CN119351338B

CN119351338B - Low-immunogenicity cell for expressing CD300LD and preparation method thereof

Info

Publication number: CN119351338B
Application number: CN202411301345.0A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Shize Biomedical Shanghai Co ltd; Shize Biomedical Suzhou Co ltd
Current assignee: Shi Ze Biopharma Shanghai Co Ltd; Shize Biomedical Suzhou Co ltd
Priority date: 2023-09-15
Filing date: 2024-09-18
Publication date: 2026-01-23
Anticipated expiration: 2044-09-18
Also published as: CN119351338A

Abstract

The invention provides a low immunogenicity cell expressing CD300LD and a preparation method thereof. The low-immunogenicity pluripotent cells can significantly reduce or escape recognition and attack of the immune system, especially attack of T cells, natural killer cells, macrophages and the like, while not changing the updating and differentiating characteristics of stem cells.

Description

Low-immunogenicity cell for expressing CD300LD and preparation method thereof

The present application claims priority from China patent application 2023111948810 with the application date 2023/9/15. The present application incorporates the entirety of the above-mentioned chinese patent application.

Technical Field

The invention belongs to the technical field of genetic engineering and stem cells, and particularly relates to a low-immunogenicity cell for expressing CD300LD and a preparation method thereof.

Background

Stem cells are a type of cells having self-renewal ability and differentiation ability into specific functional somatic cells, and are mainly classified into totipotent stem cells, pluripotent stem cells and adult stem cells according to the degree difference of stem cell characteristics. The Induced Pluripotent Stem Cells (iPSC) have the potential of unlimited proliferation, self-renewal and differentiation into various cell types, and have important application prospects in the treatment of cancers, nerve-related diseases, cardiovascular diseases and the like. But the clinical application of transplantation of allogeneic functional cells for therapy is hampered by the critical problems of immune incompatibility and immune rejection suffered by transplanted cells.

Human Major Histocompatibility Complex (MHC), human Leukocyte Antigen (HLA), is a major cause of immune incompatibility. MHC consists of a series of genes, which can be classified into class I, class II and class III. MHC-I genes are expressed in almost all tissue cell types, and transplanted cells expressing "non-hexon" MHC-I molecules will stimulate activation of CD8+ T cells to be eliminated. Cd4+ helper T cells recognize MHC-II genes from "nonhexon" cells, thus undergoing immune rejection, while class III molecules are not involved in immune activity.

In recent years, deletion expression of genes on the surfaces or in the self of MHC-I and MHC-II cells can be achieved by knocking out genes such as B2M, CIITA and the like, so that the cells have immune tolerance or escape T cell/B cell specific immune response, and immune compatible low-immunogenicity pluripotent stem cells are generated.

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 signal motif, but has a negatively charged residue (glutamate) within the transmembrane domain. Transmembrane signaling receptor activity and viral receptor activity may be enabled to participate in the immune system process.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides a novel method for obtaining low immunogenicity by modifying cells, adopts a strategy for carrying out mass screening on all mechanism molecules related to maternal-fetal tolerance and tumor escape for the first time, and finally identifies a representative novel gene CD300LD through multiple rounds of functional detection. The gene can obviously reduce or escape the recognition and attack of immune system, especially the attack of natural killer cells, T cells, macrophages and the like, and the invention provides a feasible strategy for realizing immunity and immunity of cells by transforming and developing new genes in unpredictable fields.

The invention successfully constructs B2M/CIITA bi-allele knockout positive clone DKO cells by knocking out beta-2-microglobulin (B2M) in the endoplasmic reticulum of the human pluripotent stem cells and knocking out positive regulator CIITA transcribed by MHC-II genes, and then uses a lentiviral vector to overexpress the novel gene CD300LD identified by the invention in the DKO cells, so that the obtained human pluripotent stem cells can further escape the killing of NK cells on the basis of escaping T cell attack. Meanwhile, the low-immunogenicity pluripotent stem cells retain the key biological functions of the pluripotent stem cells such as the stem cells and the differentiation capacity.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

In one aspect, the invention provides a low immunogenic pluripotent stem cell comprising reduced endogenous major histocompatibility class I antigen (MHC-I) function as compared to a parent pluripotent stem cell, reduced endogenous major histocompatibility class II antigen (MHC-II) function as compared to a parent pluripotent stem cell, and reduced susceptibility to NK cell and T cell killing as compared to a parent pluripotent stem cell, wherein the reduced susceptibility to NK cell and T cell killing is caused by increased expression of the CD300LD protein.

In some embodiments, the MHC-I function is reduced by decreasing the activity of an MHC-I protein or an MHC-I transcription regulator.

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

In one embodiment, the MHC-I transcription modulator comprises a B2M protein, a TAP1 protein, a TAP2 protein, a TAP-related glycoprotein, or an NLRC5 protein.

In one embodiment, the MHC-I function is reduced by decreasing the activity of a B2M protein.

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

In one embodiment, the MHC-II function is reduced by decreasing the activity of an MHC-II protein or MHC-II transcription regulator.

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

In one embodiment, the MHC-II transcription modulator comprises a CIITA2 protein, RFXANK protein, RFX5 protein, or RFXAP protein.

In one embodiment, the MHC-II function is reduced by decreasing the activity of CIITA protein.

In one embodiment, the CIITA protein is a human CIITA protein comprising the amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence having 90% identity to the amino acid sequence shown in SEQ ID NO. 2.

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

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

In one embodiment, the low-immunogenicity pluripotent stem cell comprises one or more alterations that reduce the activity of an endogenous B2M protein, one or more alterations that reduce the activity of an endogenous CIITA protein, and one or more alterations that cause increased expression of the CD300LD protein in the low-immunogenicity pluripotent stem cell.

In one embodiment, the low immunogenicity pluripotent stem cell further comprises one or more alterations that increase expression of a polypeptide selected from the group consisting of DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, mfge8, and SerpinB.

In one embodiment, the low immunogenicity pluripotent stem cell comprises one or more changes that inactivate two alleles of an endogenous B2M gene; one or more alterations that inactivate both alleles of the endogenous CIITA gene;

And causing one or more changes in increased CD300LD gene expression in said low immunogenicity pluripotent stem cells.

In another aspect, the present invention also provides a method of producing the above-described low-immunogenicity pluripotent stem cells of the invention, comprising decreasing endogenous major histocompatibility class I antigen (MHC-I) function in the pluripotent stem cells, decreasing endogenous major histocompatibility class II antigen (MHC-II) function in the pluripotent stem cells, and increasing expression of a protein that decreases susceptibility of the pluripotent stem cells to NK cell and T cell killing, wherein the protein is the CD300LD protein.

In one embodiment, the method comprises decreasing the activity of a B2M protein in the pluripotent stem cell, decreasing the activity of a CIITA protein in the pluripotent stem cell, and increasing the expression of a CD300LD protein in the pluripotent stem cell.

In one embodiment, the method comprises abrogating the activity of both alleles of the B2M gene in the pluripotent stem cell, abrogating the activity of both alleles of the CIITA gene in the pluripotent stem cell, and increasing the expression of the CD300LD gene in the pluripotent stem cell.

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.

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

Introducing a gene expression modification molecule, a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technique, a transcription activator-like effector nuclease (TALEN) technique, a Zinc Finger Nuclease (ZFN) technique, or a homologous recombination technique; preferably, the gene expression modification molecule comprises siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotide ASO or anti-miRNA oligonucleotide AMO.

In one embodiment, the activity of the B2M protein in the pluripotent stem cells is reduced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas 9 gene editing technology.

In one embodiment, the activity of the CIITA protein in the pluripotent stem cells is reduced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas 9 gene editing technology.

In one embodiment, the activity of the B2M protein and CIITA protein in the pluripotent stem cells is reduced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas 9 gene editing techniques.

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

In one embodiment, expression of the CD300LD protein is increased by expression of the transgene.

In a preferred embodiment, the expression of the CD300LD protein is increased by synthesizing a nucleic acid sequence encoding the CD300LD protein to construct into a lentiviral vector, and introducing at least one copy of the CD300LD gene under the control of a promoter into the pluripotent stem cell through the lentiviral vector.

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.

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.

In another aspect, the present invention also provides the use of the low-immunogenicity pluripotent stem cells of the invention or the low-immunogenicity pluripotent stem cells prepared by the method of the invention for the preparation of a medicament for preventing or treating a disease requiring cell transplantation.

In one embodiment, the disease comprises acute leukemia, chronic leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, small cell lung cancer, breast cancer, testicular cancer, neuroblastoma, ovarian cancer, melanoma, aplastic anemia, congenital immunodeficiency, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, type I diabetes, parkinson's disease, alzheimer's disease, spinal cord injury, retinal degenerative disease, cerebral apoplexy, huntington's disease, amyotrophic lateral sclerosis, atherosclerosis, hypertension, rheumatic heart disease, cardiomyopathy, cardiac arrhythmias, congenital heart disease, valvular heart disease, cardiac inflammation, myocardial infarction, heart failure, aortic aneurysm, peripheral arterial disease, type II diabetes, necrosis, hypoglycemia, hyperlipidemia, or osteoporosis.

In another aspect, the invention provides a composition comprising the low immunogenicity cell of the first aspect.

Drawings

FIG. 1 shows B2M gene knockout strategy of B2M and CIITA double knockout cell line (DKO) and results of B2M gene knockout verification by PCR.

FIG. 2 shows the knockout strategy of CIITA gene of B2M and CIITA double knockout cell line (DKO) and the results of CIITA gene knockout verification by PCR.

FIG. 3 shows the results of qPCR detection of expression of B2M and CIITA at the RNA level in B2M/CIITA double allele knock-out clone H1 cells DKO.

FIG. 4 shows the results of Western blot detection of B2M protein levels of B2M/CIITA double allele knock-out clone H1 cells DKO. FIG. 5A shows the results of FACS detection of HLA type I/II molecules on the surface of H1 cells using IFN-gamma stimulation of WT and DKO cells.

FIG. 5B shows the results of FACS detection using IFN-. Gamma.stimulated WT and DKO cells to detect HLA type I/II molecules on the surface of iPSC cells.

FIG. 6 shows the karyotype detection results of B2M/CIITA double allele knock-out clone H1 cells DKO.

Fig. 7A-7F show the results of detection of expression of a stem gene in DKO cells.

FIG. 7A is a result of detecting protein levels of the stem genes POU5F1 and NANOG in H1 cells (WT cells) and H1-derived DKO cells (DKO cells) by immunofluorescence;

FIG. 7B is a result of detecting expression of the dry genes POU5F1, NANOG and SOX2 at RNA level in H1 cells (WT cells) and H1-derived DKO cells (DKO cells) by RT-qPCR;

FIG. 7C is the results of detecting expression of the surface stem genes SSEA-4 and Tra1-81 by flow cytometry for H1 cells (WT cells) and H1-derived DKO cells (DKO cells);

FIG. 7D shows the results of detecting expression of the stem genes SSEA-4, TRA-1-60, TRA-1-81, OCT-4, SOX2 on the cell surface of iPSC-WT and iPSC-DKO by flow cytometry, and FIG. 7E shows the results of detecting protein levels of the stem genes TRA-1-60, TRA-1-81, OCT-4, SOX2, and NANOG in iPSC-WT cells by immunofluorescence;

FIG. 7F shows the results of detecting protein levels of the dry genes TRA-1-60, TRA-1-81, OCT-4, SOX2 and NANOG in iPSC-DKO cells by immunofluorescence.

Fig. 8A and 8B show the results of detection of immune escape function of WT and DKO cells by RTCA.

Fig. 8A shows the results of detection of NK and T cell killing rate of H1 cell WT and H1 cell-derived DKO cells by RTCA;

FIG. 8B shows the results of detection of killing rate of iPSC-WT and iPSC-DKO cells by NK cells and T cells by RTCA, i.e., the results of detection of immune escape function of iPSC-WT and iPSC-DKO cells on NK cells.

FIG. 9 shows a schematic structural diagram of the lentiviral vector pGC-EF1 a.

Fig. 10A and 10B show the results of validating CD47 overexpression in H1 cells dko+cd47 cells. FIG. 10A shows the results of measuring the expression level of CD47 in the H1 cell DKO+CD47 cell line by FACS, and FIG. 10B shows the results of measuring the expression level of CD47 in the H1 cell DKO+CD47 cell line by qPCR.

Fig. 11 shows the results of detection of immune escape function of WT cells and derived dko+cd47 cells of H1 cells by RTCA.

FIG. 12 shows the results of protein levels of the stem genes OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81 in iPSC-DKO+CD300LD cells constructed by immunofluorescence assay.

FIG. 13 shows the results of detecting the expression levels of the constructed iPSC-DKO+CD300LD cell surface stem genes SSEA-4, TRA-1-60, TRA-1-81 and OCT4 by flow cytometry.

Fig. 14 shows the results of tricodermic differentiation ability of iPSC-dko+cd300LD cells constructed by immunofluorescence assay.

Fig. 15 shows the results of NK cells tested by RTCA on iPSC-dko+cd300LD cell killing experiments, where B is a multiple killing statistic plot, n=6.

Fig. 16 shows the results of PBMC killing experiments on iPSC-dko+cd300LD cells detected by RTCA, where B is a multiple killing statistic plot, n=4.

Fig. 17 shows the results of an iPSC-dko+cd300LD cell killing experiment by macrophages detected by RTCA, where B is a multiple killing statistic plot, n=4.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

General definitions and terms

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

Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology related terms as used herein are terms that are widely used in the corresponding field (see, e.g., ,Molecular Cloning:A Laboratory Manual,2nd Edition,J.Sambrook et al.eds.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor 1989)., while, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, the terms "comprises," "comprising," "includes," "including," "having" and "containing" are open-ended, meaning the inclusion of the stated elements, steps or components, but not the exclusion of other non-recited elements, steps or components. The expression "consisting of" does not include any element not specified steps or components. The expression "consisting essentially of means that the scope is limited to the specified elements, steps or components, plus any optional elements, steps or components that do not significantly affect the basic and novel properties of the claimed subject matter. It is to be understood that the expression "consisting essentially of the expression" comprising "and" consisting of the expression "comprising" are encompassed within the meaning of the expression "comprising".

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

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.

Unless specifically indicated to the contrary, the numerical values or ranges set forth herein are modified by "about" to mean the enumerated or claimed values or ranges are + -20%, + -10%, + -5%, or + -3%.

Unless otherwise indicated, the identification of method steps described herein, such as 1), 2), i), ii), a), b), or the like, is merely by way of distinguishing between them and not necessarily by way of indicating that the method steps are performed in such an order.

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

As used herein, the term "pluripotent stem cells" has the potential to differentiate into any of three germ layers, endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, genitourinary tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue). The term "pluripotent stem cell" as used herein also includes "induced pluripotent stem cell" or "iPSC", a pluripotent stem cell derived from a non-pluripotent cell. Exemplary human pluripotent stem cell lines include the H1 human pluripotent stem cell line, the H9 human pluripotent stem cell line, and the iPSC human pluripotent stem cell line. Additional exemplary pluripotent stem cell lines include those obtainable by the National Institutes of Health HumanEmbryonic STEM CELL REGISTRY and Howard Hughes Medical Institute HUES sets (as described in Cowan CA,et al.Derivation of embryonic stem-cell lines from human blastocysts.N Engl J Med.2004Mar 25;350(13):1353-6.).

As used herein, the term "totipotent" refers to the ability of a cell to form a whole organism. For example, in mammals, only fertilized eggs and first cleavage stage blastomeres are totipotent. In one embodiment, the pluripotent stem cells described herein are not totipotent, nor do they form a whole organism.

As used herein, the term "low-immunogenicity cells" refers to cells that have been engineered to eliminate immune rejection using gene editing techniques to achieve low immunogenicity.

The cells may be derived 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, cows, and non-human primates. In some embodiments, the cell is from an adult or non-human mammal. In some embodiments, the cells are from neonatal humans, adults, or non-human mammals.

As used herein, the term "immune rejection" or "immune incompatibility" refers to the fact that allogeneic cells, tissues or organs, after transplantation into a recipient, are challenged by immune cells of the recipient itself, thereby failing to guarantee their normal physiological function. Human Major Histocompatibility Complex (MHC), human Leukocyte Antigen (HLA), is the leading cause of "immune rejection" or "immune incompatibility.

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

"Low immunogenic pluripotent stem cells" herein refers to pluripotent stem cells that retain the characteristics of their pluripotent stem cells and that produce reduced immune rejection when transferred into an allogeneic host. In a preferred embodiment, the low immunogenicity pluripotent stem cells do not generate an immune response. Thus, "hypoimmunogenic" refers to an immune response that is significantly reduced or eliminated compared to the immune response of the parental ("WT") stem cells prior to immune engineering. For example, such a low-immunogenicity cell may be less susceptible to immune rejection by about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or greater than 99% relative to a wild-type cell that has not been immunoengineered.

The term "Major Histocompatibility Complex (MHC)" relates to the gene complex that occurs in all vertebrates. MHC proteins or molecules play a role in the signaling between lymphocytes and antigen presenting cells in a normal immune response. Human MHC, also known as HLA, a human leukocyte antigen, is located on chromosome 6, including MHC-I and MHC-II.

The term "MHC-I" or "MHC class I" refers to a major histocompatibility complex class I protein or gene. Within the human MHC-I region are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, CD1a, CD1b and CD1c sub-regions. MHC class I proteins are present on almost all cell surfaces, including most tumor cells. MHC-I proteins are loaded with antigens, which are typically derived from endogenous proteins or intracellular pathogens, and then presented to cytotoxic T lymphocytes (CTLs, also known as cd8+ T cells). T cell receptors are capable of recognizing and binding peptides complexed with MHC class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor, capable of binding to a specific MHC/peptide complex. MHC class I molecules mediate primarily the presentation of endogenous antigens.

The term "MHC-II" or "MHC class II" refers to a major histocompatibility complex class II protein or gene. 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, macrophages and dendritic cells. MHC class II molecules mediate mainly the presentation process of exogenous antigens, which present exogenous antigen polypeptide molecules to Th cells (helper T cells), i.e. stimulate cd4+ T cells.

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.

"Knockout" herein refers to a process of rendering a particular gene inactive in the host cell in which it is located, which results in the production of no protein of interest or inactive form. As will be appreciated by those skilled in the art and further described below, this can be accomplished in a number of different ways, including removal of the nucleic acid sequence from the gene, or disruption of the sequence with other sequences, altering the reading frame, or altering regulatory components of the nucleic acid. For example, all or part of the coding region of the gene of interest may be deleted or replaced with a "nonsense" sequence, all or part of the regulatory sequence (e.g., promoter) may be deleted or replaced, the translation initiation sequence may be deleted or replaced, etc.

In this context, the terms "reduce" and "decrease" are generally used to denote a reduction by a statistically significant amount. However, for the avoidance of doubt, "reducing" includes at least a 10% reduction compared to the reference level, such as at least about 20% or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% reduction (i.e., a level that is not present compared to the reference sample), or any reduction between 10-100%.

"Knock-in" or "overexpression" herein refers to a process that directs a host cell to add genetic functions. This results in an increased level of encoded protein. As will be appreciated by those skilled in the art, this may be accomplished in several ways, including adding one or more additional copies of the gene to the host cell or altering 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.

For the avoidance of any doubt, the term "increase" is generally used herein to mean an increase in a statistically significant amount, the term "increase" means an increase of at least 10% from a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% increase or any increase between 10-100%, 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-fold or at least about 10-fold increase, or any increase between 2-fold and 10-fold or greater than 10-fold from the reference level.

The "beta-2 microglobulin" or "beta 2M" or "B2M" proteins are part of MHC class I molecules. The B2M protein can be expressed in all nucleated cells (except red blood cells) and can be non-covalently combined with the alpha chain of MHC-I molecules, attached to cell membranes and released into various tissue fluids.

"CD47 protein" or "integrin-associated protein (Integrin-associated protein, IAP)" is an important self-signal that can inhibit phagocytosis of macrophages by binding to the N-terminus of ligand signaling regulator protein alpha (SIRP alpha) on immune cells, causing immune escape.

"MHC-II transactivator (CIITA) proteins" are key molecules that regulate MHC-II expression, and the body regulates the expression level of MHC II genes, mainly by controlling CIITA expression.

CD300LD is a specific tumor immunosuppressive receptor of a polymorphonuclear marrow-derived suppressor cell (PMN-MDSC), which is a kind of neutrophils which are generated by pathological induction and widely exist in various tumors, can suppress the functions of T cells and NK equivalent cells, promote the development, infiltration and metastasis of tumors, and plays a key role in tumor immunoregulation.

As used herein, the term "isogene" refers to the genetic similarity or identity of a host organism and a cell graft, wherein there is immune compatibility, e.g., no immune response is generated.

As used herein, the term "allogeneic" refers to a genetic difference between a host organism and cell transplantation in which an immune response is generated.

As used herein, the term "B2M-/-" refers to diploid cells having inactivated B2M genes in both chromosomes.

As used herein, the term "CIITA-/-" refers to diploid cells having inactivated CIITA genes in both chromosomes.

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 by covalent or non-covalent means. Unless otherwise indicated, "polypeptide" and "protein" may be used interchangeably.

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, e.g., the parental pluripotent stem cells (WTs) described herein. The parental pluripotent stem cells of the H1 cell line in the present disclosure are also referred to as WTs, and the parental pluripotent stem cells of the iPSC cell line are also referred to as iPSC-WTs.

As used herein, the term "% identity" with respect to sequences refers to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences to be compared. The difference between the two sequences may be distributed over a local area (section) or the entire length of the sequences to be compared. The identity between two sequences is typically determined after optimal alignment of the segments or "comparison windows". The optimal alignment may be performed manually or by means of algorithms known in the art, including but not limited to the local homology algorithms described by Smith and5 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 Wisconsin Genetics Software Package, genetics Computer Group,575Science Drive,Madison,Wis. For example, the percent identity of two sequences may be determined using the BLASTN or BLASTP algorithm commonly available at the National Center for Biotechnology Information (NCBI) website.

The% identity is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100. 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% of the regions give a degree of identity. In some embodiments, the degree of identity is given to the entire length of the reference sequence. Alignment for determining sequence identity can be performed using tools known in the art, preferably using optimal sequence alignment, e.g., using Align, using standard settings, preferably EMBOSS:: needle, matrix: blosum62, gap Open 10.0, gap extension 0.5.

Herein, "nucleotide" includes deoxyribonucleotides and ribonucleotides and derivatives thereof. As used herein, a "ribonucleotide" is a constituent material of ribonucleic acid (RNA) and consists of one molecule of base, one molecule of pentose, and one molecule of phosphate, which refers to a nucleotide having a hydroxyl group at the 2' -position of the β -D-ribofuranose (β -D-ribofuranosyl) group. The "deoxyribonucleotide" is a constituent substance of deoxyribonucleic acid (DNA), and also comprises one molecule of base, one molecule of pentose and one molecule of phosphoric acid, and refers to a nucleotide in which the hydroxyl group at the 2' -position of the beta-D-ribofuranose (beta-D-ribofuranosyl) group is replaced by hydrogen, and is a main chemical component of a chromosome. "nucleotide" is generally referred to by a single letter representing a base therein, "A (a)" means deoxyadenylate or adenylate containing adenine, "C (C)" means deoxycytidylate or cytidylate containing cytosine, "G (G)" means deoxyguanylate or guanylate containing guanine, "U (U)" means uridylate containing uracil, and "T (T)" means deoxythymidylate containing thymine.

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 ordering of nucleotides in a polynucleotide. It will be appreciated by those skilled in the art that the coding strand (sense strand) of DNA can be considered to have the same nucleotide sequence as the RNA it encodes, with deoxythymidylate in the sequence of the coding strand of DNA corresponding to uridylate in the sequence of the RNA it encodes.

As used herein, the term "expression" includes transcription and/or translation of a nucleotide sequence. Thus, expression may involve the production of transcripts and/or polypeptides. The term "transcription" relates to the process of transcribing the genetic code in a DNA sequence into RNA (transcript). The term "in vitro transcription" refers to the synthesis of RNA, in particular mRNA, in vitro in a cell-free system (e.g. in a suitable cell extract) (see, e.g. Pardi N.,Muramatsu H.,Weissman D.,KarikóK.(2013).9In:Rabinovich P.(eds)Synthetic Messenger RNA and Cell Metabolism Modulation.Methods in Molecular Biology(Methods and Protocols),vol 969.Humana Press,Totowa,NJ.). vectors which may be used for the production of transcripts are also referred to as "transcription vectors", which contain the regulatory sequences required for transcription.

As used herein, "encoding" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, that can be used as a template to synthesize polymers and macromolecules in other biological processes, provided that there is a defined nucleotide sequence or a defined amino acid sequence. Thus, a gene encodes a protein, meaning that mRNA of the gene is transcribed and translated to produce the protein in a cell or other biological system.

All methods described herein can be performed in any suitable order unless otherwise indicated.

Pluripotent stem cells

In one aspect, the invention provides a low-immunogenicity pluripotent stem cell comprising:

Reduced endogenous major histocompatibility class I antigen (MHC-I) function compared to the parental pluripotent stem cells;

reduced endogenous major histocompatibility class II antigen (MHC-II) function compared to the parental pluripotent stem cell, and

Reduced susceptibility to NK cell and T cell killing compared to the parental pluripotent stem cells.

In this context, a parental pluripotent stem cell refers to a parental (also referred to herein as "WT" or "iPSC-WT") pluripotent stem cell that has not undergone a gene editing procedure to achieve low immunogenicity prior to immune engineering.

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

As will be appreciated by those of skill in the art, the reduction of function may be achieved in a variety of ways, including removal of nucleic acid sequences from genes, disruption of sequences with other sequences, or altering regulatory components of nucleic acids. For example, all or part of the coding region of the gene of interest may be deleted or replaced with a "nonsense" sequence, frame shift mutations may be made, all or part of regulatory sequences such as promoters may be deleted or replaced, translation initiation sequences may be deleted or replaced, and the like.

As will be appreciated by those skilled in the art, the decrease in MHCI (HLA I) function in pluripotent stem cells can be measured using techniques known in the art and described below, e.g., FACS techniques using labeled antibodies that bind to HLA complexes, e.g., using commercially available HLA-A, HLA-B, HLA-C antibodies that bind to human major histocompatibility HLA class I. The decrease 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 described below, for example, FACS techniques using labeled antibodies that bind to HLA complexes, for example, using commercially available HLA-DQ, HLA-DR, HLA-DP antibodies that bind to human major histocompatibility HLA class II.

In some embodiments, the MHC-I function is reduced by decreasing the activity of an MHC-I protein.

In one embodiment, the MHC class I 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.

In some embodiments, the MHC-I function is reduced by decreasing the activity of an MHC-I transcription regulator.

In some preferred embodiments, the transcriptional regulator of MHC-I may be selected from one or more of beta 2 microglobulin (B2M), antigen processing related transporter 1 (TAP 1), antigen processing related transporter 2 (TAP 2), antigen processing related Transporter (TAP) related glycoprotein (Tapasin) or NOD-like receptor family caspase recruitment domain 5 (NLRC 5).

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

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

In one embodiment, the MHC-I function is reduced by decreasing the activity of HLA-B protein.

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

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

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

In one embodiment, the MHC-I function is reduced by reducing the activity of the TAP1 protein.

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

In one embodiment, the MHC-I function is reduced by reducing the activity of the TAP2 protein.

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

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

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

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

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

In a preferred embodiment, the MHC-I function is reduced by reducing the activity of the B2M protein.

In one embodiment, the B2M protein is a human B2M protein comprising the amino acid sequence set forth in SEQ ID No. 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID No. 1.

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

In some embodiments, the MHC-II function is reduced by decreasing the activity of an MHC-II protein.

In one embodiment, the MHC class II 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.

In some embodiments, the MHC-II function is reduced by decreasing the activity of an MHC-II transcription regulator. In some preferred embodiments, the MHC-II transcription regulator may be selected from one or more of MHC-II transactivator (CIITA), regulator X-related ankyrin (RFXANK), regulator X5 (RFX 5), regulator X-related protein (RFXAP).

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

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

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

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

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

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

In one embodiment, the MHC-II function is reduced by decreasing the activity of RFXANK proteins.

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

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

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

In one embodiment, the MHC-II function is reduced by decreasing the activity of RFXAP proteins.

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

In a preferred embodiment, the MHC-II function is reduced by reducing the activity of the CIITA protein.

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

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

In a preferred embodiment, the gene is knocked out using CRISPR techniques. In some cases, CRISPR techniques are used to introduce small deletions/insertions into the coding region of a gene such that no functional protein is produced, typically as a result of a frame shift mutation, which results in the production of a stop codon such that a truncated, non-functional protein is produced.

Successful reduction of MHC-I (HLA-I when the cells are derived from human cells) and MHC-II (HLA-II when the cells are derived from human cells) functions in pluripotent stem cells can be measured using techniques known in the art, such as Western blotting using protein antibodies, FACS techniques, RT-PCR, qPCR techniques, and the like.

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

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, lentiviral constructs are used as described herein or as known in the art. As known in the art, the CD300LD gene may be integrated into the genome of a host cell under the control of a suitable promoter.

In one embodiment, the increased expression of the CD300LD protein is caused by a CD300LD transgene.

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

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

The presence of sufficient CD300LD protein expression may be determined using known techniques, such as those described in the examples, for example using western blotting, ELISA assays, or FACS assays. In general, "sufficient" in this context means an increase in CD300LD protein expression on the surface of pluripotent stem cells, which silences NK cells and T cell killing.

In yet another aspect, the invention also provides a low immunogenicity pluripotent stem cell comprising:

one or more alterations that reduce endogenous B2M protein activity;

one or more alterations that reduce the activity of endogenous CIITA proteins, and

Causing one or more changes in increased CD300LD protein expression in said low immunogenicity pluripotent stem cells.

In one embodiment, the low immunogenicity pluripotent stem cell comprises:

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

One or more changes that inactivate both alleles of an endogenous CIITA gene, and

Causing one or more changes in increased CD300LD gene expression in said low immunogenicity pluripotent stem cells.

In one embodiment, the low immunogenicity pluripotent stem cell further comprises increasing one or more changes in gene expression selected from the group consisting of DUX4, CD27, CD35, CD200, HLA-C, PD-L1, CD47, CD24, CD26, CCL21, mfge, and SerpinB.

As used herein, the term "alteration" or "genetic alteration" refers to a change that causes a cell, such as a pluripotent stem cell described herein, that can be achieved, for example, by modifying the genome or introducing a new gene fragment. In this context, modifying a genome refers to modifying a nucleic acid sequence within a cell or under cell-free conditions to produce engineered pluripotent cells and multipotent stem cells. Exemplary "altered" or "genetically altered" techniques include, but are not limited to, homologous recombination, knock-in, ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), CRISPR (clustered regularly interspaced short palindromic repeats)/Cas 9, and other site-specific nuclease techniques. These techniques enable double-stranded DNA breaks at the desired locus. These controlled double strand breaks promote homologous recombination at specific locus sites. This process focuses on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind sequences and induce double strand breaks in nucleic acid molecules. Double strand breaks are repaired by error-prone non-homologous end joining (NHEJ) or by Homologous Recombination (HR). Exemplary "altering" or "genetic alteration" techniques also include the introduction of gene expression modification molecules including, but not limited to siRNA, shRNA, microRNA, antisense RNA, antisense oligonucleotide ASO (antisense oligonucleotides), or Anti-miRNA oligonucleotide AMO (Anti-miRNA oligonucleotides).

Those skilled in the art will appreciate that many different techniques can be used to engineer the pluripotent cells and multipotent stem cells of the invention to become hypoimmunogenic.

In general, these techniques may be used alone or in combination. For example, CRISPR techniques are used to reduce expression of active B2M and/or CIITA proteins in engineered cells and viral techniques (e.g., lentiviruses) are used to knock-in CD300LD genes. Furthermore, it will be appreciated by those skilled in the art that these genes may be manipulated in different sequences using different techniques. In some embodiments, one or more changes encompassed by the low-immunogenicity pluripotent stem cells of the invention are capable of reducing endogenous major histocompatibility class I antigen (MHC-I) function. In some embodiments, one or more changes encompassed by the low-immunogenicity pluripotent stem cells of the invention are capable of reducing endogenous major histocompatibility class II antigen (MHC-II) function.

In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention can reduce susceptibility to NK cell and T cell killing.

In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention are capable of reducing endogenous B2M protein activity. In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention result in a decrease in endogenous CIITA protein activity. In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention increase CD300LD protein expression.

In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention are capable of inactivating both alleles of an endogenous B2M gene. In some embodiments, the low immunogenicity pluripotent stem cells of the invention comprise one or more changes that inactivate both alleles of an endogenous CIITA gene. In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention can increase CD300LD gene expression.

In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention enable inhibition of endogenous B2M protein expression. In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention enable inhibition of endogenous CIITA protein expression.

In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention are capable of interfering with endogenous B2M protein expression. In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention are capable of interfering with endogenous CIITA protein expression.

In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention result in reduced expression of endogenous B2M proteins. In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention result in reduced expression of endogenous CIITA proteins.

In some embodiments, one or more changes encompassed by the low immunogenicity pluripotent stem cells of the invention are capable of knocking out endogenous B2M proteins. In some embodiments, one or more changes encompassed by the low-immunogenicity pluripotent stem cells of the invention are capable of knocking out endogenous CIITA proteins.

In one embodiment, the pluripotent stem cells are altered to reduce endogenous B2M protein activity using clustered regularly interspaced short palindromic repeats/Cas ("CRISPR") techniques known in the art.

In one embodiment, the pluripotent stem cells are altered to reduce endogenous CIITA protein activity using clustered regularly interspaced short palindromic repeats/Cas ("CRISPR") techniques known in the art.

In one embodiment, the pluripotent stem cells are altered using clustered regularly interspaced short palindromic repeats/Cas ("CRISPR") techniques known in the art to inactivate both alleles of the endogenous B2M gene.

In one embodiment, the pluripotent stem cells are altered using clustered regularly interspaced short palindromic repeats/Cas ("CRISPR") techniques known in the art to inactivate both alleles of the endogenous CIITA gene.

Assays for testing whether a gene has been inactivated are 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 an inactivation change.

In one embodiment, viral techniques known in the art may be used to cause increased expression of the CD300LD gene in the low immunogenicity pluripotent stem cells. Such 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 the selected site of the cell, which is the safe harbor gene site of AAVS1, CCR5, etc. As used herein, a "safe harbor gene site" refers to a site that can be used for safe knock-in of a gene and can ensure normal stable expression of the transferred gene.

In a preferred embodiment, lentiviral vectors are used to cause increased expression of the CD300LD gene in the low immunogenicity pluripotent stem cells.

In one embodiment, the low immunogenicity pluripotent stem cell is a human pluripotent stem cell.

In one embodiment, the low immunogenicity stem cell comprises:

Reduced endogenous major histocompatibility class II antigen (MHC-II) function as compared to a parental pluripotent stem cell, and reduced susceptibility to NK cell and T cell killing as compared to a parental pluripotent stem cell.

In one embodiment, the low immunogenicity pluripotent stem cell elicits a T cell response that is lower than a T cell response elicited by a parent pluripotent stem cell that does not comprise the alteration that reduces B2M and CIITA protein activity and the alteration that causes increased expression of the CD300LD protein. In one embodiment, the T cell response is measured by measuring the killing of T cells by the low immunogenic pluripotent stem cell or the parental pluripotent stem cell by real-time label-free dynamic cell analysis (RTCA) technique.

In one embodiment, the low immunogenicity pluripotent stem cell-induced Natural Killer (NK) cell response is lower than an NK cell response elicited by B2M/CIITA double allele knock-out cloned DKO cells comprising the alteration that reduces B2M and CIITA protein activity but not comprising the alteration that causes increased expression of CD300LD protein. In one embodiment, the NK cell response is measured by determining the IFN- γ level of NK cells incubated with the low immunogenic pluripotent stem cells or DKO cells in vitro. In one embodiment, the NK cell response is measured by measuring NK cell killing of the low-immunogenicity pluripotent stem cell or DKO cell by real-time label-free dynamic cell analysis (RTCA) technique.

Methods of producing the low immunogenicity pluripotent stem cells of the invention

The invention also provides a method of producing a low immunogenicity pluripotent stem cell of the invention comprising reducing endogenous major histocompatibility class I antigen (MHC-I) function in the pluripotent stem cell, reducing endogenous major histocompatibility class II antigen (MHC-II) function in the pluripotent stem cell, and increasing expression of a protein that reduces susceptibility of the pluripotent stem cell to NK cell and T cell killing, wherein the protein is a CD300LD protein.

In some embodiments, the method comprises decreasing the activity of a B2M protein in the pluripotent stem cell, decreasing the activity of a CIITA protein in the pluripotent stem cell, and increasing the expression of a CD300LD protein in the pluripotent stem cell.

In some embodiments, the activity of a B2M protein in the pluripotent stem cell may be reduced by a technique of "altering" or "genetic alteration" as described above. In some embodiments, the activity of the CIITA protein in the pluripotent stem cells may be reduced by a technique of "altering" or "genetic alteration" as described above. Such as the introduction of gene expression modification molecules, clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) techniques, transcription activator-like effector nucleases (TALENs) techniques, zinc Finger Nucleases (ZFNs) techniques, or homologous recombination techniques. 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).

In some embodiments, the CRISPR/Cas system comprises a Cas protein or a nucleic acid sequence encoding a Cas protein and at least one to two ribonucleic acids (e.g., grnas) capable of directing and hybridizing to a target motif of a target polynucleotide sequence. In some embodiments, the CRISPR/Cas system comprises a Cas protein or a nucleic acid sequence encoding a Cas protein and a single ribonucleic acid or at least one ribonucleic acid (e.g., gRNA) pair capable of directing and hybridizing a Cas protein to a target motif of a target polynucleotide sequence.

In some embodiments, the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprise conservative amino acid substitutions. In some cases, the substitution and/or modification may prevent or reduce proteolytic degradation and/or extend the half-life of the polypeptide in the cell. In some embodiments, the Cas protein may comprise a peptide bond substitution (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 an optional amino acid (e.g., D-amino acid, β -amino acid, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein may comprise modifications to include moieties (e.g., pegylation, glycosylation, lipidation, acetylation, capping, etc.).

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 of the e.coli (e.coli) subtype (also known as CASS 2). 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 Cas protein of subtype Ypest (also referred to as CASS 3). Exemplary Cas proteins of subtype Ypest include, but are not limited to Csy1, csy2, csy3, and Csy4. In some embodiments, the Cas protein comprises Cas protein of subtype Nmeni (also referred to as CASS 4). Exemplary Cas proteins of subtype Nmeni include, but are not limited to Csn1 and Csn2. In some embodiments, the Cas protein comprises Cas protein of subtype Dvulg (also referred to as CASS 1). Exemplary Cas proteins of subtype Dvulg include, but are not limited to Csd1, csd2, and Cas5d. In some embodiments, the Cas protein comprises Cas protein of subtype Tneap (also referred to as CASS 7). Exemplary Cas proteins of subtype Tneap include, but are not limited to Cst1, cst2, cas5t. In some embodiments, the Cas protein comprises Cas protein of subtype Hmari. Exemplary Cas proteins of subtype Hmari include, but are not limited to Csh1, csh2, and Cas5h. In some embodiments, the Cas protein comprises Cas protein of subtype Apern (also referred to as CASS 5). Exemplary Cas proteins of subtype Apern include, but are not limited to Csa1, csa2, csa3, csa4, csa5, and Cas5a. In some embodiments, the Cas protein comprises Cas protein of subtype Mtube (also referred to as CASS 6). Exemplary Cas proteins of subtype Mtube 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.

In some embodiments, the Cas protein is a streptococcus pyogenes (Streptococcus pyogenes) Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a staphylococcus aureus (Streptococcus aureus) Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a streptococcus thermophilus (Streptococcus thermophilus) Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a neisseria meningitidis (NEISSERIA MENINGITIDES) Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a dense tooth helix (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. Cas9 proteins are members of a type II CRISPR system that typically includes a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas protein. The Cas9 protein (also known as CRISPR-associated endonuclease Cas9/Csn 1) is a polypeptide comprising one amino acid.

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)/Cas 9 gene editing technology.

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)/Cas 9 gene editing techniques.

In one embodiment, the expression of the CD300LD protein is increased by modification of the endogenous locus. In some embodiments, the endogenous locus is modified by a "change" or "genetic alteration" technique as described above. Such as gene knock-in, clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) techniques, transcription activator-like effector nucleases (TALENs) techniques, zinc Finger Nucleases (ZFNs) techniques, or homologous recombination techniques.

In one embodiment, expression of the CD300LD protein is increased by expression of the transgene. Transgenic expression techniques known in the art can be used to increase expression of CD300LD protein, including but not limited to viral techniques, piggybac transposon techniques, sleeping beappearance transposon techniques.

In this context, well-known recombinant techniques may be used to generate expression constructs as described herein. In certain embodiments, the nucleic acid sequence encoding the protein of interest may be operably linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequences are generally suitable for the host cell and the subject to be treated. Various types of suitable expression vectors and suitable regulatory sequences are known in the art for use in a variety of host cells. In general, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Expression constructs used herein may use constitutive or inducible promoters known in the art. The promoter may be a naturally occurring promoter, or a hybrid promoter combining elements of more than one promoter. The expression construct may be present in the cell on an episome (e.g., a plasmid), or the expression construct may be inserted into a chromosome. In a specific embodiment, the expression vector includes a selectable marker gene to allow selection of transformed host cells. Certain embodiments include expression vectors comprising a nucleotide sequence encoding a protein of interest operably linked to at least one regulatory sequence. Regulatory sequences for use herein include promoters, enhancers and other expression control elements. In certain embodiments, the expression vector is designed for the selection of the host cell to be transformed, the desired protein to be expressed, the copy number of the vector, the ability to control that copy number, or the expression of any other protein encoded by the vector, such as an antibiotic marker. In some embodiments, the promoter is an EF1a promoter.

Viral technology can be used to cause increased expression of the CD300LD gene in the low immunogenicity pluripotent stem cells. Such viral techniques include, but are not limited to, the use of retroviral vectors, lentiviral vectors, adenoviral vectors, and sendai viral vectors.

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

In one embodiment, the CD300LD protein is a human CD300LD protein.

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

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

Prevention or treatment of

The invention further provides the use of the low-immunogenicity pluripotent stem cells of the invention or the low-immunogenicity pluripotent stem cells prepared by the method of the invention in the preparation of a medicament for preventing or treating a disease requiring cell transplantation.

The low-immunogenicity pluripotent stem cells of the invention or the low-immunogenicity pluripotent stem cells prepared by the method of the invention can induce differentiation 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 method of differentiation depends on the desired cell type using known techniques. For example, cells can be differentiated in suspension and then made into a gel matrix form, such as matrigel, gelatin, or fibrin/thrombin form, to promote cell survival. Differentiation can generally be determined by assessing the presence of cell-specific markers as known in the art. For example, cells may be differentiated into cardiomyocytes, neural cells, 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, liver cells, thyroid cells, skin cells, blood cells, or epithelial cells under certain differentiation conditions.

In some embodiments, the disease is a cancer comprising a solid tumor and a hematological tumor. In some embodiments, the solid tumor comprises small cell lung cancer, breast cancer, testicular cancer, neuroblastoma, ovarian cancer, or melanoma. In some embodiments, the hematological tumor comprises acute leukemia, chronic leukemia, lymphoma, myelodysplastic syndrome, or multiple myeloma.

In one embodiment, the disease comprises aplastic anemia.

In one embodiment, the disease comprises an innate immune deficiency disease.

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

In some embodiments, the disease is a neurodegenerative disease comprising parkinson's disease, alzheimer's disease, spinal cord injury, retinal degenerative disease, stroke, huntington's disease, or amyotrophic lateral sclerosis.

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

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

Differentiation of pluripotent stem cells

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, techniques known in the art may be used to engraft the differentiated low-immunogenicity pluripotent cell derivatives, depending on the cell type and the end use of the cells.

1. Cardiomyocytes differentiated from pluripotent stem cells

The present invention provides pluripotent stem cells that can differentiate into different cardiac cell types for subsequent transplantation or implantation into a subject (e.g., recipient).

As will be appreciated by those skilled in the art, the method of differentiation depends on the desired cell type using known techniques. Exemplary cardiac cell types include, but are not limited to, cardiomyocytes, nodular 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, cardiac valve mesenchymal cells, cardiac pacing marker cells, and the like.

In some embodiments, cardiomyocyte precursors include cells capable of producing progeny that include mature (end-stage) cardiomyocytes (without dedifferentiation or reprogramming). Cardiomyocyte precursor cells can be identified generally using one or more markers selected from the GATA-4, nkx2.5 and MEF-2 transcription factor families.

In some embodiments, the cardiomyocyte is a hypoimmunogenic cardiomyocyte.

In some embodiments, cardiomyocytes as described herein are administered to a recipient subject to treat a cardiac disorder selected from pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, perinatal cardiomyopathy, inflammatory cardiomyopathy, idiopathic cardiomyopathy, other cardiomyopathy, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart disease, arterial inflammation, cardiovascular disease, myocardial infarction, myocardial ischemia, congestive heart failure, myocardial infarction, myocardial ischemia, cardiac injury, myocardial ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, coronary artery disease, conduction system dysfunction, coronary arterial hypertension, disorders, muscle dysfunction trophy, abnormal muscle mass, muscle degeneration, myocarditis, infectious myocarditis, drug-induced myocarditis, allergic myocarditis, and autoimmune membrane-induced myocarditis.

In some embodiments, a method of producing a low immunogenic cardiomyocyte population from a low immunogenic pluripotent (HIP) stem cell population by in vitro differentiation comprises (a) culturing the HIP cell population in a medium comprising a GSK inhibitor, (b) culturing the HIP cell population in a medium comprising a WNT antagonist to produce a pre-cardiomyocyte population, and (c) culturing the pre-cardiomyocyte population in a 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 of GSK inhibitor ranges from about 2mM to about 10mM. In some embodiments, the WNT antagonist is IWR1, a derivative or variant thereof. In some cases, the concentration of WNT antagonist ranges from about 2mM to about 10mM.

Other useful methods for inducing differentiation of pluripotent stem cells or multipotent stem cells into cardiomyocytes are described, for example, in US2017/0152485, US2017/0058263, US2017/0002325, US2016/0362661, US2016/0068814, US9,062,289, US7,897,389, and US7,452,718. Other methods for generating cardiac cells from induced pluripotent stem cells or multipotent stem cells are described, for example, in Xu et al, STEM CELLS AND Development,2006,15 (5): 631-9, burridge et al, CELL STEM CELL,2012,10:16-28, and Chen et al, STEM CELL RES,2015,15 (2): 365-375.

In various embodiments, the hypoimmunogenic 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, cardiomyocytes, other compounds, and the like.

WNT signaling activators include, but are not limited to CHIR99021.PCK activators include, but are not limited to, PMA. Inhibitors of WNT signaling include, but are not limited to, compounds selected from KY02111, SO3031 (KY 01-I), SO2031 (KY 02-I), SO3042 (KY 03-I), and XAV939. Src inhibitors include, but are not limited to, a419259. Such EGFR inhibitors include, but are not limited to AG1478.

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.

2. Neural cells differentiated from pluripotent stem cells

The present invention provides pluripotent stem cells that can differentiate 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 method of differentiation 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.

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's Syndrome (TS), schizophrenia, psychosis, depression, other neuropsychiatric disorders. In some embodiments, the neural cells described herein are administered to a subject to treat or ameliorate stroke.

In some embodiments, neurons and glial cells are administered to a subject suffering from Amyotrophic Lateral Sclerosis (ALS). In some embodiments, brain endothelial cells are administered to alleviate symptoms or effects of cerebral hemorrhage. In some embodiments, the dopaminergic neurons are administered to patients suffering from parkinson's disease. In some embodiments, noradrenergic neurons, gabaergic interneurons are administered to patients who have experienced seizures. In some embodiments, motor neurons, interneurons, schwann cells, oligodendrocytes, and microglia are administered to a patient experiencing spinal cord injury.

In some embodiments, brain Endothelial Cells (ECs), precursors and progenitors thereof are differentiated from pluripotent stem cells (e.g., induced pluripotent stem cells) on the surface by culturing the cells in a medium comprising one or more factors that promote production of the brain endothelial cells or nerve cells. In some cases, the medium comprises one or more of CHIR-99021, VEGF, basic FGF and Y-27632. In some embodiments, the culture medium includes a supplement designed to promote survival and function of the neural cells. In some embodiments, brain Endothelial Cells (ECs), their precursor cells and progenitor cells are differentiated from pluripotent stem cells on the surface by culturing the cells in a non-conditioned 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, differentiation is typically determined by assessing the presence of cell-specific markers, as known in the art. In some embodiments, the brain endothelial cells express or secrete a factor selected from the group consisting of CD31, VE cadherin, and combinations thereof.

In some embodiments, neurons, precursors and progenitors thereof differentiate from pluripotent stem cells by culturing the 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, a basic EGF, NGF, CNTF, SMAD inhibitor, a Wnt antagonist, an activator of SHH signaling, 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、LY550410、LY580276、LY364947、LY2109761、SB-505124、E-616452(RepSoxALK inhibitors), SD-208, SMI6, NPC-30345, K26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, dorsomorphin dihydrochloride, and derivatives thereof.

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. Wherein. In some embodiments, the SHH signaling activator is selected from the group consisting of Smoothened Agonists (SAG), SAG analogs, SHH, C25-SHH, C24-SHH, pumice, hg-Ag, and derivatives thereof.

In some embodiments, the stem cells described herein differentiate into dopaminergic neurons, including dopaminergic progenitor cells. Stem cells are cultured in differentiation medium containing supplements or additives to induce neuronal differentiation. In some embodiments, the cells are cultured in the presence of a supplement or additive to induce the floor cells. In some embodiments, the supplement or additive includes BMP inhibitor LDN193189, ALK-5 inhibitor a83-01, smoothened agonist pumice, FGF8, GSK3 inhibitor CHIR99021, glial cell line derived neurotrophic factor, GDNF, ascorbic acid, brain-derived neurotrophic factor BDNF, dbcAMP di Ding Xianxian glycoside loop monophosphate, ROCK inhibitor Y-27632, and the like.

In some embodiments, a method of producing a population of low-immunogenicity dopaminergic neurons from a population of low-immunogenicity induced pluripotent stem cells (HIP cells) by in vitro differentiation comprises (a) culturing the population of HIP cells in a first medium comprising one or more factors selected from the group consisting of sonic hedgehog (SHH), BDNF, EGF, bFGF, FGF, WNT1, retinoic acid, a GSK3 inhibitor, an ALK inhibitor, and the ROCK inhibitor can produce a population of immature dopaminergic neurons, and (b) culturing the population of immature dopaminergic neurons in a second medium different from the first medium to produce the population of dopaminergic neurons. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of GSK inhibitor ranges from about 2mM to about 10mM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or variant thereof. In some cases, the concentration of ALK inhibitor ranges from about 1mM to about 10mM. In some embodiments, the first medium and/or the second medium is free of animal serum.

Methods for differentiating pluripotent stem cells are described, for example, in Kikuchi et al, nature,2017,548,592-596, kriks et al, nature, 2011, 547-551, doi et al, stem Cell Reports,2014,2,337-50, perier et al, proc NATL ACAD SCI USA,2004,101,12543-12548, chamers et al, natBiotechnol,2009,27,275-280, kirkeby et al, cell Reports, 2012,1, 703-714. Useful descriptions of stem Cell-derived neurons and methods of 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,"Progress in Brain Research", "chapter 8-strategy for introducing stem Cell-derived dopamine neurons into the clinic-NYSTEM test", 2017, volume 230, page 15. 191-212; liu et al Nat Protoc,2013,8:1670-1679; uppaphia et al Curr Protoc Stem Cell Biol,38,2D.7.1-2D.7.47; U.S. patent application 20160115448 and US8,252,586; US8,273,570; U.S. 9,487,752 and US10,093,897 are incorporated herein by reference in their entirety.

In some embodiments, glial cells, including microglial cells, astrocytes, oligodendrocytes, ependymal cells and schwann cells, glial precursors and glial progenitor cells, are produced by differentiating pluripotent stem cells into therapeutically effective glial cells, and the like. Differentiation of the low-immunogenicity pluripotent stem cells produces low-immunogenicity neural cells, such as low-immunogenicity glial cells.

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, TGF beta inhibitor, BMP signaling inhibitor, SHH signaling activator, FGF, platelet derived growth factor PDGF, PDGFR-alpha, HGF, IGF-1, noggin. Hog (SHH), dorsomorphin, noggin, and any combination thereof. In certain instances, the BMP signaling inhibitor is LDN193189, SB431542, or a combination thereof.

In some embodiments, glial cells are determined to express a known glial cell biomarker. Useful methods for generating glial cells, their precursors and progenitors from stem cells can be found, 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;

US2017/0182097, US2017/253856, US 2018/023604, WO2017/172976, and WO2018/093681.

In some embodiments, differentiation of pluripotent stem cells is performed by exposing or contacting the cells to specific factors known to produce a specific cell lineage in order to target their differentiation to a specific, desired lineage and/or cell type.

In some embodiments, terminally differentiated cells exhibit a particular phenotypic characteristic or characteristic. In certain embodiments, the stem cells described herein differentiate into a neuroectodermal, neuronal, neuroendocrine, dopaminergic, cholinergic, serotonergic (5-HT), glutamatergic, GABAergic, adrenergic, noradrenergic, sympathetic, parasympathetic, sympathetic peripheral, or glial cell population. In some cases, the glial cell population includes a microglial cell population or a macroglial cell population (central nervous system cells: astrocytes, oligodendrocytes, ependymal cells and radial glial cells; and peripheral nervous system cells: schwann cells and satellite cells), or precursor and progenitor cells of any of the foregoing.

Protocols for generating different types of neural cells are described in PCT application No. WO2010144696, U.S. Pat. No. 9,057,053, 9,376,664, and 10,233,422. Additional description of methods for differentiating low immunogenicity pluripotent cells can be found in Deuse et al, nature Biotechnology,2019,37,252-258 and Han et al, proc NATL ACAD SCI USA,2019,116 (21), 10441-10446, for example. Methods for determining the effect of neural cell transplantation in animal models of neurological diseases or disorders are described in the references of, for example, 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, interpen, DOI 10.5772/intelopen.72862, for epilepsy-Upadhya et al, PNAS,2019,116 (l): 287-296 neural cell transplantation efficacy for spinal cord injury can be assessed in rat models of acute spinal cord injury, such as McDonald et al, nat. Med.,1999, 5:1410) and Kim et al Nature,2002,418:50. For example, a successful transplant may show that after 2-5 weeks the lesion has cells of graft origin, differentiate into astrocytes, oligodendrocytes and/or neurons, and migrate from the diseased end along the spinal cord with improved gait, coordination and weight bearing. Depending on the type of neural cells and the neurological disease or disorder to be treated, the neural cells may be administered in a manner that allows them to be transplanted into the desired tissue site and to reconstruct or regenerate the functionally defective region. For example, depending on the disease being treated, the nerve cells may be transplanted directly into a parenchymal or intrathecal site of the central nervous system. In some embodiments, any of the neural cells described herein, including brain endothelial cells, neurons, dopaminergic neurons, ependymal cells, astrocytes, microglial cells, oligodendrocytes, and schwann cells, are injected into the patient by intravenous, intraspinal, intraventricular, intrathecal, intraarterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, intraabdominal, intraocular, retrobulbar, and combinations thereof. In some embodiments, the cells are injected or deposited in the form of a bolus or continuous infusion. In certain embodiments, the neural cells are administered by injection into the brain, near the brain, and combinations thereof. For example, injection may be performed through a drill hole made in the skull of the subject. Suitable sites for administration of the neural cells to the brain include, but are not limited to, ventricles, lateral ventricles, cerebellar ponds, putamen, basal nuclei, hippocampal cortex, striatum, caudate region of the brain, and combinations thereof.

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.

3. Endothelial cells differentiated from pluripotent stem cells

The present invention provides pluripotent stem cells that can differentiate into various endothelial cell types for subsequent transplantation or implantation into a subject (e.g., recipient). As will be appreciated by those skilled in the art, the method of differentiation depends on the desired cell type using known techniques. Exemplary endothelial cell types include, but are not limited to, capillary endothelial cells, vascular endothelial cells, aortic endothelial cells, arterial endothelial cells, venous endothelial cells, renal endothelial cells, brain endothelial cells, hepatic endothelial cells, and the like.

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 injury, reperfusion injury, limb ischemia, stroke, neuropathy (e.g., peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver failure, kidney failure, etc.), diabetes, rheumatoid arthritis, osteoporosis, cerebrovascular disease, hypertension, angina and myocardial infarction due to coronary heart disease, renal vascular hypertension, renal failure due to renal arterial stenosis, lower limb lameness mites, other vascular conditions or diseases.

In some embodiments, the low immunogenicity pluripotent cells differentiate into Endothelial Colony Forming Cells (ECFCs) to form new blood vessels to address peripheral arterial disease. Techniques for differentiating endothelial cells are known. See, e.g., prasain et al, doi:10.1038/nbt.3048, which is incorporated herein by reference in its entirety, and is directed specifically to methods and reagents for the production of 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.

In some embodiments, a method of producing a population of low-immunogenic endothelial cells from a population of low-immunogenic pluripotent cells by in vitro differentiation comprises (a) culturing the population of HIP cells in a first medium comprising a GSK inhibitor, (b) culturing the population of HIP cells in a second medium comprising VEGF and bFGF to produce a population of pre-endothelial cells, and (c) culturing the population of pre-endothelial cells in a third medium comprising a ROCK inhibitor and an ALK inhibitor to produce a population of low-immunogenic endothelial cells.

In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of GSK inhibitor ranges from about 1mM to about 10mM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some cases, the concentration of ROCK inhibitor ranges from about 1pM to about 20pM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or variant thereof. In some cases, the concentration of ALK inhibitor ranges from about 0.5pM to about 10pM.

In some embodiments, the first medium comprises CHIR-99021 of from 2pM to about 10 pM. In some embodiments, the second medium comprises 50ng/ml VEGF and 10ng/ml bFGF. In other embodiments, the second medium further comprises Y-27632 and SB-431542. In various embodiments, the third medium comprises 10pM Y-27632 and 1pM SB-431542. In certain embodiments, the third medium further comprises VEGF and bFGF. In certain cases, the first medium and/or the second medium is free of insulin.

In some embodiments, the population of hypoimmunogenic endothelial cells is isolated from the non-endothelial cells. In some embodiments, the isolated population of low-immunogenicity endothelial cells is expanded prior to administration. In certain embodiments, the isolated population of low-immunogenicity endothelial cells is expanded and cryopreserved prior to administration.

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.

4. Thyroid cells differentiated from pluripotent stem cells

In some embodiments, the pluripotent stem cells can differentiate 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,2015, 11, 5; 17 (5): 527-42, which is incorporated by reference in its entirety, and particularly directed to methods and reagents for generating thyroid cells from human pluripotent stem cells, and transplantation techniques. Differentiation can be assayed as known in the art, typically by assessing the presence of thyroid cell associated or specific markers or by functional measurement.

5. Differentiation of pluripotent stem cells into hepatocytes

In some embodiments, the pluripotent stem cells may differentiate into hepatocytes to address loss of hepatocyte function or cirrhosis. There are various techniques available for differentiating HIP cells into hepatocytes, see, e.g., 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.

6. Islet cells differentiated from pluripotent stem cells

The present invention provides pluripotent stem cells that can differentiate into various islet cell types for subsequent transplantation or implantation into a subject (e.g., recipient). As will be appreciated by those skilled in the art, the method of differentiation depends on the desired cell type using known techniques. Exemplary islet cell types include, but are not limited to, islet progenitor cells, immature islet cells, mature islet cells, and the like. In some embodiments, pancreatic cells described herein are administered to a subject to treat diabetes.

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

In some embodiments, the low-immunogenicity pluripotent cells differentiate into β -like cells or islet organoids for transplantation to address type I diabetes (T1 DM). The cellular system is a promising approach to address T1DM, see Ellis et al Nat Rev Gastroenterol Hepatol. 10 months 2017, 14 (10): 612-628, incorporated herein by reference. Furthermore, pagbuca et al (Cell, 2014,159 (2): 428-39), report successful differentiation of b cells from hiPSCs, the contents of which are incorporated herein by reference in their entirety, and in particular the methods and reagents for large-scale production of functional human b cells from human pluripotent stem cells outlined therein. Furthermore, vegas et al, which produce human b cells from human pluripotent stem cells and then are packaged to avoid immune rejection by the host, vegas et al, natMed,2016,22 (3): 306-11, which is incorporated herein by reference in its entirety, particularly the methods and reagents for large-scale production of functional human b cells from human pluripotent stem cells as outlined therein.

In some embodiments, a method of producing a low immunogenic islet cell population from a low immunogenic pluripotent cell population by in vitro differentiation comprises (a) culturing the cell population in a first medium comprising one or more factors selected from the group consisting of insulin-like growth factor (IGF), transforming Growth Factor (TGF), fibroblast growth factor (EGF), epidermal Growth Factor (EGF), hepatocyte Growth Factor (HGF), sonic hedgehog (SHH), and Vascular Endothelial Growth Factor (VEGF), transforming growth factor-b (TORb) superfamily, bone morphogenic protein-2 (BMP 2), bone morphogenic protein-7 (BMP 7), GSK inhibitor, ALK inhibitor, BMP type 1 receptor inhibitor, and retinoic acid to produce an immature islet cell population, (b) culturing the immature islet cell population in a second medium different from the first medium to produce the low immune islet cell population. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of GSK inhibitor ranges from about 2mM to about 10mM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or variant thereof. In some cases, the concentration of ALK inhibitor ranges from about 1pM to about 10pM. In some embodiments, the first medium and/or the second medium is free of animal serum.

In some embodiments, the population of low-immunogenicity islet cells is isolated from non-islet cells. In some embodiments, the isolated population of low-immunogenicity islet cells is expanded prior to administration. In certain embodiments, an isolated population of low-immunogenicity islet cells is expanded and cryopreserved prior to administration.

Differentiation is typically determined by assessing the presence of b-cell related or specific markers, including but not limited to insulin, as is known in the art. Differentiation may also be measured functionally, for example, by measuring glucose metabolism, see generally Muraro et al, cell Syst.2016, 10-month 26; 3 (4): 385-394.e3, which is hereby incorporated by reference in its entirety, and particularly for the biomarkers outlined therein. Once the beta cells are produced, they can be transplanted (either as a cell suspension or within the gel matrix discussed herein) into the portal vein/liver, omentum, gastrointestinal mucosa, bone marrow, muscle, or subcutaneous sac.

Additional description of islet 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.

7. Retinal Pigment Epithelial (RPE) cells differentiated from pluripotent stem cells

The present invention provides low-immunogenicity pluripotent cells that can differentiate into various RPE cell types for subsequent transplantation or implantation into a subject (e.g., recipient). As will be appreciated by those skilled in the art, the method of differentiation depends on the desired cell type using known techniques. Exemplary RPE cell types include, but are not limited to, retinal Pigment Epithelial (RPE) cells, RPE progenitor cells, immature RPE cells, mature RPE cells, functional RPE cells, and the like.

Useful methods for differentiating pluripotent stem cells into RPE cells are described, for example, in US9,458,428 and US9,850,463, the disclosures of which are incorporated herein by reference in their entirety, including the specification. Other methods for inducing pluripotent stem cells from humans to produce RPE cells can be found, for example, in Lamba et al, PNAS,2006,103 (34): 12769-12774; melugh et al, stem cells, 2012,30 (4): 673-686; idelson et al, cell stem cells, 2009,5 (4): 396-408; rowland et al, journal of Cellular Physiology,2012,227 (2): 457-466, buchholz et al, STEM CELLS TRANS MED,2013,2 (5): 384-393, da Cruz et al, nat Biotech,2018, 36:328-337.

In some embodiments, the RPE cells described herein are administered to a subject to treat an ocular disorder selected from wet macular degeneration, dry macular degeneration, juvenile macular degeneration (e.g., stargardt disease, best disease, and juvenile retinal cleavage), leber's congenital amaurosis, retinitis pigmentosa, retinal detachment, age-related macular degeneration (AMD), early AMD, intermediate AMD, advanced AMD, non-neovascular age-related macular degeneration.

Human pluripotent stem cells have been differentiated into RPE cells using the techniques outlined in Kamao et al, stem Cell Reports 2014:2:205-18, which is incorporated herein by reference in its entirety, particularly the methods and reagents for differentiation techniques and reagents outlined therein, see also Mandai et al, N Engl J Med,2017,376:1038-1046, which is incorporated herein in its entirety for an understanding of techniques for generating and transplanting into a patient. 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, e.g., 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.

In some embodiments, a method of producing a population of low immunogenic Retinal Pigment Epithelium (RPE) cells from a population of low immunogenic pluripotent cells by in vitro differentiation comprises (a) culturing the population of low immunogenic pluripotent cells in a first medium comprising a population of pre-RPE cells selected from the group consisting of activin A, bFGF, BMP/7, DKKl, IGFl, noggin, BMP inhibitor, ALK inhibitor, ROCK inhibitor, and VEGFR inhibitor, and (b) culturing the population of pre-RPE cells in a second medium different from the first medium to produce the population of low immunogenic RPE cells. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or variant thereof. In some cases, the concentration of ALK inhibitor ranges from about 2mM to about 10mM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some cases, the concentration of ROCK inhibitor ranges from about 1pM to about 10pM. In some embodiments, the first medium and/or the second medium is free of animal serum.

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, e.g., 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 to the results section.

Additional description of RPE cells useful in the present invention can be found in WO2020/018615, the disclosure of which is incorporated herein by reference in its entirety.

8. NK cells differentiated from pluripotent stem cells

The present invention provides low immunogenicity pluripotent cells that can differentiate into Natural Killer (NK) cells. In some embodiments, a method of producing a Natural Killer (NK) cell population from a low immunogenicity pluripotent cell population by in vitro differentiation comprises (a) culturing a stem cell population in a first medium comprising a ROCK inhibitor under conditions sufficient to form the aggregate, (b) culturing the aggregate in a second medium comprising BMP-4, (C) culturing the aggregate in a third medium comprising BMP-4, FGF2, WNT pathway activator, and activin A, (d) culturing the aggregate in a fourth medium comprising FGF2, VEGF, TPO, SCF, IL-3, FLT3L, WNT C-59, and activin/node inhibitor to form a cell population comprising hematopoietic stem cells and progenitor cells (HSPC), (e) culturing the cell population in a fifth medium comprising FGF2, VEGF, TPO, SCF, IL-3, and FLT3L, (f) culturing the cell population in a sixth medium comprising IL-3, IL-7, FLT3L, IL-15, and SCF, (g) culturing the cell population in a seventh medium comprising IL-7, FLT L, IL, and SCF for a time sufficient to produce the cells.

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

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.

For therapeutic applications, cells prepared according to the disclosed methods can generally be provided in the form of pharmaceutical compositions comprising isotonic excipients and prepared under conditions sufficiently sterile for human administration. For general principles of pharmaceutical formulations of cellular compositions, see "cell therapy: stem cell transplantation, gene therapy and cellular immunotherapy", edit Morstyn & Sheridan, cambridge university press, 1996, and "Hematopic STEM CELL THERAPY", E.D.Ball, J.Lister & P.Law, churchill Livingstone,2000. The cells may be packaged in a device or container suitable for dispensing or clinical use.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The positive progress effect of the invention is that the pluripotent stem cells and the low immunogenicity pluripotent stem cells prepared by the method of the invention can exhibit excellent effects such as, but not limited to, (1) have good self-renewal and differentiation ability, (2) escape T cell killing, (3) escape NK cell killing, (4) escape PBMC cell killing, (5) escape MAC cell killing, and/or (6) escape NK cell, T cell or MAC cell killing of differentiated cells, thereby exhibiting excellent application potential.

The invention is further described by reference to the following examples. It should be understood that these embodiments are by way of example only and are not limiting of the invention. The following materials and instruments are commercially available or prepared according to methods well known in the art. The following experiments were performed according to the manufacturer's instructions or according to methods and procedures well known in the art.

The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations.

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

Example 1 construction of B2M and CIITA double knockout cell line (DKO)

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

The following example selects human induced pluripotent stem cells (iPSC cells) for the construction of the target cell line, which were prepared from human peripheral blood cells using CTS ^TM CytoTune^TM -iPS2.1Sendai Virus reprogramming kit (CTS ^TM CytoTune^TM -iPS2.1Sendai Reprogramming Kit, cat# A34546) according to the manufacturer's instructions. Human pluripotent stem cell lines H1 (Wicell, WA 01) or H9 (Wicell, WA 09) can also be used to construct target cell lines using cell culture and gene knockout reagents as shown in Table 1.

TABLE 1 cell culture and Gene knockout reagent

Beta-2-microglobulin (B2M) in endoplasmic reticulum is knocked out by CRISPR/CAS9, so that functional molecules cannot be formed on the cell surface of MHC-I, thereby escaping the killing of allogeneic CD8 ⁺ T cells, and escaping the killing of CD4+ T cells is realized by knocking out the upregulating factor CIITA transcribed by MHC-II genes, thereby reducing the expression of MHC-II molecules. Wherein the double knockout pluripotent stem cells (DKO) of the H1 cell line in the present disclosure 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.

The CRISPR/CAS9 gene knockout strategy of B2M and the gRNA sequences and identification primers used are shown in fig. 1 and table 2, the B2M exon segments were knocked out directly at both ends using B2M-gRNA1 and B2M-gRNA2 (EASYEDIT SGRNA, kunsui), and then the genomic sequences were knocked out using two pairs of PCR primers B2M-F1/R1 and B2M-F2/R2, respectively, wherein a of fig. 1 is the knockout strategy of B2M gene of B2M and CIITA double knockout cell line (DKO), B of fig. 1 is the result of B2M gene knockout verification of H1 cell line by PCR, and C of fig. 1 is the result of B2M gene knockout verification of iPSC cell line by PCR.

In addition, the CIITA CRISPR/CAS9 gene knockout strategy and the gRNA sequences and identification primers used are shown in FIG. 2 and Table 2, the CIITA exon segments were knocked out directly at both ends using CIITA-gRNA1 and CIITA-gRNA2 (EASYEDIT SGRNA, kirsrui), and then genomic sequence knockdown verification was performed using two pairs of PCR primers, CIITA-F1/R1 and CIITA-F2/R2, respectively, wherein A in FIG. 2 is the knockout strategy of CIITA gene of B2M and CIITA double knockout cell line (DKO), B in FIG. 2 is the result of CIITA gene knockdown verification of H1 cell line by PCR, and C in FIG. 2 is the result of CIITA gene knockdown verification of iPSC cell line by PCR.

TABLE 2 gRNA sequences and identifying primers

The specific operation is as follows:

1) Human induced pluripotent stem cells were cultured to 80% density on Matrigel coated 6 well plates using mTeSR1 medium supplemented with Y-27632. Digestion with TrypLE was followed by neutralization with DMEM/F12 and counting. The 2X 10 ⁶ cells were pipetted into an EP tube and the supernatant was discarded after centrifugation.

2) According to the Neon transfection System (ThermoFisher) 100. Mu.L electric rotator system, 15. Mu. g TrueCut ^TM Cas9 protein+3mu. ggRNA (B2M-gRNA 1+B2M-gRNA2+CIITA-gRNA1+CIITA-gRNA 2) was added to make up a ribonucleoprotein complex (RNP) system, and after mixing, left at room temperature for 20min.

3) Cells were resuspended with 100. Mu.L of RNP electrotransfection system and electrotransfection was performed with the Neon transfection system with parameters 1200V,30ms,1pause. The pre-warmed medium was rapidly added to the cells after electrotransformation and then plated uniformly in 1 well Matrigel coated 6 well plates.

4) Fresh mTeSR1 medium was changed daily. After single cells grow up, single clones are picked up in a 48-well plate, after cloning and amplification, genome samples are collected for PCR detection and gene editing conditions, and the PCR results are shown in figures 1 and 2. The PCR positive clones were sent to Sanger sequencing for further validation.

5) Amplification culture and cryopreservation identify positive B2M/CIITA bi-allele knock-out clones DKO.

1.2 Detection of expression of RNA levels of B2M and CIITA of DKO

H1 cells and derived DKO cell total RNA were extracted using FastPure Cell/Tissue Total RNA Isolation Kit V2 (Nuo ZAN, RC 112-01) and then RNA was inverted to cDNA using HISCRIPT III RT SuperMix for qPCR (Nuo ZAN, R323-01) according to the manufacturer's instructions.

Expression of B2M and CIITA at the RNA level was detected using qPCR on B2M/CIITA bi-allele knockdown clone H1 cell-derived DKO cells using the primers shown below.

B2M-F(SEQ ID NO:19):AAGATGAGTATGCCTGCCGT

B2M-R(SEQ ID NO:20):ATGCGGCATCTTCAAACCTC

CIITA-F(SEQ ID NO:21):CCTGGAGCTTCTTAACAGCGA

CIITA-R(SEQ ID NO:22):TGTGTCGGGTTCTGAGTAGAG

Using a Roche 480II instrument, the reaction system was as follows:

Pre-incubation,95°C,30s。

amplification,95 ℃ 10s,60 ℃ 30s,40 cycles.

Mering cut and Cooling are default procedures.

The qPCR results are shown in fig. 3, where B2M and CIITA were expressed at RNA level nearly absent from B2M/CIITA double allele knock-out clones DKO, relative to untreated wild-type human pluripotent stem cells, confirming successful knock-out of B2M and CIITA.

1.3 Detection of expression of DKO at the B2M protein level

Western blotting (Western-Blot) was used to detect expression of B2M protein levels of DKO derived from B2M/CIITA double allele knock-out clone H1 cells (B2M antibody accession number ab75853; reference antibody GAPDH accession number ab181602, both from Abcam).

The western blot results are shown in fig. 4, where B2M protein in H1 cell-derived DKO was significantly reduced relative to wild-type human pluripotent stem cells, confirming successful knockout of B2M protein.

1.4 Detection of HLA-I/II class molecules of DKO

WT and DKO were stimulated with IFN-. Gamma.s (PeproTech, cat # 300-02) to detect HLA I/II type molecules on the surfaces of H1 cells (WT) and iPSC (iPSC-WT) cell lines and their corresponding derived DKO cells (DKO cells and iPSC-DKO cells), as follows:

Cells were plated, and on the next day of liquid change, medium containing IFN-. Gamma.was added to the cells, and after 48 hours of action, the cells were digested and examined for HLA-I/II expression using a flow cytometer (Agilent Technologies, novoCyte).

The flow detection results are shown in fig. 5A and 5B, wherein fig. 5A shows the detection results of H1 cells and DKO cells, fig. 5B shows the detection results of iPSC cells and iPSC-DKO cells, all of the figures show HLA-ABC on the left to detect HLa-class I molecules, HLa-DR, DQ, DP on the right to detect HLa-class II molecules, and T cells are positive controls for detection. It was observed that both B2M/CIITA bi-allelic knockout clone H1 and iPSC cell line derived DKO cells (DKO cells and iPSC-DKO cells) were unable to express HLA-I/II class molecules in response to stimulation by IFN- γ, demonstrating reduced HLA-I and HLA-II functions of DKO cells and iPSC-DKO cells.

1.5 Nuclear detection of DKO

The obtained H1 cell positive clone (DKO) with B2M/CIITA double allele knockout is subjected to karyotyping, and the specific detection method is as follows:

Chromosomal specimens mounted on slides were treated with trypsin and stained with Giemsa's staining solution. According to the characteristics of chromosome length, the position of the fiber point, the ratio of long and short arms, the existence of a follower and the like, the metaphase chromosome is analyzed for chromosome number and morphological structure so as to determine whether the karyotype is consistent with the normal karyotype.

As shown in FIG. 6, the karyotype of the H1 cell-derived DKO cell is normal, and has no obvious change compared with the normal karyotype.

Example 2 drying and immune Functions of DKO cell lines

This example further examined whether the stem and immune functions of pluripotent stem cells were altered after knockout of the B2M/CIITA bi-allele.

2.1 Expression of Stem genes in DKO cells

Protein levels of the stem 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, expression of the stem genes in H1 cells (WT cells) and DKO cells (DKO cells) at RNA levels was detected by RT-qPCR, and expression of the corresponding proteins of the stem genes on the surfaces of H1 cells (WT cells) and iPSC-WT cells and DKO cells (DKO cells and iPSC-DKO cells) was detected by flow cytometry, which were specifically detected as follows:

immunofluorescence assay by plating WT or DKO cells in 12 well plates, pipetting off the medium after the cells grow to 60-80% density, and fixing with 4% paraformaldehyde. After cell rupture, the primary antibodies of other dry gene proteins such as POU5F1 and NANOG are used for overnight incubation at 4 ℃, the secondary antibodies with fluorescent markers are incubated at room temperature after the primary antibodies are washed away, and then a fluorescence microscope (Nikon Ts 2R-FL) is used for photographing.

RT-qPCR detection:

using a Roche 480II instrument, the reaction system was as follows:

Pre-incubation,95°C,30s。

amplification,95 ℃ 10s,60 ℃ 30s,40 cycles.

Mering cut and Cooling are default procedures.

Wherein the Mesenchymal Stem Cells (MSCs) are negative controls for the expression of the dry gene.

Flow cytometry:

After the cells are collected, the antibodies are incubated in an EP tube at 4 ℃ in a dark place for 30min, then a proper fluorescence acquisition channel is selected according to the antibody information, and fluorescence is detected on the machine. All antibodies were from BD Biosciences.

FIG. 7A shows the results of detecting the protein levels of the stem genes POU5F1 and NANOG in H1 cells (WT cells) and H1-derived DKO cells (DKO cells) by immunofluorescence, FIG. 7B shows the results of detecting the expression of the stem genes POU5F1, NANOG and SOX2 at the RNA level in H1 cells (WT cells) and H1-derived DKO cells (DKO cells) by RT-qPCR, FIG. 7C shows the results of detecting the expression of the stem genes SSEA-4 and Tra1-81 on the surfaces of H1 cells (WT cells) and H1-derived DKO cells (DKO cells) by flow cytometry, FIG. 7D shows the results of detecting the expression of the stem genes SSEA-4, TRA-1-60, TRA-1-81, OCT-4, SOX2 by immunofluorescence, FIG. 7E shows the results of detecting the stem genes TRA-1-60, OCT-1-4, SOX-1-81, and SOX-1-81 by immunofluorescence, and the results of detecting the levels of the stem genes SSSC-4, TRA-1-81 by flow cytometry.

The immunofluorescence assay results are shown in fig. 7A, both H1 cells WT and H1 cell-derived DKO cells expressed the dry genes POU5F1 and NANOG at the protein level, and the expression of dry genes POU5F1 and NANOG in H1 cell-derived DKO cells was not significantly different from that of WT cells. The results of RT-qPCR detection are shown in FIG. 7B, the H1 cell WT and H1 cell-derived DKO cells expressed the dry genes POU5F1, NANOG and SOX2 at the RNA level, and the expression of the dry genes POU5F1, NANOG and SOX2 in the H1 cell-derived DKO cells was not significantly different from that of the H1 cell WT cells. The results of flow cytometry are shown in FIG. 7C, wherein the results of flow cytometry detection are shown in FIG. 7C, the surfaces of H1 cells WT and H1 cell-derived DKO cells highly express the dry gene corresponding proteins SSEA-4 (WT 100% and DKO 99.98%) and Tra1-81 (WT 96.75% and DKO 99.13%), FIG. 7D is the results of flow cytometry detection for the expression of iPSC-WT and iPSC-DKO cell surface dry genes SSEA-4, TRA-1-60, TRA-1-81, OCT-4 and SOX2, the surfaces of iPSC-cells WT and iPSC cell-derived DKO cells highly express the dry gene corresponding proteins, FIG. 7E shows the results of detecting the protein levels of the dry genes TRA-1-60, TRA-1-81, OCT-4, SOX2 and NANOG in iPSC-WT cells by immunofluorescence, and FIG. 7F shows the results of detecting the protein levels of the dry genes TRA-1-60, TRA-1-81, OCT-4, SOX2 and NANOG in iPSC-DKO cells by immunofluorescence.

2.2 Differentiation ability of DKO cells

This example examined the differentiation ability of DKO cells. The specific detection method comprises subcutaneously injecting 100 μl of suspension containing 5×10 ⁵ DKO cells into immunodeficient mice (SCID Beige, vetong Liwa), taking out after teratoma volume is greater than 1.5cm ³, and performing paraffin section and hematoxylin eosin staining.

Staining results show that the H1 cell-derived DKO cells with B2M/CIITA double allele knockdown can form teratomas in vivo and differentiate into cells with three germ layers of inner, middle and outer germ layers, and the H1 cell-derived DKO cells have normal three germ layer differentiation capacity.

2.3 Immune function of DKO cells

The change in immune function of H1 cells and iPSC cells and derived DKO cells was detected by performing a killing experiment of T cells and NK cells using xCELLigence RTCA Instrument. The reagents used for the killing experiments are shown in table 3.

Specific assays were performed by resuspension of the same amount of WT and DKO cell lines using Essenal 8 medium containing human IL-2, and seeding in 96-well E-plates coated with matrigel, adding activated T cells (XC 11228, available from SAILYBIO) or NK cells (XC 11013, available from SAILYBIO). Before use, the T cells will be subjected to CD3, CD4 and CD8 streaming detection, and before use, the NK cells will be subjected to CD16 and CD56 streaming detection, so that the functions of the T cells and the NK cells used are ensured. RTCA test data were analyzed using xcelligent software to calculate kill rate and escape function.

TABLE 3 reagents for killing experiments

Name of the name	Goods number (Cat No.)	Specification of specification	Manufacturer' s
				PE anti-human CD4	980804	500 Μl/tube	Biolegend
APC anti-human CD8	980904	500 Μl/tube	Biolegend
				FITC anti-human CD3	300440	500 Times	Biolegend
APC anti-human CD16	301012	100 Times	Biolegend
				PE anti-human CD56(NCAM)	318306	100 Times	Biolegend
human IL-2	202-1L-050/CF	50μg	R&D
				Y-27632 2HCl	S1049	5mg	Selleck
Matrigel	354277	5ml	Gibco
				Essenal 8 ^TM culture medium	A1517001	500ml	Gibco
E-Plate VIEW 96 PET	300601030	6 Blocks/box	Agilent

The result of RTCA is shown in FIGS. 8A and 8B, in which WT cells escape NK cell killing due to HLA-I expression, but are killed by T cells. DKO cells can escape T cell killing while being more sensitive to NK cell killing. Wherein the upper three graphs of FIG. 8A are results of detection of NK cell killing rate against H1 cell WT and H1 cell-derived DKO cells by RTCA, i.e., detection of immune escape function against NK cells by H1 cell WT and H1 cell-derived DKO cells, and the second three graphs of FIG. 8A are results of detection of T cell killing rate against H1 cell WT and H1 cell-derived DKO cells by RTCA, i.e., detection of immune escape function against T cells by WT cells and H1 cell-derived DKO cells. FIG. 8B is a top three graphs showing the results of detecting the killing rate of NK cells against iPSC-WT and iPSC-DKO cells by RTCA, i.e., the results of detecting the immune escape function of iPSC-WT and iPSC-DKO cells against NK cells, and FIG. 8B is a second three graphs showing the results of detecting the killing rate of T cells against iPSC-WT and iPSC-DKO cells by RTCA, i.e., the results of detecting the immune escape function of iPSC-WT and iPSC-DKO cells against T cells.

EXAMPLE 3 construction of DKO+CD47 cell line and verification of immune function

3.1 Construction of H1 cell DKO+CD47 cell line

This example uses lentiviral vectors to overexpress CD47 (NM-198793) in the DKO cells obtained in example 1, the amino acid sequence of which is shown in SEQ ID NO. 5.

The nucleic acid sequence encoding the CD47 protein (SEQ ID NO: 6) was constructed in a lentiviral vector (pGC-EF 1 a) that was driven by EF1a and harbored a puromycin selection marker, the structure of the pGC-EF1a vector being shown in FIG. 9. The specific operation method is as follows:

The lentiviral vector was digested with BamHI/NheI, and a nucleic acid sequence encoding the CD47 protein (SEQ ID NO: 6) was ligated into the lentiviral vector, and after ligation was successful, the inserted sequence was verified for correctness by Sanger sequencing and virus packaging was performed. The lentiviral vector was transfected into DKO cells constructed in example 1, and the cells were changed to medium with puromycin after 24 hours and then to medium with puromycin after 48 hours for selection.

Flow cytometric detection (CD 47 antibodies were purchased from FACS: biolegend, cat# 323108) and qPCR detection were performed on the constructed stable transgenic cells DKO+CD47. The qPCR primer was CD47-F AGAAGGTGAAACGATCATCGAGC (SEQ ID NO: 27), CD47-R CTCATCCATACCACCGGATCT (SEQ ID NO: 28).

The results of the assay are shown in fig. 10A and 10B, and the expression level of CD47 was significantly higher in the constructed dko+cd47 cell line than in WT cells. And performing cell expansion and subsequent functional detection on the DKO+CD47 cell line after verification.

3.2DKO+CD47 verification of immune Functions

The use of RTCA to detect whether an overexpressed dko+cd47 cell line can escape NK cell killing while escaping T cell killing was successful is described in example 2.3.

RTCA assay as shown in fig. 11, NK cells can effectively kill DKO cells, while WT and dko+cd47 overexpressing cells can escape NK killing.

Example 4 construction of DKO+CD300LD cell line and detection of Stem and differentiation Capacity

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

Directly synthesizing nucleic acid sequence for encoding CD300LD protein (the amino acid sequence of CD300LD protein is shown as SEQ ID NO:3 or 4).

CD300LD amino acid sequence:

>NCBI Reference Sequence:NP_001108624.1

CMRF35-like molecule 5precursor[Homo sapiens]

MWLSPALLLLILPGYSIAAKITGPTTVNGSEQGSLTVQCAYGSGWETYLKWRCQGADWNYCNILVKTNGSEQEVKKNRVSIRDNQKNHVFTVTMENLKRDDADSYWCGTERPGIDLGVKVQVTINPGTQTAVSEWTTTTASLAFTAAATQKTSSPLTRSPLKSTHFLFLFLLELPLLLSMLGTVLWVNRPQRRS(SEQ ID NO:3)

>NCBI Reference Sequence:XP_047291002.1

CMRF35-like molecule 5isoform X1[Homo sapiens]

MNLRFPGYSIAAKITGPTTVNGSEQGSLTVQCAYGSGWETYLKWRCQGADWNYCNILVKTNG SEQEVKKNRVSIRDNQKNHVFTVTMENLKRDDADSYWCGTERPGIDLGVKVQVTINPGTQTAVSE WTTTTASLAFTAAATQKTSSPLTRSPLKSTHFLFLFLLELPLLLSMLGTVLWVNRPQRRS(SEQ ID NO:4)

The nucleic acid sequence encoding the CD300LD protein was constructed in a lentiviral vector (pGC-EF 1 a) with puromycin selection marker, as described in example 3.1, started by EF1a, and the structure of the pGC-EF1a vector is shown in FIG. 9. The specific procedure was as follows, the vector was digested with BamHI/NheI, and the nucleic acid sequence of the above synthesized CD300LD was ligated into lentiviral vector. After successful ligation, sanger sequencing was used to verify the correctness of the insert and virus packaging was performed. The lentiviral vector was transfected into iPSC-DKO cells constructed in example 1, and the cells were changed to medium with puromycin after 24 hours and then to medium with puromycin after 48 hours for selection.

And (3) carrying out over-expression detection on the constructed iPSC-DKO+CD300LD cell line. The qRT-PCR assay for mRNA overexpression levels, iPSC-WT cells as negative control, and primers as shown in Table 4. FACS detects the over-expression level of the protein, iPSC-WT cells are negative control cells, isotype are detection signal negative controls.

TABLE 4 qPCR detection primers for CD300LD

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

The detection result shows that the constructed iPSC-DKO+CD300LD cell line has high expression of CD300 LD.

4.2 Expression of the Dry Gene in the iPSC-DKO+CD300LD cell line

The expression of the stem gene in dko+cd300LD cell lines was examined by immunofluorescence and flow cytometry, confirming that the constructed cells had stem properties, see example 2.1 for specific detection methods. The result of the immunofluorescence detection of the dry gene is shown in FIG. 12, and the result of the flow detection of the dry gene is shown in FIG. 13. The immunofluorescence detection result shows that the iPSC-DKO+CD300LD cells of the constructed iPSC express dry genes OCT4, NANOG, SOX2, TRA-1-60 and TRA-1-81 at the protein level. Flow cytometry results show that the cell surface of iPSC-DKO+CD300LD highly expresses dry genes SSEA-4, TRA-1-60, tra1-81 and OCT4.

4.3 Trigerm layer differentiation Capacity of iPSC-DKO+CD300LD cell line

Tricodermic differentiation capacity assays were performed using dko+cd300LD cells. To generate mesogenic, endogenic and ectodermal cells, dissociated iPSC-dko+cd300ld single cells were resuspended using three germ layer media supplemented with Y27632, respectively, and appropriate amounts of cells were attached to matrigel coated well plates with cell climbing plates. After 24 hours, the medium is replaced by a preheated differentiation medium, and medium, inner and outer three germ layer cells can be obtained from day to day seven. The expression of the three germ layers marker protein is detected by immunofluorescence to detect that the iPSC-DKO+CD300LD cell line has the three germ layers differentiation capability. The results of the expression detection of the three germ layers marker proteins are shown in FIG. 14. The immunofluorescence test results show that the iPSC-DKO+CD300LD cells express ectodermal marker proteins, namely PAX6 and GAD1, mesodermal marker proteins, namely Brachyury and NCAM, and endodermal marker proteins, namely SOX17 and FOXA2 at the protein level. EXAMPLE 5 immune escape function of iPSC-DKO+CD300LD cell line

To verify the escape function of iPSC-dko+cd300LD cells on different immune cells, experiments were performed using NK cells, PBNK (T cell+nk cell mix), and macrophages (Macrophage). The escape function of iPSC-dko+cd300LD cells on different immune cells was detected by RTCA. For specific detection methods see example 2.3.

5.1IPSC-DKO+CD300LD cells against NK cell killing

NK cell killing assays were performed on the XCelligence platform (ACEA BioSciences). Various types of iPSC cells (iPSC-WT cells, iPSC-DKO cells, iPSC-DKO+CD300LD cells) were resuspended in 100. Mu.l of cell-specific medium and plated onto Matrigel (Sigma-Aldrich) coated 96-well E-plates (ACEA Biosciences). After the cell index value reached 1, NK cells were added at an E:T ratio of 1:1. Data were normalized and analyzed using RTCA software (ACEA). The killing rate test results are shown in fig. 15, and a of fig. 15 and B of fig. 15 show the results of NK cell killing experiments on iPSC-dko+cd300ld cell by RTCA, where B of fig. 15 is a multiple killing statistical plot, 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 killing of NK.

5.2IPSC-DKO+CD300LD cells against PBMC cell killing

NK activating factors are added into PBMC in advance, the NK cell proportion and the killing performance of T cells in the PBMC are improved, an RTCA experiment is carried out by taking activated mixed lymphocytes (PBMC) as effector cells, and the immune escape capacity of iPSC-DKO+CD300LD cells is comprehensively judged (method reference PMID: 33309274). The specific procedure was to conduct PBMC cell killing assays 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. Mu.l of cell-specific medium and plated onto Matrigel (Sigma-Aldrich) coated 96-well E-plates (ACEA Biosciences). After the cell index value reached 1, PBMC cells were added at an E:T ratio of 2:1. Data were normalized and analyzed using RTCA software (ACEA). The killing rate test results are shown in fig. 16, and a of fig. 16 and B of fig. 16 show the results of the PBMC killing experiment of iPSC-dko+cd300ld cells by RTCA test, where B of fig. 16 is a multiple killing statistical plot, n=4. The results show that iPSC-dko+cd300LD cells can significantly escape killing of PBMCs.

5.3IPSC-DKO+CD300LD cells against macrophage killing

MAC cell killing assays were performed on the XCelligence platform (ACEA BioSciences). Various types of iPSC cells (iPSC-WT cells, iPSC-DKO cells, iPSC-DKO+CD300LD cells) were resuspended in 100. Mu.l of cell-specific medium and plated onto Matrigel (Sigma-Aldrich) coated 96-well E-plates (ACEA Biosciences). After the cell index value reaches 1, MAC cells are added at an E:T ratio of 2:1. Data were normalized and analyzed using RTCA software (aces), and the killing rate test results are shown in fig. 17, with a of fig. 17 and B of fig. 17 showing the results of an iPSC-dko+cd300LD cell killing experiment by macrophages tested by RTCA, where B of fig. 17 is a graph of multiple killing statistics, n=4. The results show that iPSC-dko+cd300LD cells can significantly escape killing of PBMCs.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

Claims

1. A low immunogenicity pluripotent stem cell comprising:

reduced endogenous major histocompatibility class I antigen MHC-I function compared to the parental pluripotent stem cells;

reduced endogenous major histocompatibility class II antigen MHC-II function compared to the parental pluripotent stem cell, and

A reduced sensitivity to NK cell killing compared to a parental pluripotent stem cell, wherein the reduced sensitivity to NK cell killing is caused by increased expression of the CD300LD protein;

the MHC-I function is reduced by reducing the activity of the B2M protein, one or more alterations that reduce the activity of the endogenous B2M protein;

the MHC-II function is reduced by reducing the activity of the CIITA protein, one or more alterations that reduce the activity of the endogenous CIITA protein, and

One or more changes in the expression of an increased CD300LD protein in said low immunogenicity pluripotent stem cell, the amino acid sequence of said CD300LD protein being shown in SEQ ID No. 3.

2. The low immunogenicity pluripotent stem cell of claim 1, comprising:

3. The low immunogenicity pluripotent stem cell according to claim 1, wherein the B2M protein is human B2M protein having an amino acid sequence shown in SEQ ID No. 1.

4. The low immunogenicity pluripotent stem cell according to claim 1, wherein the CIITA protein is a human CIITA protein having an amino acid sequence as shown in SEQ ID No. 2.

5. A method of producing the low immunogenicity pluripotent stem cell of any one of claims 1 to 4, comprising decreasing the activity of a B2M protein in the pluripotent stem cell;

Decreasing the activity of CIITA protein in said pluripotent stem cells, and

Increasing expression of CD300LD protein in said pluripotent stem cells.

6. The method of claim 5, the method comprising:

Abrogating the activity of both alleles of the B2M gene in the pluripotent stem cell;

Eliminating the activity of both alleles of the CIITA gene in said pluripotent stem cells, and

Increasing expression of the CD300LD gene in said pluripotent stem cells.

7. The method of claim 5, wherein the activity of B2M protein in the pluripotent stem cell and/or the activity of CIITA protein in the pluripotent stem cell is reduced by a technique selected from the group consisting of:

Introducing a gene expression modification molecule, a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technique, a transcription activator-like effector nuclease (TALEN) technique, a Zinc Finger Nuclease (ZFN) technique, or a homologous recombination technique.

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

9. The method of claim 8, wherein the activity of the B2M protein in the pluripotent stem cell is reduced by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas 9 gene editing technique and/or the activity of the CIITA protein in the pluripotent stem cell is reduced by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas 9 gene editing technique.

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

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

12. The method of claim 11, wherein introducing at least one copy of the CD300LD gene under the control of a promoter into the pluripotent stem cell increases expression of the CD300LD protein by synthesizing a nucleic acid sequence encoding the CD300LD protein to construct into a lentiviral vector, and then passing through the lentiviral vector.

13. The method of claim 12, wherein the nucleic acid sequence encoding CD300LD protein is set forth as SEQ ID No. 25.