on the surface of constructed iPSC-DKO+CD300LD cells by flow cytometry.

[0064] Fig.14 The figure shows the results of immunofluorescence detection of the three-germ layer differentiation ability of the constructed iPSC-DKO+CD300LD cells.

[0065] Fig.15 The results of the NK cell killing experiment on iPSC-DKO+CD300LD cells detected by RTCA are shown, wherein B is a multiple killing statistical diagram, N=6.

[0066] Fig.16 The results of the RTCA-detected killing experiment of PBMC on iPSC-DKO+CD300LD cells are shown, wherein B is a multiple killing statistical graph, N=4.

[0067] Fig.17 The results of the macrophage killing experiment on iPSC-DKO+CD300LD cells detected by RTCA are shown, wherein B is a multiple killing statistics chart, N=4. DETAILED DESCRIPTION

[0068] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0069] General Definitions and Terminology

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

[0071] Unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology related terms used herein are widely used terms in the corresponding fields (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). At the same time, in order to better understand the present invention, the definitions and explanations of the related terms are provided below.

[0072] As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended, meaning the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of" excludes any element, step, or component not specified. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of" are encompassed within the meaning of the expression "comprising."

[0073] As used herein, the singular forms "a", "an" or "the" include plural references unless the context indicates otherwise. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0074] 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 separate value is incorporated into the specification as if it were individually recited herein.

[0075] Unless expressly stated to the contrary, numerical values ​​or ranges set forth herein are modified by "about" to mean ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0076] Unless otherwise specified, in the method steps described herein, labels such as 1), 2), ..., i), ii), ..., a), b), ... are merely examples of distinction and do not imply that the method steps described are performed in such an order.

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

[0078] As used herein, the term "pluripotent stem cell" has the potential to differentiate into any of the following three germ layers: endoderm (e.g., gastric junction, 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 cells" or "iPSC", a pluripotent stem cell derived from a non-pluripotent cell. Exemplary human pluripotent stem cell lines include H1 human pluripotent stem cell lines, H9 human pluripotent stem cell lines, and iPSC human pluripotent stem cell lines. Additional exemplary pluripotent stem cell lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and Howard Hughes Medical Institute HUES collection (such as Cowan CA, et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med. 2004 Mar 25; 350 (13): 1353-6. described).

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

[0080] As used herein, the term "low immunogenic cells" refers to cells that are modified using gene editing technology to eliminate immune rejection and achieve low immunogenicity.

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

[0082] As used herein, the term "immune rejection" or "immune incompatibility" refers to the fact that foreign cells, tissues or organs will be attacked by the recipient's own immune cells after being transplanted into the recipient, thereby failing to ensure their normal physiological functions. The human major histocompatibility complex (MHC), i.e., human leukocyte antigen (HLA), is the main cause of "immune rejection" or "immune incompatibility".

[0083] As used herein, the term "subject" or "patient" refers to any animal, such as a domesticated animal, a zoo animal, or a human. A "subject" or "patient" can be a mammal, such as a dog, a cat, a bird, livestock, or a human. Specific examples of "subjects" and "patients" include, but are not limited to, individuals (particularly humans) with diseases or conditions associated with the liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood, bones, bone marrow, etc.

[0084] " Hypoimmunogenic pluripotent stem cells " herein refer to pluripotent stem cells, which retain the characteristics of their pluripotent stem cells, and produce a reduced immune rejection reaction when transferred to an allogeneic host. In a preferred embodiment, low immunogenic pluripotent stem cells do not produce an immune response. Therefore, " low immunogenicity " refers to an immune response that is significantly reduced or eliminated compared to the immune response of the parent (" WT ") stem cell before immune modification. For example, relative to wild-type cells that have not been immune modified, such low immunogenic cells may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more than 99% less likely to produce immune rejection.

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

[0086] The term "MHC-I" or "MHC class I" refers to the major histocompatibility complex class I proteins or genes. Within the human MHC-I region, there are the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, CD1a, CD1b, and CD1c 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 peptides complexed with MHC-I class molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that 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" refers to major histocompatibility complex class II proteins or genes. MHC II includes 5 proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR. MHC class II proteins are mainly expressed on antigen presenting cells such as B cells, monocytes and macrophages, dendritic cells, etc. MHC class II molecules mainly mediate the presentation process of exogenous antigens. They present exogenous antigen polypeptide molecules to Th cells (helper T cells), that is, stimulate CD4+ T cells.

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

[0089] "Knockout" herein refers to the process of making a specific gene inactive in the host cell in which it is located, which results in the non-production of the target protein or an inactive form. As will be appreciated by those skilled in the art and described further below, this can be achieved in a variety of different ways, including removing the nucleic acid sequence from the gene, or interrupting the sequence with other sequences, changing the reading frame, or changing the regulatory elements 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, all or part of the regulatory sequence (e.g., a promoter) can be removed or replaced, the translation initiation sequence can be removed or replaced, etc.

[0090] In this article, the terms "reduce" and "reduce" are generally used to represent a statistically significant amount of reduction. However, for the avoidance of doubt, "reduce", "reduce" 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% compared to a reference level, 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 a reference sample), or any reduction between 10-100%.

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

[0092] As used herein, the term "increase" is generally used to indicate an increase by a statistically significant amount; to avoid any doubt, the term "increase" refers to 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 a 100% increase or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or 10 at least about 10-fold compared to a reference level, or any increase between 2-fold and 10-fold or greater than 10-fold.

[0093] "Beta-2 microglobulin" or "β2M" or "B2M" protein is a component of MHC-I class molecules. B2M protein is expressed in all nucleated cells (except red blood cells) and 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 the phagocytosis of macrophages and cause immune escape by binding to the N-terminus of the ligand signal regulatory protein α (SIRPα) on immune cells.

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

[0096] CD300LD is a tumor immunosuppressive receptor specific for polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC). PMN-MDSC is a type of neutrophil granulocyte induced by pathology and widely present in various tumors. It can inhibit the function of effector cells such as T cells and NK cells, promote tumor development, infiltration and metastasis, and play a key role in tumor immunoregulation.

[0097] As used herein, the term "syngeneic" refers to the genetic similarity or identity of the host organism and the cell transplant wherein there is immunological compatibility; eg, no immune response is generated.

[0098] As used herein, the term "allogeneic" refers to the genetic differences of the host organism and the cells transplanted into which an immune response is generated.

[0099] As used herein, the term "B2M- / -" refers to a diploid cell having an inactivated B2M gene in both chromosomes.

[0100] As used herein, the term "CIITA- / -" refers to a diploid cell having an inactivated CIITA gene in both chromosomes.

[0101] As used herein, the term "polypeptide" refers to a polymer comprising two or more amino acids covalently linked by peptide bonds. A "protein" may comprise one or more polypeptides, wherein the polypeptides interact with each other covalently or non-covalently. Unless otherwise indicated, "polypeptide" and "protein" may be 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 a gene editing procedure to achieve low immunogenicity, for example, the parental pluripotent stem cells (WT) described herein. The parental pluripotent stem cells of the H1 cell line in the present disclosure are also referred to as WT, and the parental pluripotent stem cells of the iPSC cell line are also referred to as iPSC-WT.

[0103] As used herein, the term "% identity" with respect to a sequence refers to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The differences between the two sequences can be distributed over local regions (segments) or over the entire length of the sequences to be compared. The identity between the two sequences is usually determined after optimal alignment of a segment or "comparison window". Optimal alignment can be performed manually, or by means of algorithms known in the art, including but not limited to the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search method described by 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 in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percent identity of two sequences can be determined using the publicly available BLASTN or BLASTP algorithms available on the website of the National Center for Biotechnology Information (NCBI).

[0104] By determining the number of identical positions corresponding to sequences to be compared, divide this number by the number of positions compared (for example, the number of positions in the reference sequence), and multiply this result by 100, obtain % homogeneity. In some embodiments, 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% region gives homogeneity degree. In some embodiments, the whole length of the reference sequence is given homogeneity degree. Can use tools known in the art to determine the comparison of sequence homogeneity, preferably utilize optimal sequence comparison, for example, utilize Align, utilize standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0105] In this article, "nucleotide" includes deoxyribonucleotides and ribonucleotides and their derivatives. As used herein, "ribonucleotide" is a constituent substance of ribonucleic acid (RNA), consisting of a molecule of base, a molecule of pentose, and a molecule of phosphoric acid, which refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotide" is a constituent substance of deoxyribonucleic acid (DNA), also consisting of a molecule of base, a molecule of pentose, and a molecule of phosphoric acid, which refers to a nucleotide in which the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen, and is the main chemical component of chromosomes. "Nucleotide" is usually referred to by a single letter representing the base: "A (a)" refers to deoxyadenosine or adenylic acid containing adenine, "C (c)" refers to deoxycytidine or cytidine containing cytosine, "G (g)" refers to deoxyguanosine or guanylate containing guanine, "U (u)" refers to uridylate containing uracil, and "T (t)" refers to deoxythymidylate containing thymine.

[0106] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence", "nucleic acid sequence" and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. It should be understood by those skilled in the art that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, and the deoxythymidylic acid in the DNA coding strand sequence corresponds to the uridine acid in the RNA sequence it encodes.

[0107] As used herein, the term "expression" includes 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 (transcript). The term "in vitro transcription" refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). 9 In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). The vector that can be used to produce transcripts is also called a "transcription vector", which contains the regulatory sequence required for transcription. The term "transcription" encompasses "in vitro transcription".

[0108] As used herein, "encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA can be used as a template to synthesize polymers and macromolecules in other biological processes, as long as there is a clear nucleotide sequence or a clear amino acid sequence. Therefore, a gene encodes a protein when 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 can be performed in any suitable order.

[0110] Pluripotent stem cells

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

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

[0113] Reduced endogenous major histocompatibility class II antigen (MHC-II) function compared to the parental pluripotent stem cell; and

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

[0115] Herein, parental pluripotent stem cells refer to parental (also referred to herein as “WT” or “iPSC-WT”) pluripotent stem cells before immune modification that have not undergone a gene editing procedure to achieve low immunogenicity.

[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 will be appreciated by those skilled in the art, reduction of function can be achieved in a variety of ways, including removal of nucleic acid sequences from a gene, interruption of a sequence with another sequence, or alteration of a regulatory component of a nucleic acid. For example, all or part of the coding region of a target gene can be removed or replaced with a "nonsense" sequence, frameshift mutations can be performed, all or part of a regulatory sequence such as a promoter can be removed or replaced, a translation initiation sequence can be deleted or replaced, etc.

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

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

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

[0121] In some embodiments, the MHC-I function is reduced by reducing the activity of an MHC-I transcriptional regulator.

[0122] In some preferred embodiments, the MHC-I transcriptional regulator can be selected from: one or more of: β2 microglobulin (B2M), transporter associated with antigen processing 1 (TAP1), transporter associated with antigen processing 2 (TAP2), transporter associated with antigen processing (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 reducing 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 reducing the activity of the HLA-B protein.

[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 reducing 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 reducing the activity of 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 reducing 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 reducing 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 reducing 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 reducing the activity of the B2M protein.

[0138] In one embodiment, the B2M protein is a human B2M protein, which comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 reducing the activity of MHC class II proteins.

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

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

[0143] In one embodiment, the MHC-II function is reduced by reducing 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 reducing 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 reducing 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 reducing 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 reducing 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 reducing 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 reducing the activity of the CIITA protein.

[0156] In one embodiment, the CIITA protein is a human CIITA protein, which comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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, the gene is knocked out using CRISPR technology. In some cases, CRISPR technology is used to introduce small deletions / insertions into the coding region of a gene so that no functional protein is produced, usually as a result of a frameshift mutation, which results in the generation of a stop codon, resulting in a truncated, non-functional protein.

[0159] The successful reduction of MHC-I (HLA-I when the cells are derived from human cells) function 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 using protein antibodies, FACS technology, RT-PCR, qPCR technology, etc.

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

[0161] Thus, 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, a lentiviral construct is used as described herein or known in the art. As 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 a CD300LD transgene.

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

[0164] In one embodiment, the CD300LD protein is a human CD300LD protein, which comprises the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 example using Western blot, ELISA assay or FACS assay. Generally, "sufficient" in this context means an increase in CD300LD protein expression on the surface of pluripotent stem cells, which silences killing by NK cells and T cells.

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

[0167] One or more changes that reduce the activity of endogenous B2M protein;

[0168] One or more changes that reduce the activity of endogenous CIITA protein; and

[0169] One or more changes result in increased expression of CD300LD protein in the low immunogenic pluripotent stem cells.

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

[0171] one or more alterations that inactivate both alleles of the endogenous B2M gene;

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

[0173] One or more alterations result in increased CD300LD gene expression in the low immunogenic pluripotent stem cells.

[0174] In one embodiment, the low immunogenic pluripotent stem cells further comprise: one or more changes that increase the expression level 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 "change" or "genetic change" refers to causing a cell, such as a change in a pluripotent stem cell as described herein, which can be achieved, for example, by modifying a genome or introducing a new gene fragment. In this article, modifying a genome refers to modifying a nucleic acid sequence in a cell or under a cell-free condition to produce a transformed pluripotent cell and a pluripotent stem cell. Exemplary "changes" or "genetic changes" include, but are not limited to, homologous recombination, knock-in, ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), CRISPR (clustered regularly spaced short palindromic repeats) / Cas9 and other site-specific nuclease technologies. These technologies enable double-stranded DNA breaks to be performed at the desired gene locus. These controlled double-strand breaks promote homologous recombination at specific gene locus points. The process focuses on targeting specific sequences of nucleic acid molecules with endonucleases, such as chromosomes, and the endonucleases recognize and bind sequences and induce double-strand breaks in nucleic acid molecules. Double-strand breaks are repaired by fallible non-homologous end joining (NHEJ) or by homologous recombination (HR). Exemplary "alteration" or "genetic alteration" techniques also include the introduction of gene expression modifying molecules, including but not limited to siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotides ASO (antisense oligonucleotides) or anti-miRNA oligonucleotides AMO (Anti-miRNA oligonucleotides).

[0176] It will be appreciated by those skilled in the art that many different techniques may be used to engineer the pluripotent cells and pluripotent stem cells of the present invention to render them less immunogenic.

[0177] Typically, these techniques can be used alone or in combination. For example, CRISPR technology is used to reduce the expression of active B2M and / or CIITA proteins in transformed cells, and the CD300LD gene is knocked in using viral technology (e.g., lentivirus). In addition, it should be understood by those skilled in the art that these genes can be operated in different orders using different techniques. In some embodiments, one or more changes included in the low immunogenicity pluripotent stem cells of the present invention can reduce endogenous major histocompatibility class I antigen (MHC-I) function. In some embodiments, one or more changes included in the low immunogenicity pluripotent stem cells of the present invention can reduce endogenous major histocompatibility class II antigen (MHC-II) function.

[0178] In some embodiments, the reduced immunogenic pluripotent stem cells of the present invention comprise one or more changes that reduce sensitivity to NK cell and T cell killing.

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

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

[0181] In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that inhibit the expression of endogenous B2M protein. In some embodiments, the low immunogenic pluripotent stem cells of the present invention comprise one or more changes that inhibit the expression of endogenous CIITA protein.

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

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

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

[0185] In one embodiment, the pluripotent stem cells are altered using Clustered Regularly Interspaced Short Palindromic Repeats / Cas ("CRISPR") technology known in the art to reduce the activity of endogenous B2M protein.

[0186] In one embodiment, the pluripotent stem cells are altered using Clustered Regularly Interspaced 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 to inactivate both alleles of the endogenous B2M gene using Clustered Regularly Interspaced Short Palindromic Repeats / Cas ("CRISPR") technology known in the art.

[0188] In one embodiment, the pluripotent stem cells are altered to inactivate both alleles of the endogenous CIITA gene using Clustered Regularly Interspaced Short Palindromic Repeats / Cas ("CRISPR") technology known in the art.

[0189] The assay for testing whether a gene has been inactivated is known and described herein. In one embodiment, the assay is a Western blot of cell lysates probed with antibodies to B2M protein or CIITA protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) confirms the presence of inactivation changes.

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

[0191] In a preferred embodiment, a lentiviral vector is used to induce increased expression of the CD300LD gene in the low immunogenic 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, which comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:1.

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

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

[0196] In one embodiment, the CD300LD protein is a human CD300LD protein, which comprises the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to 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 antigen (MHC-I) function compared to parental pluripotent stem cells;

[0199] reduced endogenous major histocompatibility class II antigen (MHC-II) function compared to the parental pluripotent stem cell; and reduced sensitivity to NK cell and T cell killing compared to the parental pluripotent stem cell.

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

[0201] In one embodiment, the natural killer (NK) cell response triggered by the low immunogenic pluripotent stem cells is lower than the NK cell response triggered by the cloned DKO cells with B2M / CIITA biallelic knockout, and the DKO cells contain the changes that reduce the activity of B2M and CIITA proteins but do not contain the changes that cause increased CD300LD protein expression. In one embodiment, the NK cell response is measured by measuring the IFN-γ level 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 measuring the lethality of NK cells to the low immunogenic pluripotent stem cells or DKO cells by real-time label-free dynamic cell analysis (RTCA).

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

[0203] The present invention also provides a method for producing the low immunogenic pluripotent stem cells of the present invention, the method comprising: reducing the endogenous major histocompatibility class I antigen (MHC-I) function in the pluripotent stem cells; reducing the endogenous major histocompatibility class II antigen (MHC-II) function 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 comprises: 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 comprises: eliminating the activity of both alleles of the B2M gene in the pluripotent stem cells; eliminating the activity of both 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 cell can be reduced by the technique of "changing" or "genetic change" as described above. In some embodiments, the activity of the CIITA protein in the pluripotent stem cell can be reduced by the technique of "changing" or "genetic change" as described above. The technology, for example, introduces gene expression modification molecules, clustered regularly interspaced short palindromic repeats (CRISPR) technology, transcription activator-like effector nuclease (TALEN) technology, zinc finger nuclease (ZFN) technology or homologous recombination technology. In a preferred embodiment, the gene expression modification molecule comprises siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotide ASO (antisenseoligonucleotides) or anti-miRNA oligonucleotide AMO (Anti-miRNA oligonucleotides).

[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 to two ribonucleic acids (e.g., gRNA), which can guide the Cas protein to a target motif of a target polynucleotide sequence and hybridize with the target motif. 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, which can guide the Cas protein to a target motif of a target polynucleotide sequence and hybridize with the target motif.

[0208] In some embodiments, the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, one or more amino acid substitutions comprise conservative amino acid substitutions. In some cases, substitutions and / or modifications can prevent or reduce proteolytic degradation and / or extend the half-life of polypeptides in cells. In some embodiments, the Cas protein may comprise peptide bond replacements (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, lipidation, acetylation, 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 a Cas protein (also referred to as CASS2) of an Escherichia coli (E. coli) subtype. Exemplary Cas proteins of E. coli subtypes include, but are not limited to, Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, the Cas protein comprises a Cas protein (also referred to as CASS3) of a Ypest subtype. Exemplary Cas proteins of Ypest subtypes include, but are not limited to, Csy1, Csy2, Csy3, and Csy4. In some embodiments, the Cas protein comprises a Cas protein (also referred to as CASS4) of an Nmeni subtype. Exemplary Cas proteins of Nmeni subtypes include, but are not limited to, Csn1 and Csn2. In some embodiments, the Cas protein comprises a Cas protein (also referred to as CASS1) of a Dvulg subtype. Exemplary Cas proteins of Dvulg subtypes include, but are not limited to, Csd1, Csd2, and Cas5d. In some embodiments, the Cas protein comprises a Cas protein of the Tneap subtype (also known as CASS7). Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cst1, Cst2, and Cas5t. In some embodiments, the Cas protein comprises a Cas protein of the Hmari subtype. Exemplary Cas proteins of the Hmari subtype include, but are not limited to, Csh1, Csh2, and Cas5h. In some embodiments, the Cas protein comprises a Cas protein of the Apern subtype (also known as CASS5). Exemplary Cas proteins of the Apern subtype include, but are not limited to, Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, the Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6). Exemplary Cas proteins of the Mtube subtype 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 a Streptococcus pyogenes Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Streptococcus aureus Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Streptococcus thermophilus Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Neisseria meningitides Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Treponema denticola Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Cas9 protein from any bacterial species or a functional portion thereof. The Cas9 protein is a member of a type II CRISPR system, which typically includes a trans-encoded small RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas protein. The Cas9 protein (also referred to as a CRISPR-associated endonuclease Cas9 / Csn1) is a polypeptide comprising amino acids.

[0211] In one embodiment, the activity of B2M protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 gene editing technology.

[0212] In one embodiment, the activity of both alleles of the B2M gene in the pluripotent stem cells is eliminated by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 gene editing technology.

[0213] In one embodiment, the activity of CIITA protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 gene editing technology.

[0214] In one embodiment, the activity of both alleles of the CIITA gene in the pluripotent stem cells is eliminated by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 gene editing technology.

[0215] In one embodiment, the expression of CD300LD protein is increased by modification of the endogenous locus. In some embodiments, the endogenous locus is modified by the technique of "alteration" or "genetic alteration" as described above. The technique, for example, gene knock-in, clustered regularly interspaced short palindromic repeats (CRISPR) technology, transcription activator-like effector nuclease (TALEN) technology, zinc finger nuclease (ZFN) technology or homologous recombination technology.

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

[0217] In this article, known recombinant techniques can be used to produce expression constructs as described herein. In certain embodiments, the nucleic acid sequence encoding the target protein can be operably connected to one or more regulatory nucleotide sequences in the expression construct. The regulatory nucleotide sequence is 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 a variety of host cells. Generally, one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader sequences or signal sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancers or activator sequences. The expression construct used herein can use constitutive or inducible promoters known in the art. The promoter can be a naturally occurring promoter, or a hybrid promoter combining elements of more than one promoter. The expression construct can be present in the cell on an episome (e.g., a plasmid), or the expression construct can be inserted into a chromosome. In a specific embodiment, the expression vector includes a selectable marker gene to allow selection of transformed host cells. Some embodiments include an expression vector comprising a nucleotide sequence encoding the target protein operably connected to at least one regulatory sequence. The regulatory sequences used herein include promoters, enhancers, and other expression control elements. In certain embodiments, the expression vector is designed to select the host cell to be transformed, the desired protein to be expressed, the copy number of the vector, the ability to control the 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 techniques can be used to induce increased expression of the CD300LD gene in the low immunogenic pluripotent stem cells. The viral techniques 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 CD300LD protein is synthesized and constructed into a lentiviral vector, and then at least one copy of the CD300LD gene under the control of a promoter is introduced into the pluripotent stem cells via the lentiviral vector to increase the expression of CD300LD protein.

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

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

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

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

[0224] Prevention or treatment

[0225] The present invention further provides 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 preparing a drug for preventing or treating a disease requiring cell transplantation.

[0226] The low immunogenicity pluripotent stem cells of the present invention or the low immunogenicity pluripotent stem cells prepared by the method of the present invention can be induced to differentiate into different cells, which can be used for different prevention or treatment purposes, to prevent or treat different diseases. As will be appreciated by those skilled in the art, the differentiation method depends on the desired cell type using known technology. For example, the cell suspension differentiation can be made into a gel matrix form, such as matrigel, gelatin or fibrin / thrombin form, to promote cell survival. Usually differentiation can be determined as known in the art by assessing the presence of cell-specific markers. For example, cells can be differentiated into cardiomyocytes, neurons, glial cells, endothelial cells, T cells, NK cells, NKT cells, macrophages, hematopoietic progenitor cells, mesenchymal cells, 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, and the cancer comprises solid tumors and blood tumors. In some embodiments, the solid tumor comprises small cell lung cancer, breast cancer, testicular cancer, neuroblastoma, ovarian cancer or melanoma. In some embodiments, the blood tumor comprises acute leukemia, chronic leukemia, lymphoma, myelodysplastic syndrome or multiple myeloma.

[0228] In one embodiment, the disease comprises aplastic anemia.

[0229] In one embodiment, the disease comprises a congenital immunodeficiency disease.

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

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

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

[0233] In some embodiments, the disease is a metabolism-related disease comprising type II diabetes, scurvy, hypoglycemia, hyperlipidemia, or osteoporosis.

[0234] Differentiation of pluripotent stem cells

[0235] The present invention provides pluripotent stem cells that can be differentiated into different cell types for subsequent transplantation into a recipient subject. As will be appreciated by those skilled in the art, differentiated low immunogenic pluripotent cell derivatives can be transplanted using techniques known in the art, depending on the cell type and the ultimate use of these cells.

[0236] 1. Cardiomyocytes differentiate from pluripotent stem cells

[0237] The present invention provides pluripotent stem cells that can be differentiated into different cardiac cell types for subsequent transplantation or implantation into a subject (eg, a recipient).

[0238] As will be appreciated by those skilled in the art, the differentiation method depends on the desired cell type using known techniques. Exemplary cardiac cell types include, but are not limited to, cardiomyocytes, nodal cardiomyocytes, conducting 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, heart valve interstitial cells, cardiac pacemaker marker cells, etc.

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

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

[0241] In some embodiments, the cardiomyocytes described herein are administered to a recipient subject to treat a cardiac disorder selected from the group consisting of pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, idiopathic cardiomyopathy, other cardiomyopathies, myocardial ischemia-reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina, stroke, Wet heart disease, arterial inflammation, cardiovascular disease, myocardial infarction, myocardial ischemia, congestive heart failure, myocardial infarction, myocardial ischemia, heart damage, 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 trophy, muscle mass abnormalities, muscle degeneration, myocarditis, infectious myocarditis, drug- or toxin-induced muscle abnormalities, allergic myocarditis, and autoimmune endocarditis.

[0242] In some embodiments, the method of producing a low immunogenic cardiomyocyte population from a low immunogenic pluripotent (HIP) stem cell population by in vitro differentiation includes: (a) culturing a HIP cell population in a culture medium comprising a GSK inhibitor; (b) culturing a HIP cell population in a culture medium comprising a WNT antagonist to produce a pre-cardiomyocyte population; (c) culturing a pre-cardiomyocyte population in a culture medium comprising insulin to produce 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 about 2mM to about 10mM. 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 about 2mM to about 10mM.

[0243] Other useful methods for differentiating induced pluripotent stem cells or multipotent stem cells into cardiomyocytes are described in, for example, US2017 / 0152485; US2017 / 0058263; US2017 / 0002325; US2016 / 0362661; US2016 / 0068814; US9,062,289; US7,897,389; and US7,452,718. Other methods for generating cardiac cells from induced pluripotent stem cells or multipotent stem cells are described in, for example, 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 embodiments, low immunogenic cardiomyocytes can be cultured in a medium comprising BMP pathway inhibitors, WNT signaling activators, WNT signaling inhibitors, WNT agonists, WNT antagonists, Src inhibitors, EGFR inhibitors, PCK activators, cytokines, growth factors, cardiomyogenic agents, other compounds, etc.

[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 agents for generating cardiomyocytes from iPSCs include activin A, BMP-4, Wnt3a, VEGF, soluble Frizzled, cyclosporin A, angiotensin II, phenylephrine, ascorbic acid, dimethyl sulfoxide, 5-aza-2'-deoxycytidine, and the like.

[0247] 2. Differentiation of pluripotent stem cells into neural cells

[0248] The present invention provides pluripotent stem cells that can be differentiated into different neural cell types for subsequent transplantation or implantation into a recipient subject. As will be appreciated by those skilled in the art, 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, and the like.

[0249] In some embodiments, the neural cells are administered to a subject to treat Parkinson's disease, Huntington's disease, multiple sclerosis, other neurodegenerative diseases or disorders, attention deficit hyperactivity disorder (ADHD), Tourette syndrome (TS), schizophrenia, psychosis, depression, other neuropsychiatric disorders. In some embodiments, the neural cells described herein are administered to a subject 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, brain 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, noradrenergic neurons, GABAergic interneurons are administered to patients who have experienced epileptic 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, by culturing cells in a culture medium comprising one or more factors that promote the production of brain endothelial cells or neural cells, brain endothelial cells (EC), their precursors and progenitor cells are differentiated from multipotent stem cells (e.g., induced pluripotent stem cells) on the surface. In some cases, the culture medium comprises one or more of the following: CHIR-99021, VEGF, basic FGF and Y-27632. In some embodiments, the culture medium includes a supplement designed to promote the survival and function of neural cells. In some embodiments, brain endothelial cells (EC), their precursors and progenitor cells are differentiated from multipotent stem cells on the surface by culturing cells in non-conditional or conditioned medium. In some cases, the culture medium comprises factors or small molecules that promote or promote differentiation. In some embodiments, the medium comprises 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 comprises one or more extracellular matrix proteins. In some cases, as known in the art, differentiation is usually determined by assessing the presence of cell-specific markers. In some embodiments, the brain endothelial cells express or secrete a factor selected from CD31, VE-cadherin, and a combination thereof.

[0252] In some embodiments, neurons, their precursors and progenitor cells are differentiated from pluripotent stem cells by culturing cells in a medium comprising 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 derivatives thereof. In some embodiments, the SHH signaling activator is selected from the group consisting of Smoothened agonist (SAG), SAG analogs, SHH, C25-SHH, C24-SHH, purmorphamine, Hg-Ag, and derivatives thereof.

[0254] In some embodiments, stem cells described herein differentiate into dopaminergic neurons, including dopaminergic progenitor cells. Stem cells are cultured in a differentiation medium comprising a supplement or additive to induce neuronal differentiation. In some embodiments, cells are cultured in the presence of a supplement or additive to induce floor plate cells. In some embodiments, supplements or additives include BMP inhibitor LDN193189, ALK-5 inhibitor A83-01, Smoothened agonist purmorphamine, FGF8, GSK3 inhibitor CHIR99021, neurotrophic factors derived from glial cell lines, GDNF, ascorbic acid, brain-derived neurotrophic factors BDNF, dibutyryladenosine cyclic monophosphate dbcAMP, ROCK inhibitor Y-27632, etc.

[0255] In some embodiments, a method for generating a low immunogenic dopaminergic neuron population from a low immunogenic induced pluripotent stem cell (HIP cell) population by in vitro differentiation includes (a) culturing a HIP cell population in a first culture medium, wherein the first culture medium comprises one or more factors selected from sonic hedgehog factor (SHH), BDNF, EGF, bFGF, FGF8, WNT1, retinoic acid, GSK3 inhibitor, ALK inhibitor, and ROCK inhibitor to produce a population of immature dopaminergic neurons; (b) culturing an immature dopaminergic neuron population in a second culture medium different from the first culture medium to produce a 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 2mM to about 10mM. 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 1mM to about 10mM. In some embodiments, the first culture medium and / or the second culture medium does not contain animal serum.

[0256] Methods for differentiating pluripotent stem cells are described in, for example, 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 neurons derived from stem cells and methods for their preparation can be found, for example, in 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, "Chapter 8 - Strategies for bringing stem cell-derived dopamine neurons into the clinic - the NYSTEM trial," in Progress in Brain Research, 2017, Vol. 230, p. 15. 191-212; Liu et al., Nat Protoc, 2013, 8:1670-1679; Upadhya et al., Curr Protoc Stem Cell Biol, 38, 2D.7.1-2D.7.47; the contents of U.S. patent applications 20160115448 and US8,252,586; US8,273,570; US9,487,752 and US10,093,897 are incorporated herein by reference in their entirety.

[0257] In some embodiments, glial cells are produced by differentiating pluripotent stem cells into therapeutically effective glial cells, including microglia, astrocytes, oligodendrocytes, ependymal cells and Schwann cells, glial precursors and glial progenitor cells. Differentiation of low immunogenic pluripotent stem cells produces low immunogenic neural cells, such as low immunogenic glial cells.

[0258] In some embodiments, glial cells, precursors and progenitors thereof are produced by culturing pluripotent stem cells in a medium comprising one or more agents selected from retinoic acid, IL-34, M-CSF, FLT3 ligand, GM-CSF, CCL2, TGFβ inhibitor, BMP signaling inhibitor, SHH signaling activator, FGF, platelet-derived growth factor PDGF, PDGFR-α, HGF, IGF-1, noggin. 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 embodiments, it is determined that the glial cells express known glial cell biomarkers. Useful methods for generating glial cells, their precursor cells and progenitor cells from stem cells can be found in, for example, 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 embodiments, differentiation of pluripotent stem cells is performed by exposing or contacting the cells with specific factors known to give rise to specific cell lineages in order to target their differentiation to specific, desired lineages and / or cell types.

[0262] In some embodiments, terminally differentiated cells show special phenotypic properties or characteristics.In certain embodiments, stem cell differentiation as described herein is into neuroectoderm, neuron, neuroendocrine, dopaminergic, cholinergic, serotonergic (5-HT), glutamatergic, GABAergic, adrenergic, noradrenergic, sympathetic neuron, parasympathetic neuron, sympathetic peripheral neuron or glial cell group.In some cases, glial cell colony includes microglia cell colony or macroglia (central nervous system cells: astrocytes, oligodendrocytes, ependymal cells and radial glial cells; With peripheral nervous system cells: Schwann cells and satellite cells) cell colony, or the precursor and progenitor cell of any aforementioned cell.

[0263] Protocols for generating different types of neural cells are described in PCT Application No. WO2010144696, U.S. Patent Nos. 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 effects of neural cell transplantation in animal models of neurological diseases or disorders 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, for example, 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, successful transplantation may show that there are transplanted source cells in the lesion after 2-5 weeks, which differentiate into astrocytes, oligodendrocytes and / or neurons, and migrate along the spinal cord from the lesion end, and gait, coordination and weight bearing are improved. According to the type of nerve cells and the neurological disease or disorder to be treated, a specific animal model is selected, and the nerve cells can be used in a manner that allows them to be transplanted to the expected tissue site and to rebuild or regenerate the functional defect area. For example, according to the disease treated, the nerve cells can be directly transplanted to the substance or intrathecal position of the central nervous system. In some embodiments, any nerve cell described herein, including brain endothelial cells, neurons, dopaminergic neurons, ependymal cells, astrocytes, microglia, oligodendrocytes and Schwann cells, is injected, and enters the patient's body by intravenous, intraspinal, intraventricular, intrathecal, intraarterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, intraabdominal, intraocular, posterior to the ball and a combination thereof. In some embodiments, cells are injected or deposited in the form of push or continuous infusion. In certain embodiments, the neural cells are administered by injection into the brain, near the brain, and combinations thereof. For example, the injection can be performed through a burr hole made in the skull of the subject.Suitable sites for administering neural cells to the brain include, but are not limited to, the cerebral ventricles, lateral ventricles, cerebellar cisterna, putamen, basal ganglia, hippocampal cortex, striatum, caudate region of the brain, and combinations thereof.

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

[0265] 3. Differentiation of endothelial cells from pluripotent stem cells

[0266] The present invention provides pluripotent stem cells that can be differentiated into various endothelial cell types for subsequent transplantation or implantation into a subject (e.g., a recipient). As will be appreciated by those skilled in the art, 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, kidney endothelial cells, brain endothelial cells, liver endothelial cells, etc.

[0267] In some embodiments, the endothelial cells described herein are administered to a recipient subject to treat a vascular disorder 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 damage, reperfusion injury, limb ischemia, stroke, neuropathy (e.g., peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver failure, renal failure, etc.), diabetes, rheumatoid arthritis, osteoporosis, cerebrovascular disease, hypertension, angina pectoris and myocardial infarction caused by coronary heart disease, renovascular hypertension, renal failure caused by renal artery stenosis, leg claudication mites, and other vascular conditions or diseases.

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

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

[0270] 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 about 1 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 about 1 pM to about 20 pM. 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 about 0.5 pM to about 10 pM.

[0271] In some embodiments, the first culture medium comprises 2 pM to about 10 pM CHIR-99021. In some embodiments, the second culture medium comprises 50 ng / ml VEGF and 10 ng / ml bFGF. In other embodiments, the second culture medium further comprises Y-27632 and SB-431542. In various embodiments, the third culture medium comprises 10 pM Y-27632 and 1 pM SB-431542. In certain embodiments, the third culture medium further comprises VEGF and bFGF. In certain instances, the first culture medium and / or the second culture medium does not contain insulin.

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

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

[0274] 4. Thyroid cells differentiate from pluripotent stem cells

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

[0276] 5. Differentiation of pluripotent stem cells into hepatocytes

[0277] In some embodiments, pluripotent stem cells can be differentiated into hepatocytes to address hepatocyte function loss or cirrhosis. There are a variety of techniques 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 are particularly directed to methods and reagents for differentiation. As known in the art, differentiation is usually determined 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 measured functionally, 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] The present invention provides pluripotent stem cells that can be differentiated into various islet cell types for subsequent transplantation or implantation into a subject (e.g., a recipient). As will be appreciated by those skilled in the art, differentiation methods depend 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, the islet cells are derived from the low immunogenic pluripotent cells described herein. Useful methods for differentiating pluripotent stem cells into islet cells are described in, for example, US9,683,215; US9,157,062; and US8,927,280.

[0281] In some embodiments, low immunogenic pluripotent cells are differentiated into beta-like cells or pancreatic islet organoids for transplantation to solve type I diabetes (T1DM). Cell systems are a promising approach to solving T1DM, see Nat Rev Gastroenterol Hepatol by Ellis et al. October 2017; 14(10): 612-628, incorporated herein by reference. In addition, Pagbuca et al. (Cell, 2014, 159(2): 428-39) reported successful differentiation of b cells from hiPSC, the contents of which are incorporated herein by reference in their entirety, particularly the methods and reagents for large-scale production of functional human b cells from human pluripotent stem cells outlined therein). In addition, Vegas et al. generated human B cells from human pluripotent stem cells and then encapsulated to avoid host immune rejection; Vegas et al., Nat Med, 2016, 22(3):306-11, the entire contents of which are incorporated herein by reference, especially the methods and reagents outlined therein for large-scale production of functional human B cells from human pluripotent stem cells.

[0282] In some embodiments, the method of producing a low immunogenic islet cell population from a low immunogenic multipotent cell population by in vitro differentiation includes: (a) culturing a HIP cell population in a first culture medium comprising one or more selected from insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (SHH) factors and vascular endothelial growth factor (VEGF), transforming growth factor-b (TORb) superfamily, bone morphogenetic protein-2 (BMP2), bone morphogenetic protein-7 (BMP7), GSK inhibitors, ALK inhibitors, BMP type 1 receptor inhibitors and retinoic acid to produce immature islet cell populations; (b) culturing immature islet cell populations in a second culture medium different from the first culture medium to produce low immune islet cell populations. 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 about 2mM to about 10mM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some cases, the concentration of the ALK inhibitor ranges from about 1 pM to about 10 pM. In some embodiments, the first culture medium and / or the second culture medium does not contain animal serum.

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

[0284] As known in the art, differentiation is usually determined by assessing the presence of b cell-related or specific markers (including but not limited to insulin). Differentiation can also be measured functionally, such as measuring glucose metabolism, generally referring to Muraro et al., Cell Syst. October 26, 2016; 3 (4): 385-394.e3, hereby incorporated by reference in its entirety, and particularly for the biomarkers outlined therein. Once beta cells are produced, they can be transplanted (either as a cell suspension or in a gel matrix discussed herein) to the portal vein / liver, omentum, gastrointestinal mucosa, bone marrow, muscle or subcutaneous sac.

[0285] Additional description of pancreatic islet cells comprising dopaminergic neurons for use in the present 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] The invention provides low immunogenicity pluripotent cells, which can be differentiated into various RPE cell types, for subsequently transplanting or being implanted in a subject (e.g., a recipient). As will be appreciated by those skilled in the art, differentiation methods depend on the desired cell type using known techniques. Exemplary RPE cell types include but are not limited to retinal pigment epithelium (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 in, for example, US9, 458, 428 and US9, 850, 463, and these disclosures are incorporated herein by reference in their entirety, including specification sheets. Other methods for producing RPE cells from human induced pluripotent stem cells can be found in, for example, Lamba et al., PNAS, 2006, 103 (34): 12769-12774; Melough et al., stem cell, 2012, 30 (4): 673-686; Idelson et al., cell stem cell, 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 embodiments, the RPE cells described herein are administered to a subject to treat an ocular condition selected from wet macular degeneration, dry macular degeneration, juvenile macular degeneration (e.g., Stargardt's disease, Best disease, and juvenile retinoschisis), Leber's congenital melanoblastoma, retinitis pigmentosa, retinal detachment, age-related macular degeneration (AMD), early AMD, intermediate AMD, late AMD, non-neovascular age-related macular degeneration.

[0290] Human pluripotent stem cells have been differentiated into RPE cells using Kamao et al., Stem Cell Reports 2014: 2: The technology outlined in 205-18, the document is incorporated herein by reference in its entirety, particularly the method and reagent 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 to understand the technology for generating RPE cell sheets and being transplanted into patients. Differentiation can be measured as known in the art, usually by assessing the presence of RPE related and / or specific markers or by functional measurement. 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 particularly for the markers outlined in the first paragraph of the results section.

[0291] In some embodiments, the method of producing a low immunogenic retinal pigment epithelium (RPE) cell group from a low immunogenic multipotent cell group by in vitro differentiation includes: (a) cultivating a low immunogenic multipotent cell group in a first culture medium, wherein the first culture medium includes a pre-RPE cell group selected from activin A, bFGF, BMP4 / 7, DKK1, IGF1, noggin, BMP inhibitor, ALK inhibitor, ROCK inhibitor and VEGFR inhibitor; (b) culturing a pre-RPE cell group in a second culture medium different from the first culture medium to produce a low immunogenic RPE cell group. In some embodiments, the ALK inhibitor is SB-431542, its derivative or its variant. In some cases, the concentration range of the ALK inhibitor is about 2mM to about 10mM. In some embodiments, the ROCK inhibitor is Y-27632, its derivative or its variant. In some cases, the concentration range of the ROCK inhibitor is about 1pM to about 10pM. In some embodiments, the first culture medium and / or the second culture medium do not contain animal serum.

[0292] Differentiation can be determined as known in the art, typically by assessing the presence of RPE-related and / or specific markers or by functional measurement.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 in particular for the results section.

[0293] Additional description of RPE cells for use in the present 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] The present invention provides low immunogenic pluripotent cells that can be differentiated into natural killer (NK) cells. In some embodiments, the method of generating a natural killer (NK) cell population from a low immunogenic pluripotent cell population by in vitro differentiation comprises: (a) culturing a stem cell population in a first culture medium comprising a ROCK inhibitor under conditions sufficient to form aggregates; (b) culturing the aggregates in a second culture medium comprising BMP-4; (c) culturing the aggregates in a third culture medium comprising BMP-4, FGF2, a WNT pathway activator, and activin A; (d) culturing the aggregates in a third culture medium comprising FGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT (e) culturing the cell population in a fifth culture medium comprising FGF2, VEGF, TPO, SCF, IL-3 and FLT3L; (f) culturing the cell population in a sixth culture medium comprising IL-3, IL-7, FLT3L, IL-15 and SCF; and (g) culturing the cell population in a seventh culture medium comprising IL-7, FLT3L, IL-15 and SCF for a time sufficient to produce NK cells.

[0296] In some embodiments, the first culture medium comprises 10 μM of ROCK inhibitor. In some embodiments, the second culture medium comprises 30 ng / mL BMP-4. In some embodiments, the second culture medium comprises 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 include 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 includes 5 ng / mL IL-3. In some embodiments, the eighth culture medium includes IL-7, FLT3L, IL-15, SCF, and nicotinamide.

[0297] Additional description of NK cells for use in the present 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 can generally be provided in the form of a pharmaceutical composition comprising an isotonic excipient and prepared under sufficiently sterile conditions for human administration. For general principles of pharmaceutical preparations of cell compositions, see "Cell Therapy: Stem Cell Transplantation, Gene Therapy and Cellular Immunotherapy", edited by Morstyn & Sheridan, Cambridge University Press, 1996; and "Hematopoietic Stem Cell Therapy", ED Ball, J. Lister & P. ​​Law, Churchill Livingstone, 2000. The cells can be packaged in a device or container suitable for distribution or clinical use.

[0299] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0300] The reagents and raw materials used in the present invention are commercially available.

[0301] The positive and progressive effects of the present invention are that the pluripotent stem cells of the present invention and the low-immunogenic 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 abilities; (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; thereby exhibiting excellent application potential.

[0302] The present invention is further described by reference to the following examples. It should be understood that these examples are intended to be illustrative only and are not intended to limit the present invention. The following materials and instruments are commercially available or prepared according to methods known in the art. The following experiments were performed according to the manufacturer's instructions or according to methods and procedures known in the art.

[0303] The experimental methods for which specific conditions are not specified in the following examples are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

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

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

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

[0307] In the following examples, human induced pluripotent stem cells (iPSC cells) were used to construct the target cell line. The human induced pluripotent stem cells (iPSC cells) used were prepared according to the manufacturer's instructions using CTS TM CytoTune TM -iPS 2.1 Sendai virus reprogramming kit (CTS TM CytoTune TM-iPS2.1Sendai Reprogramming Kit, Catalog No.: A34546) is prepared from human peripheral blood cells. Human pluripotent stem cell lines H1 (Wicell, WA01) or H9 (Wicell, WA09) can also be used to construct target cell lines, and the cell culture and gene knockout reagents used are shown in Table 1.

[0308] Table 1. Cell culture and gene knockout reagents

[0309]

[0310] CRISPR / CAS9 was used to knock out β-2-microglobulin (B2M) in the endoplasmic reticulum, so that MHC-I on the cell surface could not form functional molecules, thereby escaping allogeneic CD8 + The killing of T cells; the killing of escaped CD4+T cells is achieved by knocking out CIITA, a positive regulator of MHC-II gene transcription, thereby reducing the expression of MHC-II class molecules. In the present 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] The CRISPR / CAS9 gene knockout strategy of B2M and the gRNA sequences and primers used are as follows Figure 1 As shown in Table 2, B2M-gRNA1 and B2M-gRNA2 (EasyEdit sgRNA, GenScript) were used to directly knock out the B2M exon segment at both ends, and 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 is the knockout strategy of the B2M gene in the B2M and CIITA double knockout cell lines (DKO). Figure 1 B is the result of PCR-based validation of B2M gene knockout in H1 cell line. Figure 1 C shows the result of gene knockout verification of iPSC cell line B2M by PCR.

[0312] In addition, CIITA's CRISPR / CAS9 gene knockout strategy and the gRNA sequences and identification primers used are as follows Figure 2 As shown in Table 2, CIITA-gRNA1 and CIITA-gRNA2 (EasyEdit sgRNA, GenScript) were used to directly knock out the CIITA exon segment at both ends, and then the genome sequence knockout was verified using two pairs of PCR primers CIITA-F1 / R1 and CIITA-F2 / R2, respectively. Figure 2A is the knockout strategy of CIITA gene in B2M and CIITA double knockout cell lines (DKO). Figure 2 B is the result of PCR-based validation of the CIITA gene knockout in the H1 cell line. Figure 2 C shows the result of gene knockout verification of CIITA in iPSC cell line by PCR.

[0313] Table 2. gRNA sequences and identification primers

[0314]

[0315]

[0316] The specific operations are as follows:

[0317] 1) Human induced pluripotent stem cells were cultured in mTeSR1 medium supplemented with Y-27632 on a Matrigel-coated 6-well plate to 80% density. After digestion with TrypLE, DMEM / F12 was added for neutralization and counting. 2×10 6 The cells were placed in EP tubes and the supernatant was discarded after centrifugation.

[0318] 2) According to the Neon transfection system (ThermoFisher), 100 μL electroporation system was added with 15 μg TrueCut TM Cas9Protein+3μgRNA(B2M-gRNA1+B2M-gRNA2+CIITA-gRNA1+CIITA-gRNA2)composes a ribonucleoprotein complex (RNP) system, which is mixed and placed at room temperature for 20 minutes.

[0319] 3) Resuspend the cells with 100 μL RNP electroporation system and electroporate with Neon transfection system. The electroporation parameters are 1200V, 30ms, 1pause. Quickly add preheated culture medium to the electroporated cells and then evenly inoculate them in one well of a 6-well plate coated with Matrigel.

[0320] 4) Replace the mTeSR1 medium with fresh one every day. When the single cells grow up, pick a single clone in a 48-well plate. After the clone is expanded, collect the genome sample for PCR detection of gene editing. The PCR results are as follows: Figure 1 and Figure 2 PCR positive clones were sent to the company for Sanger sequencing for further verification.

[0321] 5) Expand, culture and freeze the positive B2M / CIITA biallelic knockout clones.

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

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

[0324] qPCR was used to detect the expression of B2M and CIITA at the RNA level in DKO cells derived from cloned H1 cells with B2M / CIITA biallelic knockout. 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 Roche 480II instrument, the reaction system is as follows:

[0330] Pre-incubation, 95℃, 30s.

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

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

[0333] qPCR results 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 clones DKO was almost absent, confirming the successful knockout of B2M and CIITA.

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

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

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

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

[0338] IFN-γ (PeproTech, Cat#300-02) was used to stimulate WT and DKO to detect HLA class I / II molecules on the surface of H1 cells (WT) and iPSC (iPSC-WT) cell lines and their corresponding derived DKO cells (DKO cells and iPSC-DKO cells). The specific detection method is as follows:

[0339] The cells were plated, and the medium containing IFN-γ was added to the cells when the medium was changed on the next day. After 48 hours, the cells were digested and the expression of HLA-I / II was detected using a flow cytometer (Agilent Technologies, NovoCyte).

[0340] The flow cytometry results are as follows Figure 5A and Figure 5B As shown, Figure 5A Results of the H1 cell and DKO cell tests. Figure 5B The results of iPSC cell and iPSC-DKO cell detection, and all the figures have HLA-ABC on the left to detect HLA-I molecules; HLA-DR, DQ, DP on the right to detect HLA-II molecules; T cells are the positive control for detection. It was observed that the clone H1 with B2M / CIITA biallelic knockout and DKO cells derived from iPSC cell lines (DKO cells and iPSC-DKO cells) could not express HLA-I / II molecules in response to IFN-γ stimulation, proving that the HLA-I and HLA-II functions of DKO cells and iPSC-DKO cells were reduced.

[0341] 1.5DKO Karyotype Detection

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

[0343] The chromosome specimen fixed on the slide is treated with trypsin and then stained with Giemsa stain. Based on the chromosome length, centromere position, long-short arm ratio, satellite presence and other characteristics, the chromosome number and morphological structure of the metaphase chromosome are analyzed to determine whether its karyotype is consistent with the normal karyotype.

[0344] The results of karyotyping are as follows Figure 6 As shown, the karyotype of H1 cell-derived DKO cells was normal, with no significant changes compared with the normal karyotype.

[0345] Example 2. Stemness and immune function of DKO cell lines

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

[0347] 2.1 Expression of stemness genes in DKO cells

[0348] The protein levels of stemness genes 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 stemness genes at the RNA level in H1 cells (WT cells) and DKO cells (DKO cells) was detected by RT-qPCR, and the expression of the corresponding proteins of stemness genes on the surface of H1 cells (WT cells) and 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 detection: WT or DKO cells were plated in 12-well plates, and the medium was discarded after the cells grew to 60-80% density, and 4% paraformaldehyde was added for fixation. After the cell membrane was broken, the primary antibodies of POU5F1 and NANOG and other stem gene proteins were incubated at 4°C overnight, and the primary antibodies were washed away and incubated at room temperature with fluorescently labeled secondary antibodies, and then photographed using a fluorescence microscope (Nikon Ts2R-FL).

[0350] RT-qPCR detection:

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

[0352] Pre-incubation, 95℃, 30s.

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

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

[0355] Mesenchymal stem cells (MSC) were used as a negative control for stemness gene expression.

[0356] Flow cytometry:

[0357] After the cells were collected, the antibodies were incubated in EP tubes at 4°C in the dark for 30 min, and then the appropriate fluorescence acquisition channel was selected according to the antibody information, and the fluorescence was detected on the machine. All antibodies were from BD Biosciences.

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

[0359] Immunofluorescence test results Fig. 7A As shown in Figure 2, both H1 cell WT and H1 cell-derived DKO cells expressed stemness genes POU5F1 and NANOG at the protein level, and there was no significant difference in the expression of stemness genes POU5F1 and NANOG in H1 cell-derived DKO cells compared with WT cells. Figure 7B As shown in Figure 2, H1 cell WT and H1 cell-derived DKO cells expressed stemness genes POU5F1, NANOG, and SOX2 at the RNA level, and there was no significant difference in the expression of stemness genes POU5F1, NANOG, and SOX2 in H1 cell-derived DKO cells compared with H1 cell WT cells. Figure 7CAs shown, both H1 cell WT and H1 cell-derived DKO cells highly expressed stemness gene corresponding proteins SSEA-4 (WT 100% and DKO 99.98%) and Tra1-81 (WT 96.75% and DKO 99.13%) on their cell surfaces. Fig.7D The results of flow cytometry detection of 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 showed that both iPSC-WT and iPSC-derived DKO cells highly expressed the corresponding proteins of stemness genes on their surfaces. Fig. 7E The results of immunofluorescence detection of the protein levels of stemness genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in iPSC-WT cells. The corresponding proteins of stemness genes are highly expressed on the surface of iPSC-WT cells. Figure 7F The results of immunofluorescence detection of the protein levels of stemness genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in iPSC-DKO cells show that the corresponding proteins of stemness genes are highly expressed on the surface of iPSC-derived DKO cells.

[0360] Differentiation ability of 2.2DKO cells

[0361] In this example, the differentiation ability of DKO cells was detected. The specific detection method was as follows: 100 μL of 5×10 5 DKO cell suspension, wait until the teratoma volume is larger than 1.5cm 3 The sections were then removed and subjected to paraffin sectioning and hematoxylin and eosin staining.

[0362] The staining results showed that DKO cells derived from H1 cells with B2M / CIITA biallelic knockout could form teratomas in vivo and differentiate into cells of the three germ layers: endogenous, meso and ectogenetic. DKO cells derived from H1 cells had normal differentiation ability of the three germ layers.

[0363] 2.3DKO Cell Immune Function

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

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

[0366] Table 3. Reagents used in the killing experiment

[0367] name Cat No. Specification factory PE anti-human CD4 980804 500μl / tube Biolegend APC anti-human CD8 980904 500μl / 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 50μg R&D Y-27632 2HCl S1049 5mg Selleck Matrigel 354277 5ml Gibco <![CDATA[Essential 8 TM Culture medium]]> A1517001 500ml Gibco E-Plate VIEW 96 PET 300601030 6 pieces / box Agilent

[0368] RTCA results are as follows Fig. 8A and Figure 8B As shown in the figure, WT cells escape NK cell killing due to the expression of HLA-I, but will be killed by T cells. DKO cells can escape T cell killing and are more sensitive to NK cell killing. Fig. 8A The three figures above are the results of RTCA detection of the killing rate of NK cells against H1 cell WT and H1 cell-derived DKO cells, that is, the detection results of the immune escape function of H1 cell WT and H1 cell-derived DKO cells against NK cells; Fig. 8A The three figures in the second row are the results of RTCA detection of the killing rate of T cells against H1 cell WT and H1 cell-derived DKO cells, that is, the detection results of the immune escape function of WT cells and H1 cell-derived DKO cells against T cells. Figure 8B The three figures above are 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 figures in the second row are 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 of DKO+CD47 cell line and verification of immune function

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

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

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

[0373] The lentiviral vector was digested with BamHI / NheI, and the nucleic acid sequence encoding CD47 protein (SEQ ID NO: 6) was connected to the lentiviral vector. After successful connection, Sanger sequencing was used to verify the correctness of the inserted sequence and viral packaging was performed. The lentiviral vector was transfected into the DKO cells constructed in Example 1, and the medium was changed after 24 hours, and after 48 hours, the medium was changed to a medium with puromycin for screening.

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

[0375] Test results such as Fig. 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 subsequently functionally tested.

[0376] 3.2 Verification of the immune function of DKO+CD47

[0377] RTCA was used to detect whether the overexpressed DKO+CD47 cell line could successfully escape the killing of NK cells while escaping the killing of T cells. For the specific detection method, see Example 2.3.

[0378] RTCA test Fig.11 As shown, NK cells can effectively kill DKO cells, while WT and DKO+CD47 overexpressing cells can escape NK killing.

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

[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) is 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) driven by EF1a and carrying a puromycin selection marker. The structure of the pGC-EF1a vector is as follows: Fig. 9As shown. The specific operation is as follows: the vector is cut with BamHI / NheI, and the nucleic acid sequence of the above-synthesized CD300LD is connected to the lentiviral vector. After successful connection, Sanger sequencing is used to verify the correctness of the inserted sequence and perform viral packaging. The lentiviral vector is transfected into the iPSC-DKO cells constructed in Example 1, and the medium is changed after 24 hours, and after 48 hours, it is replaced with a medium containing puromycin for screening.

[0390] Overexpression detection was performed on the constructed iPSC-DKO+CD300LD cell line. qRT-PCR was used to detect the overexpression level of mRNA, iPSC-WT cells were used as negative control, and the primers used were shown in Table 4. FACS was used to detect the overexpression level of protein, iPSC-WT cells were used as negative control cells, and isotype was used as a negative control for the detection signal.

[0391] Table 4. qPCR detection primers for CD300LD

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

[0393] The detection results showed that CD300LD was highly expressed in the constructed iPSC-DKO+CD300LD cell line.

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

[0395] The expression of stemness genes in the DKO+CD300LD cell line was detected by immunofluorescence and flow cytometry to confirm that the constructed cells had stemness. The specific detection method is shown in Example 2.1. The results of stemness gene immunofluorescence detection are shown in Fig.12 As shown, the results of stem gene flow cytometry test are shown in Fig.13 As shown. Immunofluorescence detection 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 the stemness genes SSEA-4, TRA-1-60, Tra1-81, and OCT4 were highly expressed on the surface of iPSC-DKO+CD300LD cells.

[0396] 4.3 Differentiation ability of the three germ layers of iPSC-DKO+CD300LD cell line

[0397] Use DKO+CD300LD cells to detect the three-germ layer differentiation ability. In order to generate mesodermal, endoderm, and ectoderm cells, it is necessary to resuspend the dissociated iPSC-DKO+CD300LD single cells respectively using the three germ layer culture media supplemented with Y27632, and attach an appropriate amount of cells to the matrix gel-coated well plate with a cell crawler. After 24 hours, replace it with preheated differentiation medium, and change the medium every day until the seventh day to obtain mesodermal, endoderm, and ectoderm cells. The expression of the three germ layer marker proteins was detected by immunofluorescence to detect whether the iPSC-DKO+CD300LD cell line has the ability to differentiate into the three germ layers. The expression results of the three germ layer marker proteins are shown in Fig.14 As shown. The results of immunofluorescence detection showed that iPSC-DKO+CD300LD cells expressed ectoderm marker proteins: PAX6 and GAD1; mesoderm marker proteins: Brachyury and NCAM; 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, experiments were performed using NK cells, PBNK (T cell + NK cell mixture), and macrophages. The escape function of iPSC-DKO+CD300LD cells against different immune cells was detected by RTCA. For specific detection methods, see Example 2.3.

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

[0400] NK cell killing assay was performed on the XCelligence platform (ACEA BioSciences). Each type of iPSC cell (iPSC-WT cells, iPSC-DKO cells, iPSC-DKO+CD300LD cells) was resuspended in 100 μl of cell-specific culture medium and plated on a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, NK cells were added at an E:T ratio of 1:1. The data were normalized and analyzed using RTCA software (ACEA). The killing rate test results are shown in Fig.15 As shown, Fig.15 A and Fig.15 B shows the results of the NK cell killing experiment on iPSC-DKO+CD300LD cells detected by RTCA, wherein Fig.15B is a multiple killing statistics chart, DKO cell line N = 6, DKO + CD300LD cell line N = 5, WT cell line N = 7. The results show that iPSC DKO + CD300LD cells can significantly escape NK killing.

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

[0402] NK activation factors were added to PBMC in advance to increase the proportion of NK cells in PBMC and the killing performance of T cells. The activated mixed lymphocytes (PBMCs) were used as effector cells for RTCA experiments to comprehensively evaluate the immune escape ability of iPSC-DKO+CD300LD cells (method reference PMID: 33309274). The specific operation was to perform PBMC cell killing assay 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 plated on a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, PBMC cells were added at an E:T ratio of 2:1. The data were standardized and analyzed using RTCA software (ACEA). The killing rate detection results are shown in the figure. Fig.16 As shown, Fig.16 A and Fig.16 B shows the results of the RTCA-detected PBMC killing experiment on iPSC-DKO+CD300LD cells, wherein Fig.16 B is a multiple killing statistics chart, N = 4. The results show that iPSC-DKO+CD300LD cells can significantly escape the killing of PBMC.

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

[0404] MAC cell killing assay was performed on the XCelligence platform (ACEA BioSciences). Each type of iPSC cell (iPSC-WT cells, iPSC-DKO cells, iPSC-DKO+CD300LD cells) was resuspended in 100 μl of cell-specific culture medium and plated on a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, MAC cells were added at an E:T ratio of 2:1. The data were normalized and analyzed using RTCA software (ACEA). The killing rate test results are shown in Fig.17 As shown, Fig.17 A and Fig.17 B shows the results of the macrophage killing experiment on iPSC-DKO+CD300LD cells detected by RTCA, wherein Fig.17 B is a multiple killing statistics chart, N = 4. The results show that iPSC-DKO+CD300LD cells can significantly escape the killing of PBMC.

[0405] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

[0406] Sequence Listing

[0407]

[0408]

[0409]

Claims

1. A low-immunogenicity pluripotent stem cell, comprising: reduced endogenous major histocompatibility class I antigen (MHC-I) function compared to parental pluripotent stem cells; Reduced endogenous major histocompatibility class II antigen (MHC-II) function compared to the parental pluripotent stem cell; and Reduced sensitivity to T cell and NK cell killing compared to a parental pluripotent stem cell, wherein the reduced sensitivity to T cell and NK cell killing is caused by increased expression of CD300LD protein.

2. The low immunogenic pluripotent stem cell according to claim 1, characterized in that The low immunogenic pluripotent stem cells also include modifications to increase the expression of one or more of the following polypeptides: DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8, and SerpinB9.

3. The low immunogenic pluripotent stem cell according to claim 1 or 2, wherein the MHC-I function is reduced by reducing the activity of MHC-I class proteins or MHC-I transcriptional regulators. 4 . The low immunogenic pluripotent stem cell according to claim 3 , wherein the MHC-I class protein comprises an HLA-A protein, an HLA-B protein or an HLA-C protein. 5 . The low immunogenic pluripotent stem cell according to claim 3 , wherein the MHC-I transcriptional regulator comprises a B2M protein, a TAP1 protein, a TAP2 protein, a TAP-associated glycoprotein or a NLRC5 protein. The low immunogenic pluripotent stem cell according to claim 3 , wherein the MHC-I function is reduced by reducing the activity of the B2M protein. 7 . The low immunogenic pluripotent stem cell according to claim 6 , wherein the B2M protein is a human B2M protein comprising an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 90% identity with the amino acid sequence as shown in SEQ ID NO:

1.

8. The low immunogenic pluripotent stem cell according to any one of claims 1 to 7, wherein the MHC-II function is reduced by reducing the activity of MHC-II class proteins or MHC-II transcriptional regulators. 9 . The low immunogenic pluripotent stem cell according to claim 8 , wherein the MHC-II class protein comprises an HLA-DR protein, an HLA-DQ protein or an HLA-DP protein. 10 . The low immunogenic pluripotent stem cell according to claim 8 , wherein the MHC-II transcriptional regulator comprises CIITA2 protein, RFXANK protein, RFX5 protein or RFXAP protein. The low immunogenic pluripotent stem cell according to claim 8 , wherein the MHC-II function is reduced by reducing the activity of CIITA protein. 12 . The low immunogenic pluripotent stem cell according to claim 11 , wherein 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% identity with the amino acid sequence shown in SEQ ID NO:

2.

13. The low immunogenic pluripotent stem cell according to any one of claims 1 to 12, wherein the CD300LD protein is a human CD300LD protein comprising an amino acid sequence as shown in SEQ ID NO: 3 or 4 or an amino acid sequence having at least 90% identity with the amino acid sequence as shown in SEQ ID NO: 3 or 4.

14. The low immunogenic pluripotent stem cell according to claim 1, comprising: One or more changes that reduce the activity of endogenous B2M protein; One or more changes that reduce the activity of endogenous CIITA protein; and One or more changes result in increased expression of CD300LD protein in the low immunogenic pluripotent stem cells.

15. The low immunogenic pluripotent stem cell according to claim 14, comprising: one or more alterations that inactivate both alleles of the endogenous B2M gene; One or more alterations that inactivate both alleles of the endogenous CIITA gene; and One or more alterations result in increased CD300LD gene expression in the low immunogenic pluripotent stem cells.

16. A method for producing the low immunogenic pluripotent stem cell according to any one of claims 1 to 15, the method comprising: reducing endogenous major histocompatibility class I antigen (MHC-I) function in the pluripotent stem cells; reducing endogenous major histocompatibility class II antigen (MHC-II) function in the pluripotent stem cells; and The expression of a protein that reduces the sensitivity of the pluripotent stem cells to NK cell killing is increased, wherein the protein is CD300LD protein.

17. The method of claim 16, comprising: reducing the activity of B2M protein in the pluripotent stem cells; Reducing the activity of CIITA protein in the pluripotent stem cells; and The expression of CD300LD protein in the pluripotent stem cells is increased.

18. The method of claim 17, comprising: 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 The expression of the CD300LD gene in the pluripotent stem cells is increased.

19. The method according to any one of claims 16 to 18, characterized in that The low immunogenic pluripotent stem cells also express one or more gene sequences selected from DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, Mfge8 and SerpinB9.

20. The method according to any one of claims 16 to 19, wherein the activity of the B2M protein in the pluripotent stem cells is reduced and / or the activity of the CIITA protein in the pluripotent stem cells is reduced by a technique selected from the following: Introduce gene expression modifying molecules, clustered regularly interspaced short palindromic repeats (CRISPR) technology, transcription activator-like effector nuclease (TALEN) technology, zinc finger nuclease (ZFN) technology or homologous recombination technology; preferably, the gene expression modifying molecules comprise siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotide ASO or anti-miRNA oligonucleotide AMO.

21. A method as claimed in claim 20, wherein the activity of B2M protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 gene editing technology; and / or the activity of CIITA protein in the pluripotent stem cells is reduced by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 gene editing technology.

22. The method of any one of claims 16-20, wherein the expression of CD300LD protein is increased by modification of an endogenous locus.

23. The method according to any one of claims 16 to 20, wherein the expression of CD300LD protein is increased by expression of a transgene; preferably, the nucleic acid sequence encoding the CD300LD protein is synthesized and constructed into a lentiviral vector, and then at least one copy of the CD300LD gene under the control of a promoter is introduced into the pluripotent stem cells through the lentiviral vector to increase the expression of the CD300LD protein.

24. The method of claim 23, wherein the nucleic acid sequence encoding the CD300LD protein comprises the nucleic acid sequence shown in SEQ ID NO: 25 or a nucleic acid sequence having at least 80% identity to the nucleic acid sequence shown in SEQ ID NO:

25.

25. The method of claim 23, wherein the nucleic acid sequence encoding the CD300LD protein comprises the nucleic acid sequence shown in SEQ ID NO: 26 or a nucleic acid sequence having at least 80% identity to the nucleic acid sequence shown in SEQ ID NO:

26.

26. Use of the low immunogenic pluripotent stem cells according to any one of claims 1 to 15 or the low immunogenic pluripotent stem cells prepared by the method of any one of claims 16 to 25 in the preparation of a medicament for preventing or treating a disease requiring cell transplantation; preferably, 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 degeneration, stroke, Huntington's disease, amyotrophic lateral sclerosis, atherosclerosis, hypertension, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, myocardial infarction, heart failure, aortic aneurysm, peripheral arterial disease, type II diabetes, scurvy, hypoglycemia, hyperlipidemia or osteoporosis.

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