HLA class II-deficient cells, HLA class I-deficient cells, and their use, which have the ability to express HLA class II protein.

Abstract

To provide cells applicable for more effective and less expensive cell-based therapies that are not impeded by rejection.SOLUTION: The invention relates to isolated human cells having genetically engineered disruption in endogenous human leukocyte antigen (HLA) class II-related genes.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to HLA class II-deficient cells, HLA class I-deficient cells, and their use, all of which have the ability to express HLA class II proteins. [Background technology]

[0002] Human pluripotent stem cells have the potential to treat diseases affecting almost any organ system. However, there are significant limitations to the clinical use of human pluripotent stem cells and their derivatives, namely, recipient rejection of transplanted cells due to differences in major histocompatibility complexes.

[0003] The major histocompatibility complex (MHC) is a multi-component molecule found on the cell surface of all vertebrates, mediating interactions between leukocytes and other leukocytes or other cells. The MHC gene family is divided into three classes: Class I, Class II, and Class III. In humans, MHC is called human leukocyte antigen (HLA). HLA class II molecules (HLA-II) are transmembrane proteins found only on professional antigen-presenting cells (APCs), including macrophages, dendritic cells, and B cells. In addition, parenchymal organs can sometimes express HLA class II genes involved in immune rejection. HLA class I (HLA-I) proteins are expressed in all nucleated cells and consist of an HLA class I heavy chain (or α chain) and β2 microglobulin (B2M). HLA class I proteins present peptides to CD8+ cytotoxic T cells on the cell surface. To date, six HLA class I α chains have been identified, including three classical α chains (HLA-A, HLA-B, and HLA-C) and three non-classical α chains (HLA-E, HLA-F, and HLA-G). The peptide bond specificity on the peptide bond groove of HLA class I molecules is determined by the α chain. Cellular immunity is mediated when CD8+ T cells recognize peptides presented by HLA class I molecules.

[0004] Both HLA class II and class I molecules are heterodimers. Class I molecules consist of an α chain (or heavy chain) and β2 microglobuin (B2M), while class II molecules consist of two homologous subunits: an α subunit and a β subunit.

[0005] HLA class II (HLA-II) molecules or proteins present peptide antigens derived from extracellular proteins, including extracellular pathogen proteins, on the cell surface, while HLA class I proteins present peptides derived from intracellular proteins or pathogens. HLA class II proteins loaded on the cell surface interact with CD4+ helper T cells. This interaction triggers phagocytic cell recruitment, local inflammation, and / or humoral responses through the activation of B cells. To date, several HLA class II loci have been identified, including HLA-DM (HLA-DMA and HLA-DMB, encoding HLA-DMα and HLA-DMβ chains, respectively), HLA-DO (HLA-DOA and HLA-DOB, encoding HLA-DOα and HLA-DOβ chains, respectively), HLA-DP (HLA-DPA and HLA-DPB, encoding HLA-DPα and HLA-DPβ chains, respectively), HLA-DQ (HLA-DQA and HLA-DQB, encoding HLA-DQα and HLA-DQβ chains, respectively), and HLA-DR (HLA-DRA and HLA-DRB, encoding HLA-DRα and HLA-DRβ chains, respectively).

[0006] In the context of transplantation, HLA class I and / or class II proteins from allogeneic sources act as exogenous antigens. Recognition of non-self HLA class I and / or class II proteins is a significant obstacle in the use of pluripotent stem cells for transplantation or replacement therapy. [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, while individually tailored stem cell preparations or HLA-diversified stem cell banks may address current transplantation challenges, these require characterizing multiple cell lines, differentiating them into therapeutic cell products, and obtaining approval for human administration. This time-consuming, technically challenging, and expensive process is a major factor hindering stem cell-based therapies from progressing to clinical trials. Consequently, there is a need for more effective and less expensive cell-based therapies that are not hindered by rejection. [Means for solving the problem]

[0008] In a first embodiment, the present invention provides isolated cells in which a human leukocyte antigen (HLA) class II-related gene has been genetically engineered to be disrupted, wherein the cells are primate cells. In one embodiment, the HLA class II-related gene is a regulatory factor X-associated ankyrin-containing protein (RFXANK). The following are selected from the group consisting of HLA-DPA (α chain), HLA-DPB (β chain), HLA-DQA, HLA-DQB, HLA-DRA, HLA-DRB, HLA-DMA, HLA-DMB, HLA-DOA, and HLA-DOB. In another embodiment, the cells include genetically engineered disruption of at least two, at least three, or all four of the HLA class II related genes. In yet another embodiment, the HLA class II related gene is regulatory factor X-related ankyrin-containing protein (RFXANK). In yet another embodiment, the cells include genetically engineered disruption of all copies of the HLA class II related gene. In another embodiment, the cell further comprises one or more recombinant immunomodulatory genes, each having the ability to express immunomodulatory polypeptides in human cells. In yet another embodiment, the one or more immunomodulatory genes comprise a polynucleotide capable of encoding an HLA II protein. In yet another embodiment, the one or more immunomodulatory genes comprise a polynucleotide capable of encoding a single-strand fusion HLA class II protein. In yet another embodiment, the cell further comprises genetically engineered disruption of the β2-microglobulin (B2M) gene.

[0009] In another embodiment, the present invention provides an isolated cell comprising (a) a genetically engineered disruption of the β2 microglobulin (B2M) gene and (b) one or more polynucleotides capable of encoding an HLA class II protein or a single-strand fusion HLA class II protein; wherein the cell is a primate cell.

[0010] In one embodiment of any of these aspects, the cell includes the genetically engineered disruption of all copies of the B2M gene. In a further embodiment, the HLA II gene encodes an HLA protein selected from the group consisting of HLA-DMα, HLA-DMβ, HLA-DOα, HLA-DOβ, HLA-DPα, HLA-DPβ, HLA-DQα, HLA-DQβ, HLA-DRα, and HLA-DRβ chains.

[0011] In another embodiment, the single-stranded fusion HLA class II protein comprises at least a portion of the HLA class II gene α chain covalently bonded to at least a portion of the HLA class II gene β chain, where the HLA class II gene is selected from the group consisting of HLA-DP, HLA-DQ, HLA-DR, HLA-DM, and HLA-DO. In yet another embodiment, the single-stranded fusion HLA class II protein comprises a plurality of different single-stranded fusion HLA class II proteins. In yet another embodiment, the single-stranded fusion HLA class II protein comprises at least a portion of the HLA-DQα chain and at least a portion of the HLA-DQβ chain. In yet another embodiment, the single-stranded fusion HLA class II protein comprises the HLA-DQα chain allele HLA-DQA1 * At least a portion of 01 and the HLA-DQβ chain allele HLA-DQB1 * Includes at least a portion of 02.

[0012] In further embodiments of any aspect of the cells of the present invention, an HLA protein or a single-strand fusion HLA class II protein presents a first target peptide antigen on the cell surface. In one such embodiment, the first target peptide antigen is covalently bound to the single-strand fusion HLA class II protein.

[0013] In another embodiment, the cell further comprises a polynucleotide capable of encoding a single-strand fusion HLA class I protein. In one such embodiment, the single-strand fusion HLA class I protein comprises at least a portion of B2M covalently bound to at least a portion of an HLA class Iα chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In another such embodiment, the single-strand fusion HLA class I protein comprises at least a portion of B2M covalently bound to at least a portion of HLA-A. In a further embodiment, the single-strand fusion HLA class I protein comprises at least a portion of B2M covalently bound to at least a portion of HLA-A0201. In another embodiment, the cell further expresses a second target peptide antigen presented by the single-strand fusion HLA class I protein on its cell surface. For example, the second target peptide antigen may be covalently bound to the single-strand fusion HLA class I protein.

[0014] In another embodiment of any aspect of the cells of the present invention, the cells further comprise one or more recombinant genes capable of encoding suicide gene products. For example, the suicide gene product may comprise a protein selected from the group consisting of thymidine kinases and apoptosis signaling proteins.

[0015] Cells of any form may have a normal karyotype and may be non-transformed cells. The cells may be stem cells, such as hematopoietic stem cells, embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, hepatic stem cells, neural stem cells, pancreatic stem cells, or mesenchymal stem cells. The stem cells may be differentiated, such as dendritic cells, islet cells, hepatocytes, muscle cells, keratinocytes, nerve cells, hematopoietic cells, lymphocytes, erythrocytes, platelets, skeletal muscle cells, ophthalmic cells, mesenchymal cells, fibroblasts, lung cells, gastrointestinal (GI) duct cells, vascular cells, endocrine cells, adipocytes, or cardiomyocytes. The cells may be human cells.

[0016] In another embodiment, the present invention provides a vaccine comprising cells of any one embodiment or combination of embodiments of the cells of the present invention, wherein the vaccine comprises at least one target peptide antigen on the cell surface, and the vaccine can induce an immune response specific to the target peptide antigen in primates.

[0017] In a further embodiment, the present invention provides a method of transplantation in a patient in need, the method comprising the step of administering to the patient an effective amount of cells or a vaccine in any embodiment or combination of embodiments of the cells of the present invention. In one such embodiment, the patient may be immunocompetent. In another embodiment, the cells or vaccine may comprise differentiated cells. [Brief explanation of the drawing]

[0018] [Figure 1] The diagram shows the structures of two exemplary adeno-associated virus (AAV) gene targeting vectors, which are designed to insert either the TKNeo gene (AAV-RFXANK-ETKNpA) or the HyTK gene (AAV-RFXANK-HyTK), controlled by the EF1α promoter (EF), into exon 3 of the RFXANK gene (also shown below their vectors). Cells targeted by the TKNeo or HyTK vectors can be isolated by selecting vector-infected cells with G418 or hygromycin (Hygro), respectively. Subsequently, Cre recombinase expression and selection with ganciclovir (GCV) allow for the isolation of clones from which the TKNeo or HyTK gene has been removed, leaving behind two inactivated RFXANK alleles with stop codons, loxP sites, and polyadenylation sites (StopX3-loxP-pA) in all three reading frames. loxP is the recombination site for Cre recombinase. ITR is the reverse terminal repeat of the vector. Similar vectors can be designed targeting other genes. [Figure 2](A) is a schematic diagram of a targeting strategy for infecting human embryonic stem cells with the construct AAV-RFXANK-ETKNpA. (B) is a photograph of a stained gel showing polymerase chain reaction (PCR) products, which were obtained after infection of human embryonic stem cells with AAV-RFXANK-ETKNpA and PCR, in which forward primer homologous to the neomycin sequence of the selection cassette and reverse primer homologous to the RFXANK gene outside the target homologous arm were used, as indicated by the arrows above. [Modes for carrying out the invention]

[0019] All cited references are incorporated herein by reference in their entirety. Unless otherwise specified in this application, the techniques used are those described in several well-known references, such as "Molecular Cloning: A Laboratory Manual" (by Sambrook et al., 1989, Cold Spring Harbor Laboratory Press), "Gene Expression Technology" (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press, San Diego, California), "Guide to Protein Purification" (Methods in Enzymology, edited by MP Deutscher, 1990, Academic Press, Inc.); and "PCR Protocols: A Guide to Methods and Applications." Applications) (Innis et al., 1990, Academic Press, San Diego, California), Culture of Animal Cells: A Manual of Basic Technique, 2nd edition (RI Freshney, 1987, Liss, Inc., New York, New York), Gene Transfer and Expression Protocols Refer to either "Protocols)", pp. 109-128, edited by E.J. Murray, Humana Press Inc., Clifton, New Jersey, or the Ambion 1998 catalog (Ambion, Austin, Texas).

[0020] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "And" is used synonymously with "or" herein unless stated otherwise specifically.

[0021] All embodiments of any aspect of the present invention can be used in combination unless the context clearly dictates otherwise. In a first aspect, the present invention provides an isolated cell comprising a genetically engineered disruption in a human leukocyte antigen (HLA) class II-related gene, wherein the cell is a primate cell. As used herein, the HLA class II-related gene, in a broad sense, refers to a gene encoding a protein involved in HLA class II-mediated immune responses. Thus, HLA class II-related genes include genes encoding HLA class II molecules such as HLA-DM (SEQ ID NOs: 48, 50), HLA-DO (SEQ ID NOs: 52, 54), HLA-DP (SEQ ID NOs: 36, 38), HLA-DQ (SEQ ID NOs: 40, 42), and HLA-DR (SEQ ID NOs: 44, 46). Exemplary HLA class II gene / protein sequences can be referenced in publicly available databases based on GenBank or IMGT / HLA database numbers of NM_033554.3 (SEQ ID NOs: 36, 37) for HLA-DPA, HLA00514 (SEQ ID NOs: 38, 39) for HLA-DPB, HLA00601 (SEQ ID NOs: 40, 41) for HLA-DQA, HLA00622 (SEQ ID NOs: 42, 43) for HLA-DQB, NM_019111 (SEQ ID NOs: 44, 45) for HLA-DRA, HLA00664 (SEQ ID NOs: 46, 47) for HLA-DRB, NM_00612 (SEQ ID NOs: 48, 49) for HLA-DMA, NM_002118 (SEQ ID NOs: 50, 51) for HLA-DMB, NM_002119 (SEQ ID NOs: 52, 53) for HLA-DOA, and NM_002120 (SEQ ID NOs: 54, 55) for HLA-DOB.

[0022] In addition, HLA class II-related genes include, without limitation, genes encoding HLA class II regulatory proteins that regulate the expression of HLA class II molecules, including regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor V (RFX5), regulatory factor X-related protein (RFXAP), and class II transactivator (CIITA). For example, regulatory factor X-related ankyrin-containing protein (RFXANK), together with regulatory factor X-related protein and regulatory factor V, forms a complex that binds to the X-box motif of the HLA class II gene promoter and activates the transcription of the HLA class II gene. Sequences of exemplary HLA class II-related genes that regulate the expression of HLA class II molecules can be referenced in publicly available databases based on the following GenBank accession numbers: RFXANK: NM_134440.1 (SEQ ID NOs. 24, 25) and NM_003721.2 (SEQ ID NOs. 26, 27); RFX5: NM_000449.3 (SEQ ID NOs. 28, 29) and NM_001025603.1 (SEQ ID NOs. 30, 31); RFXAP: NM_000538.3 (SEQ ID NOs. 32, 33); and CIITA: NM_000246.3 (SEQ ID NOs. 34, 35). All sequences disclosed based on these GenBank accession numbers are incorporated herein by reference.

[0023] In certain embodiments, the present invention provides isolated primate cells, preferably human cells, that include genetically engineered disruption of at least one HLA class II-related gene as defined herein. In certain detailed embodiments, the cells include genetically engineered disruption of all copies of at least one HLA class II-related gene. In certain other embodiments, the cells include multiple genetically engineered disruptions of multiple HLA class II-related genes.

[0024] In certain embodiments, the HLA class II-related genes are selected from the group consisting of regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor V (RFX5), regulatory factor X-related protein (RFXAP), and class II transactivator (CIITA). In certain detailed embodiments, the cells involve at least one genetically engineered disruption of at least one, at least two, at least three, or all of the HLA class II-related genes selected from the group consisting of regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor V (RFX5), regulatory factor X-related protein (RFXAP), and class II transactivator (CIITA). Any combination of these four HLA class II-related genes is within the scope of the present invention as a gene disruption target for creating HLA class II-deficient cells.

[0025] In certain other embodiments, the cells contain regulatory factor X-related ankyrin-containing protein (RFXANK) (SEQ ID NOs. 24-27), regulatory factor V (RFX5) (SEQ ID NOs. 28-31), regulatory factor X-related protein (RFXAP) (SEQ ID NOs. 32, 33), class II transactivator (CIITA) (SEQ ID NOs. 34, 35), HLA-DPA (α chain) (SEQ ID NOs. 36, 37), HLA-DPB (β chain) (SEQ ID NOs. 38, 39), HL This involves the genetically engineered disruption of at least one HLA class II-related gene selected from the group consisting of A-DQA (sequences 40, 41), HLA-DQB (sequences 42, 43), HLA-DRA (sequences 44, 45), HLA-DRB (sequences 46-47), HLA-DMA (sequences 48, 49), HLA-DMB (sequences 50, 51), HLA-DOA (sequences 52, 53), and HLA-DOB (sequences 54, 55).

[0026] Genetic disruption includes, without limitation, deletion, insertion, substitution, and truncation of a target HLA class II-related gene that results in no expression of the target gene, or the expression of a truncated or mutant protein that is non-functional or has significantly reduced function compared to the wild-type protein. In certain embodiments, genetic disruption of an HLA class II-related gene leads to the expression of a truncated HLA class II-related protein. In certain detailed embodiments, the HLA class II-related gene is RFXANK (SEQ ID NOs. 24-27). In certain other detailed embodiments, the HLA-related genes are selected from the group consisting of HLA-DPA (α-chain) (SEQ ID NOs. 36, 37), HLA-DPB (β-chain) (SEQ ID NOs. 38, 39), HLA-DQA (SEQ ID NOs. 40, 41), HLA-DQB (SEQ ID NOs. 42, 43), HLA-DRA (SEQ ID NOs. 44, 45), HLA-DRB (SEQ ID NOs. 46-47), HLA-DMA (SEQ ID NOs. 48, 49), HLA-DMB (SEQ ID NOs. 50, 51), HLA-DOA (SEQ ID NOs. 52, 53), and HLA-DOB (SEQ ID NOs. 54, 55). In certain further embodiments, the cells involve genetically engineered disruption of all copies of the HLA class II-related genes.

[0027] In another embodiment, the present invention provides HLA class I and HLA class II deficient cells. In a particular embodiment, the present invention provides primate cells, preferably human cells, that include genetically engineered disruption of HLA class II-related genes and further include genetically engineered disruption of the β2 microglobulin (B2M) gene (SEQ ID NO: 1). In another detailed embodiment, the cells further include genetically engineered disruption of all copies of the B2M gene (SEQ ID NO: 1). In a particular embodiment, genetic disruption of the B2M (SEQ ID NO: 1) gene results in the absence or lack of expression of the B2M protein (SEQ ID NO: 2). In another detailed embodiment, the cells further include genetically engineered disruption of all copies of the B2M gene. In a particular embodiment, genetic disruption of the B2M gene results in the absence or lack of expression of the B2M protein. Since B2M is a common component of all HLA class I proteins, this disruption prevents the expression of all native HLA class I proteins on the cell surface. Thus, in this aspect of the present invention, HLA class I / class II deficient cells are provided. The B2M coding sequence is shown in Sequence ID 1 (GenBank accession number NM_004048), and the B2M protein sequence is shown in Sequence ID 2. Genes can have many single nucleotide polymorphisms (SNPs); as those skilled in the art will understand, the human cells and methods of the present invention are applicable to any such B2M gene and SNP.

[0028] Any suitable technique can be used to introduce genetically engineered disruption (in HLA class II-related genes, B2M genes, or any other suitable genes); exemplary gene disruption techniques are disclosed throughout this application and, based on the teachings herein and teachings known in the art, are within the scope of the art for those skilled in the art. Other exemplary techniques can be seen, for example, from U.S. Patent Application Publication No. 2008 / 0219956, published on September 11, 2008, which is incorporated herein by reference in whole. These techniques may optionally include the step of removing non-human DNA sequences from cells after disruption of HLA class II-related genes and optionally B2M genes.

[0029] One such technique utilizes an adeno-associated virus gene targeting vector, which may include removing the transgene used for targeting using techniques such as those described below, or by removing the transgene used for targeting using Cre-mediated loxP recombination techniques or other suitable recombination techniques. See Khan et al., 2011, Protocol, Vol. 6, pp. 482-501 (which is incorporated by reference in whole). Exemplary targeting vectors and exemplary vector diagrams are also disclosed herein. The use of various techniques for producing HLA class II cells, preferably human cells, according to the present invention is within the scope of the art for those skilled in the art, based on teachings known herein and in the art.

[0030] In certain embodiments, the cell genome of HLA class II-deficient cells may contain 100 or fewer, 50 or fewer, or 30 or fewer non-human DNA sequence nucleotides. In certain other embodiments, the cell genome may contain 6, 5, 4, 3, 2, 1, or 0 non-human DNA sequence nucleotides. An exemplary gene disruption strategy for the RFXANK gene is shown in Figure 1. Non-human DNA sequences can be removed by a second targeting round deleting the HyTK or TKNeo transgene in the initial vector, or by Cre-mediated loxP recombination.

[0031] In certain other embodiments, HLA class II or HLA class I / class II deficient cells further comprise one or more recombinant immunomodulatory genes. Suitable immunomodulatory genes include, without limitation, genes encoding viral proteins that inhibit antigen presentation, microRNA genes, genes encoding HLA class II proteins, or genes encoding single-chain (SC) fusion HLA class II proteins. The terms “single-chain fusion HLA class II protein,” “single-chain fusion HLA class II molecule,” or “single-chain fusion HLA class II antigen” refer to a fusion protein comprising at least a portion of an HLA class II α chain directly or covalently bound via a linker sequence to at least a portion of an HLA class II β chain, or a class II α or β chain bound to a peptide antigen, or bound class II α and β chains also bound to a peptide antigen. On the other hand, the terms “HLA class II protein,” “HLA class II molecule,” or “HLA class II antigen” refer to a non-covalently associated heterodimer of HLA class II α and HLA β chains expressed on the surface of wild-type cells. In embodiments where the gene encodes an HLA class II protein (as opposed to a single-strand fusion HLA class II protein), the gene is under the control of a promoter not involved in normal class II expression in the cell. In one embodiment, the gene is expressed as an episome; in another embodiment, the gene is integrated into the cell's genome. In either embodiment, the gene is operably bound (i.e., under transcriptional control) to a promoter not involved in normal class II expression in the cell. Any suitable promoter may be used, as can be determined by those skilled in the art based on the specifically intended design and use of the construct and cell.

[0032] In another embodiment, the present invention provides isolated cells comprising (a) a genetically engineered disruption of the β2 microglobulin (B2M) gene and (b) one or more polynucleotides capable of encoding an HLA II protein (α-chain or β-chain) or a single-strand fusion HLA class II protein; wherein the cells are primate cells. The cells according to this embodiment of the invention are HLA class I deficient cells.

[0033] HLA class II-deficient cells, HLA class I-deficient cells, or HLA class I / class II-deficient cells can be used as pluripotent donor cells. In certain detailed embodiments, HLA class II-deficient cells, HLA class I-deficient cells, or HLA class I / class II-deficient cells are hematopoietic cells or dendritic cells used for transplantation. In addition, parenchymal organ cells may sometimes express HLA class II genes involved in immune rejection. Therefore, in certain advantageous embodiments, the present invention provides HLA class II, HLA class I-deficient cells, or HLA class I / class II-deficient cells to be transplanted to treat diseases or injuries related to parenchymal organs.

[0034] In any particular detailed embodiment of the cells of the present invention, the HLAα and β chains are selected from the group consisting of the α and β chains of HLA-DM, HLA-DR, HLA-DP, HLA-DQ, and HLA-DO. The α and β chains may, but do not necessarily, originate from the same HLA class II gene. For example, an HLA class II protein or single-strand fusion HLA class II protein may contain at least a portion of the HLA-DQα chain and at least a portion of the HLA-DQβ chain (also referred to as a dimeric construct). HLA class II proteins and single-strand fusion HLA class II proteins containing mismatched HLA class II alleles are also intended. In a particular detailed embodiment, the HLA class II protein or single-strand fusion HLA class II protein may be the HLA-DQα chain allele HLA-DQA1 * At least a portion of 01 (sequence number 41) and the HLA-DQβ chain allele HLA-DQB1 *It may include at least a portion of 02 (SEQ ID NO: 43). In certain preferred embodiments, the leader sequence (or signal peptide) of the second portion of the fusion protein is removed in the fusion construct. For example, HLA-DQB1 at the C-terminus. * HLA-DQA1 at the N-terminus is covalently bonded to at least a portion of 02 (sequence number 43). * In single-stranded fusion HLA class II proteins containing at least a portion of 01 (SEQ ID NO: 41), HLA-DQA1 * The 01 (sequence number 41) leader sequence remains in the construct, HLA-DQB1 * The 02 (SEQ ID NO: 43) leader sequence is removed from the construct. In certain other embodiments, the cell further expresses at least two, at least three, or at least four or more different single-strand fusion HLA class II proteins. In certain detailed embodiments, the HLA class II protein or single-strand fusion HLA class II protein also comprises a first target peptide antigen occupying the peptide binding site of the HLA class II protein or single-strand fusion HLA class II protein, where this peptide antigen is covalently bound to the HLA class II protein or single-strand fusion HLA class II protein (also referred to as a trimer construct). In certain other embodiments, the covalently bound peptide antigen is cleaved at an incorporated protease cleavage site, and the cleaved peptide antigen may bind to the peptide binding site of the single-strand fusion HLA-II protein for presentation.

[0035] Therefore, HLA class II, HLA class I deficient cells, or HLA class I / class II deficient cells also include cells having genetically engineered disruption in all copies of the HLA class II gene (e.g., disruption in all copies of the HLA-DQ α and / or β chains), where one HLA class II allele is genetically engineered to express the desired HLA class II protein or single-strand fusion HLA class II protein instead of the wild-type HLA class II protein (i.e., gene-targeted knock-in in one HLA-II allele). Taking HLA-DQ as an example, in certain embodiments, HLA-DQ- / - Cells express HLA-DQ protein only in the context of a single-chain fusion HLA-DQ protein from the HLA-DQ locus. In certain advantageous embodiments, the expression of the single-chain fusion HLA class II protein is regulated by endogenous HLA-DQ regulatory sequences located at the HLA-DQ locus.

[0036] In related embodiments, HLA class II, HLA class I-deficient cells, or HLA class I / class II-deficient cells further include cells having a genetically engineered disruption in all copies of a particular HLA-II gene, where all alleles of that particular HLA-II gene are genetically engineered to express a single-chain fusion HLA class II protein instead of the wild-type HLA-II protein (i.e., gene targeting knock-in at all HLA-II alleles). HLA class II, HLA class I-deficient cells, or HLA class I / class II-deficient cells having such a genetic disruption express a particular HLA-II protein only in the context of a single-chain fusion HLA class II protein from the locus of all alleles of that particular HLA-II gene.

[0037] The present invention envisions HLA class II proteins and single-strand fusion HLA class II proteins comprising sequence variants and fragments of HLA class II α and β chains, wherein such HLA class II proteins or single-strand fusion constructs still possess normal HLA class II function, such as forming appropriate secondary structures of heterodimers on the cell surface, presenting peptides at peptide binding sites, interacting with CD4+ helper T cells, and eliciting HLA class II-mediated immune responses. In certain embodiments, the variants share at least 75%, 78%, 80%, 81%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence homology with naturally occurring HLA class II α or β chain sequences, wherein the variants possess normal HLA class II function. In certain other embodiments, the variants share at least 75%, 80%, 81%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence homology with the HLA class IIα or β chain sequences as shown in SEQ ID NOs. 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55.

[0038] Furthermore, HLA class II, HLA class I-deficient cells, or HLA class I / class II-deficient cells can be manipulated to recombinantly express single-strand fusion HLA class I protein on a B2M- / - genetic background. HLA class I-deficient cells or HLA class I / class II-deficient cells that recombinantly express single-strand fusion HLA class I protein still lack normal B2M function in that they do not express wild-type B2M protein (SEQ ID NO: 2), which can form heterodimers that non-covalently associate with any HLA class Iα chain on the cell surface.

[0039] The terms "single-strand fusion HLA class I protein," "single-strand fusion HLA class I molecule," or "single-strand fusion HLA class I antigen" refer to a fusion protein that contains at least a portion of a B2M protein directly or covalently bound to at least a portion of an HLA-Iα chain via a linker sequence. On the other hand, the terms "HLA class I protein," "HLA class I molecule," or "HLA class I antigen" refer to a heterodimer formed by the non-covalent association of B2M and HLAα chains expressed on the surface of wild-type cells.

[0040] As used herein, the terms “HLA class I α chain” or “HLA-I heavy chain” refer to the α chain of an HLA class I heterodimer. HLA class I heavy chains include, without limitation, HLA class I α chains HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. Representative DNA and protein sequences are provided for HLA-A (GenBank number K02883.1, SEQ ID NO. 3; UniProt number P01892, SEQ ID NO. 4), HLA-B (NM_005514, SEQ ID NO. 5; NP_005505; SEQ ID NO. 6), HLA-C (NM_002117, SEQ ID NO. 7; NP_002108, SEQ ID NO. 8), HLA-E (NM_005516, SEQ ID NO. 9; NP_005507, SEQ ID NO. 10), HLA-F (NM_018950, SEQ ID NO. 11; NP_061823, SEQ ID NO. 12), and HLA-G (NM_002127, SEQ ID NO. 13; NP_002118, SEQ ID NO. 14).

[0041] In addition, while the term "HLA class I or II protein / molecule" is known to refer to MHC class I or II protein / molecule in humans, the terms HLA and MHC are sometimes used synonymously throughout this application: for example, the terms HLA class I or HLA class II protein may also be used to refer to the primate equivalents of HLA class I or HLA class II protein, respectively, in primates. Those skilled in the art will be able to infer the meaning of these terms from the context.

[0042] Therefore, HLA class I-deficient cells or HLA class I / class II-deficient cells also include cells in which all copies of the B2M gene have been genetically engineered to express a single-strand fusion HLA class I protein instead of the wild-type B2M protein (i.e., gene-targeted knock-in in a single B2M allele). B2M- / - cells with such a genetic background express B2M only in the context of a single-strand fusion HLA class I protein from the B2M locus. In certain advantageous embodiments, the expression of the single-strand fusion HLA class I protein is regulated by an endogenous B2M regulatory sequence located at the B2M locus.

[0043] In related embodiments, HLA class I-deficient cells or HLA class I / class II-deficient cells further encompass cells having genetically engineered disruption in all copies of the B2M gene, where all B2M alleles are genetically engineered to express single-stranded fusion HLA class I protein instead of wild-type B2M protein (i.e., gene-targeted knock-in in all B2M alleles). HLA class I-deficient cells or HLA class I / class II-deficient cells with such genetic disruption express B2M only in the context of single-stranded fusion HLA class I protein from all allele loci of the B2M gene. In certain embodiments, cells are genetically engineered to express homogeneous single-stranded fusion HLA class I protein from all allele loci of the B2M gene; on the other hand, in other embodiments, cells are genetically engineered to express heterogeneous single-stranded fusion HLA class I protein from different loci of the B2M gene.

[0044] In certain embodiments, the single-stranded fusion HLA class I protein comprises at least a portion of B2M (SEQ ID NO: 2) and at least a portion of HLA-A (SEQ ID NO: 4), HLA-B (SEQ ID NO: 6), HLA-C (SEQ ID NO: 8), HLA-E (SEQ ID NO: 10), HLA-F (SEQ ID NO: 12), or HLA-G (SEQ ID NO: 14) (also referred to as a dimer construct). In certain preferred embodiments, the HLAα chain contained in the single-stranded fusion HLA class I protein does not contain the leader sequence (or signal sequence) of the HLA class Iα chain (leaderless HLAα chain). In certain other embodiments, the single-stranded fusion HLA class I protein comprises at least a portion of B2M (SEQ ID NO: 2) and at least a portion of HLA-C (SEQ ID NO: 8), HLA-E (SEQ ID NO: 10), or HLA-G (SEQ ID NO: 14). In certain further embodiments, the single-strand fusion HLA class I protein comprises at least a portion of B2M (SEQ ID NO: 2) and at least a portion of HLA-A (SEQ ID NO: 4), HLA-E (SEQ ID NO: 10), or HLA-G (SEQ ID NO: 14). In certain preferred embodiments, the single-strand fusion HLA class I protein includes a leader sequence (or signal peptide) covalently bonded to at least a portion of B2M and at least a portion of the HLAα chain to ensure proper folding of the single-strand fusion on the cell surface. The leader sequence may be the leader sequence of the B2M protein, the leader sequence of the HLAα chain protein, or the leader sequence of another secretary protein. In certain detailed embodiments, the single-strand fusion HLA class I protein comprises a B2M protein from which its leader sequence has been removed. In certain other detailed embodiments, the single-strand fusion HLA class I protein comprises an HLAα chain protein from which its leader sequence has been removed. Certain HLA class Iα chains are highly polymorphic. As those skilled in the art will understand, the human cells and methods of the present invention are applicable to any such HLAα chain and its polymorphism.

[0045] The present invention envisions single-strand fusion HLA class I proteins comprising sequence variants and fragments of the B2M and / or HLAα chain, wherein such single-strand fusion constructs still possess normal HLA class I function, such as forming appropriate secondary structures of heterodimers on the cell surface, presenting peptides in peptide binding grooves, and binding to inhibitory receptors on the surface of NK cells. In certain embodiments, the variants share at least 75%, 80%, 81%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence homology with naturally occurring HLA heavy chain and B2M sequences, wherein the variants possess normal HLA class I function. In certain other embodiments, the variants share at least 75%, 80%, 81%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence homology with the B2M or HLA heavy chain sequence as shown in SEQ ID NOs. 2, 4, 6, 8, 10, 12, or 14.

[0046] In certain detailed embodiments, the HLA-A variant shares at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence homology with SEQ ID NO: 4. In certain other detailed embodiments, the HLA-B variant shares at least 81%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence homology with SEQ ID NO: 6. In certain further embodiments, the HLA-C variant shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence homology with SEQ ID NO: 8. In further embodiments, the HLA-E variant shares at least 97%, 98%, 99%, or complete sequence homology with SEQ ID NO: 10. In certain detailed embodiments, the HLA-F variant shares at least 99%, or complete sequence homology with SEQ ID NO: 12. In certain other embodiments, the HLA-G variant shares at least 98%, 99%, or complete sequence homology with SEQ ID NO: 14.

[0047] In certain other embodiments, the single-stranded fusion HLA class I protein comprises a full-length B2M (SEQ ID NO: 2) (including its leader sequence) and an HLAα chain without a leader sequence (leaderless HLAα chain); on the other hand, in certain other embodiments, the single-stranded fusion HLA class I protein comprises a B2M (SEQ ID NO: 2) protein without a leader sequence. For example, B2M- / - cells expressing any combination of two, three, or more heterogeneous single-strand fusion HLA class I proteins, such as expressing an SC fusion containing HLA-A (SEQ ID NO: 4) (or leaderless HLA-A) and an SC fusion containing HLA-C (SEQ ID NO: 8) (or leaderless HLA-C), or an SC fusion containing HLA-A (SEQ ID NO: 4) (or leaderless HLA-A) and an SC fusion containing HLA-E (SEQ ID NO: 10) (or leaderless HLA-E), or an SC fusion containing HLA-B (SEQ ID NO: 6) (or leaderless HLA-B), an SC fusion containing HLA-E (SEQ ID NO: 10) (or leaderless HLA-E), and an SC fusion containing HLA-G (SEQ ID NO: 14) (or leaderless HLA-G), are all intended by and within the scope of the present invention.

[0048] Natural killer (NK) cells are part of the innate immune response. Several pathogens can downregulate HLA class I protein expression in infected cells. NK cells monitor infection by recognizing and inducing apoptosis in cells that do not express HLA class I protein. Inhibitory receptors on the surface of NK cells recognize the HLA class I α chain allele, thereby preventing NK-mediated apoptosis in uninfected normal cells. Therefore, in certain detailed embodiments, single-strand fusion HLA-I proteins inhibit NK-mediated death of cells that do not express endogenous HLA class I protein by binding to inhibitory receptors on NK cells. For example, HLA-E is a ligand for the CD94 / NKG2 receptor on NK cells that inhibits NK cell-mediated apoptosis. Therefore, in certain detailed embodiments, B2M- / - cells express a single-strand fusion HLA class I protein comprising at least a portion of B2M and at least a portion of HLA-E. In addition, HLA-G is typically expressed on the surface of placental cell trophoblasts that do not express HLA-A, B, or C, and protects these cells from NK cell-mediated lysis by interacting with the inhibitory ILT2 (LIR1) receptor on NK cells (Pazmany et al., 1996, Science, Vol. 274, pp. 792-795). Therefore, in certain other preferred embodiments, B2M- / - cells express a single-strand fusion HLA class I protein comprising at least a portion of B2M and at least a portion of HLA-G.

[0049] In certain detailed embodiments, the single-strand fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A0201. HLA-A0201 (SEQ ID NO: 4) is a common HLA class I allele found in a large proportion of the US population. Therefore, in certain advantageous embodiments, isolated cells express a single-strand fusion HLA class I protein comprising at least a portion of B2M and at least a portion of HLA-A0201 on a B2M- / - genetic background, where the isolated cells exhibit immunocompatibility with a large proportion of the US population. Other suitable common alleles that may be used include, without limitation, HLA-A0101, HLA-A0301, HLA-B0702, HLA-B0801, HLA-C0401, HLA-C0701, and HLA-C0702. In certain preferred embodiments, the HLA allele comprises at least a portion of HLA-A0201 (SEQ ID NO: 4), HLA-B0702 (SEQ ID NO: 6), or HLA-C0401 (SEQ ID NO: 8).

[0050] In certain further embodiments, the single-strand fusion HLA class I protein also includes a second target peptide antigen that occupies the peptide binding groove of the single-strand fusion HLA class I protein, where this peptide antigen is covalently bound to the single-strand fusion HLA class I protein (also referred to as a trimer construct). An example of a trimer construct is shown in Figure 2. The HLA-bGBE construct in Figure 2 includes B2M and HLA-E covalently bound to a peptide antigen (but not limited to, an HLA-G signaling peptide as described in the figure) (SEQ ID NO: 23) designed to occupy the peptide binding groove of the single-strand fusion HLA class I protein. In certain other embodiments, the covalently bound peptide antigen is cleaved at an incorporated protease cleavage site, and the cleaved peptide antigen can bind to the peptide binding groove of the single-strand fusion HLA-I protein for presentation.

[0051] In certain alternative embodiments, the peptide antigen occupying the peptide binding groove of a single-strand fusion HLA class I protein is produced by an intracellular antigen processing pathway, where the peptide antigen is produced by the proteasome, transported to the single-strand fusion HLA class I protein in the endoplasmic reticulum, and loaded thereon. In certain detailed embodiments, the peptide antigen includes a peptide of a tumor antigen. In certain other embodiments, the peptide antigen includes a peptide of a pathogen-derived protein, including, without limitation, bacteria, viruses, fungi, and parasites. In further embodiments, the peptide antigen includes a peptide of a tumor antigen. In certain detailed embodiments, HLA class I-deficient cells or HLA class I / class II-deficient cells express a single-strand fusion HLA class I protein covalently bound to a peptide that does not contain autoantigens or nascent antigens for the patient. Designing single-strand fusion HLA class I proteins and the peptide antigens presented thereon to modulate the immune response that may be induced in the recipient is within the capabilities of those skilled in the art.

[0052] Isolated HLA class II-deficient cells, HLA class I-deficient cells, or HLA class I / class II-deficient cells expressing an HLA class II protein or single-strand fusion HLA class II protein, or optionally a single-strand fusion HLA class I protein, which contains a specific peptide antigen covalently or non-covalently bound to a single-strand fusion protein, can be used, for example, to induce an immune response in a recipient. Accordingly, in a relevant embodiment, the present invention provides a vaccine comprising the isolated cells of the present invention, wherein the vaccine has the ability to induce an immune response specific to a target peptide antigen in a recipient. The immune response includes, without limitation, a cellular immune response and / or a humoral immune response. The vaccine may comprise stem cells or differentiated cells; in certain detailed embodiments, the cells are differentiated dendritic cells. In certain other embodiments, the cells further express cytokines. Any suitable cytokine can be used; in certain detailed embodiments, the cytokine is IL2 or IFN-γ. In certain preferred embodiments, the cells are human cells and the recipient is human. Accordingly, in a further embodiment, the present invention provides a kit comprising the vaccine of the present invention and optionally an immune adjuvant.

[0053] Single-strand fusion HLA class I proteins, HLA class II proteins, or single-strand fusion HLA class II proteins can be expressed from expression vectors that allow for transient or, more preferably, stable expression of the protein in cells. Exemplary suitable expression vectors are known in the art. One such example is a retroviral vector, which has the ability to be integrated into the cell genome and provides long-term stable expression of exogenous genes. In certain detailed embodiments, the viral vector is derived from human foam virus, a type of retrovirus. Other suitable viral vectors include, without limitation, vectors derived from retroviruses, adenoviruses, adeno-associated viruses, lentiviruses, herpes simplex viruses, vaccinia viruses, and poxviruses.

[0054] In certain preferred embodiments, a polynucleotide capable of encoding a single-strand fusion HLA class I or class II protein, or an HLA class II protein, is incorporated into the cell's chromosome, preferably the B2M or HLA class II locus, for stable expression. Therefore, in certain preferred embodiments, the B2M locus is disrupted by inserting a polynucleotide capable of encoding a single-strand fusion HLA class I protein into the B2M locus, thereby replacing the expression of the endogenous wild-type B2M protein. Similarly, in certain other preferred embodiments, a specific HLA-II locus is disrupted by inserting a polynucleotide capable of encoding an HLA class II protein or a single-strand fusion HLA class II protein into the HLA-II locus, thereby replacing the expression of the endogenous wild-type HLA-II protein. As a result of such gene targeting, the formation of wild-type HLA class I protein and specific HLA class II proteins becomes impossible, however, a selected HLA class II protein or a single-strand fusion HLA class I or class II protein can be expressed on the surface of cells that are originally HLA class II deficient. Other expression vectors are also being considered, and the selection of a suitable expression vector is within the capabilities of those skilled in the art.

[0055] "Isolated cells" may be any suitable cell type for a given purpose. For example, cells may be pluripotent stem cells or differentiated cells. "Stem cells" broadly encompass any cells that have further differentiation potential. "Pluripotent stem cells" refer to stem cells that have the potential to differentiate into any of the three germ layers, namely the endoderm, mesoderm, or ectoderm. On the other hand, "adult stem cells" are multipotent in that they can produce only a limited number of cell types. "Embryonic stem (ES) cells" refer to pluripotent stem cells derived from the inner cell mass of a blastocyst, which is an early embryo. "Induced pluripotent stem cells (iPS cells)" are pluripotent stem cells artificially obtained by artificially inducing and expressing specific genes from non-pluripotent cells, typically adult somatic cells.

[0056] In another embodiment, the present invention provides a method for transplantation in a patient in need, the method comprising the step of administering an effective amount of the cells of the present invention to the patient for transplantation. Since HLA class II-deficient cells and / or HLA class I-deficient cells do not express wild-type HLA class II protein (and optionally HLA class I protein as well) on their cell surface, the cells induce little to no immune response in the patient when administered to the patient. Therefore, transplantation using HLA class II-deficient cells and / or HLA class I-deficient cells reduces the need for immunosuppressive drug therapy. Thus, in certain preferred embodiments, the patient is immunocompetent. In certain other embodiments, the cells are syngeneic; on the other hand, in other embodiments, the cells are allogeneic.

[0057] In certain further embodiments, the cells of the present invention are pluripotent stem cells; on the other hand, in other embodiments, the cells of the present invention are differentiated cells. In certain preferred embodiments, the cells are human cells, and the patient is a human patient. In certain detailed embodiments, the transplantation method comprises the step of administering an effective amount of pluripotent stem cells or differentiated cells to a human. In certain preferred embodiments, the cells of the present invention further express one or more manipulated single-strand fusion HLA class II proteins, and optionally also express single-strand fusion HLA class I proteins. In certain other embodiments, the cells can avoid NK cell-mediated death and induce minimal or no immune response in the recipient after transplantation.

[0058] Transplantation, replacement therapy, or regenerative therapy refers to the treatment of a disease condition by administering cells or tissues to a patient to compensate for or replace a deficient cellular function in a target organ. In certain detailed embodiments, the need for transplantation arises as a result of physical or pathological injury to a tissue or organ. In certain other detailed embodiments, the need for transplantation arises as a result of one or more genetic abnormalities or mutations in a patient, and the transplantation of cells of the present invention compensates for or replaces the deficient cellular function in the patient without the need to correct the underlying genetic mutation in the patient through gene therapy. In certain further embodiments, transplantation includes, without limitation, hematopoietic stem cell transplantation, or transplantation of cells taken up by organs such as the liver, kidney, pancreas, lung, brain, muscle, heart, gastrointestinal tract, nervous system, skin, bone, bone marrow, fat, connective tissue, immune system, or blood vessels. In certain detailed embodiments, the target organ is a parenchymal organ.

[0059] In certain detailed embodiments, the cells administered to the recipient may or may not be taken up by the organs in need of such treatment. In certain embodiments, the cells of the present invention differentiate into a desired cell type before or after transplantation and provide the necessary cellular functions without being taken up by the tissue at the transplantation site. For example, in a certain embodiment for treating diabetes, the cells of the present invention are transplanted into a diabetic patient as either pluripotent stem cells or differentiated pancreatic β-islet cells. The transplanted cells do not need to reconstruct a functional pancreas: they simply need to secrete insulin in response to glucose levels. In certain detailed embodiments, the cells are transplanted to an ectopic site and are not fully taken up by the pancreas. Transplantation of pluripotent cells of the present invention, differentiated cells of the present invention, or tissues differentiated and grown ex vivo from cells of the present invention are all intended by the present invention. In certain preferred embodiments, the cells are human cells, and the patient is a human patient. In certain other preferred embodiments, the cells of the present invention express one or more single-strand fusion HLA class II proteins, and optionally also express single-strand fusion HLA class I proteins.

[0060] In a further embodiment, the present invention provides a method for treating a disease condition in a patient in need, the method comprising the steps of administering an effective amount of the cells of the present invention to the patient, thereby treating the disease condition, wherein the disease condition is diabetes, autoimmune disease, cancer, infection, anemia, cytopenia, myocardial infarction, heart failure, skeletal or joint disease, osteogenesis imperfecta, or burns. In certain detailed embodiments, the disease condition is caused by pathological or physical injury to a tissue or organ. In certain embodiments, the cells of the present invention are stem cells; on the other hand, in other embodiments, the cells of the present invention are differentiated cells. In certain preferred embodiments, the cells are human cells, and the patient is a human patient. In certain detailed embodiments, the human cells are differentiated cells. Transplantation of tissue grown ex vivo from the cells of the present invention is also contemplated by the present invention. In certain preferred embodiments, the cells of the present invention further express one or more single-strand fusion HLA class II proteins, and optionally also express one or more single-strand fusion HLA class I proteins. In certain embodiments, the cells are syngeneic; on the other hand, in other embodiments, the cells are allogeneic.

[0061] In certain detailed embodiments, the cells are differentiated cells, including, but not limited to, dendritic cells, lymphocytes, erythrocytes, platelets, hematopoietic cells, islet cells, hepatocytes, muscle cells, keratinocytes, cardiomyocytes, nerve cells, skeletal muscle cells, ophthalmic cells, mesenchymal cells, fibroblasts, lung cells, GI duct cells, vascular cells, endocrine cells, and adipocytes. In certain other detailed embodiments, the present invention provides a method for treating disease conditions in parenchymal organs. In certain embodiments, the cells of the present invention used to treat disease conditions express one or more single-strand fusion HLA class I proteins, and optionally also express one or more single-strand fusion HLA class I proteins.

[0062] To “treat” a patient with a disease or disability means to achieve one or more of the following: (a) reducing the severity of the disease; (b) halting the progression of the disease or disability; (c) preventing the worsening of the disease or disability; (d) suppressing or preventing recurrence of the disease or disability in a patient who has previously had the disease or disability; (e) causing regression of the disease or disability; (f) improving or eliminating the symptoms of the disease or disability; and (f) improving survival. In a particular preferred embodiment, the disease or disability is a disease or disability that can be treated by tissue or cell transplantation.

[0063] The effective dose of the isolated cells of this invention for transplantation or treatment of a disease depends on numerous factors, including the type of tissue, the severity of the disease, the transplant response, the reason for transplantation, and the patient's age and overall health. The effective dose can be determined by a skilled researcher or clinician through routine clinical practice. Because the immunogenicity of the transplanted cells is reduced, a relatively large amount of cells may be tolerated by the patient to achieve the desired therapeutic effect. Alternatively, cells can be transplanted repeatedly at intervals until the desired therapeutic effect is achieved.

[0064] The administration route of the cells of the present invention is not limited to any particular method. Exemplary delivery routes include, without limitation, intravenous, intramuscular, subdermal, intraperitoneal, transdermal, intradermal, and subcutaneous routes. The cells of the present invention may also be administered locally by injection. For example, the cells can be injected into injured joints, broken bones, infarcted areas, ischemic areas, or areas surrounding them.

[0065] In certain detailed embodiments, cells are administered using a delivery device, including a syringe, without limitation. For example, cells can be suspended in a solution or pharmaceutical composition contained in such a delivery device. The “solution” or “pharmaceutical composition” comprises a physiologically compatible buffer and, optionally, a pharmaceutically acceptable carrier or diluent that preserves the viability of the cells of the present invention. The use of such carriers and diluents is well known in the art. Examples of solutions include, but are not limited to, physiologically compatible buffers such as Hanks’ solution, Ringer’s solution, or buffered saline. Cells can be stored in the solution or pharmaceutical composition for short-term storage without loss of viability. In certain detailed embodiments, cells are frozen according to cryopreservation methods well known in the art for long-term storage without loss of viability.

[0066] The aqueous injection suspension may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran, but it is still fluid in that it can be easily delivered by syringe injection. The solution is preferably sterile, stable under manufacturing and storage conditions, and free from microbial contamination by the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. The cells contained in the solution may be stem cells or differentiated cells as described herein, in a pharmaceutically acceptable carrier or diluent and, optionally, in other components noted above.

[0067] The cells may be administered systemically (e.g., intravenously) or locally (e.g., directly to abnormal myocardium under the guidance of echocardiography, or directly to accessible damaged tissue or organ during open surgery). For injection, the cells may be in an injectable suspension formulation or in a liquid form in an injectable biocompatible medium that becomes semi-solid at the site of the damaged tissue. Syringes, controllable endoscopic delivery devices, or other similar devices may be used, provided that the needle lumen has a diameter sufficient to avoid physical damage to the cells during delivery (e.g., at least 30 gauge or more).

[0068] In certain other embodiments, cells may be implanted using a solid support, such as a planar or three-dimensional matrix. The matrix or planar is surgically implanted at a suitable site in the patient. For example, a patient requiring a pancreatic graft may have differentiated cells on a solid support surgically implanted in pancreatic tissue. Exemplary solid supports include, but are not limited to, patches, gel matrices (such as GELFOAM® from Pharmacia-Upjohn), polyvinyl alcohol sponge (PVA)-collagen gel implants (such as IVALON, Unipoint Industries, High Point, North Carolina), and other similar or equivalent devices. Various other encapsulation techniques can be used with the cells of the present invention (e.g., International Publication No. 91 / 10470; International Publication No. 91 / 10425; U.S. Patent No. 5,837,234; U.S. Patent No. 5,011,472; U.S. Patent No. 4,892,538).

[0069] The cells of the present invention can differentiate into various cell types across three lineages, including, without limitation, hematopoietic cells, mesenchymal cells, pancreatic endoderm cells, cardiac cells, and keratinocytes. In certain embodiments, differentiated cells further express HLA class II protein or single-strand fusion HLA class II protein, and optionally also express single-strand fusion HLA class I protein. Generally, each cell type can be analyzed for HLA class II, and optionally class I protein expression, reactivity with human T cells and NK cells, appropriate differentiation markers, and xenotransplantation in immunodeficient mice to investigate its in vivo developmental potential. The following is a brief consideration of each differentiated cell type.

[0070] In certain embodiments, the cells of the present invention can differentiate into hematopoietic cells for the treatment of various hematopoietic disorders currently treated by bone marrow transplantation. Patients receiving blood transfusions may become refractory to platelet transfusions due to HLA incompatibility. Patients with anemia or cytopenia may be treated by delivering red blood cells, platelets, or neutrophils derived from the cells of the present invention to treat bleeding or infection.

[0071] Furthermore, the stem cell-derived dendritic cells of the present invention are antigen-presenting cells that can be used as cellular vaccines when appropriately manipulated. In certain embodiments, cells of the present invention, manipulated to express HLA class II protein or single-strand fusion HLA class II protein, and optionally also express single-strand fusion HLA class I protein and unique peptide antigens, are used for vaccination against specific pathogens or tumor antigens. In certain other embodiments, differentiated HLA class II-deficient, HLA class I-deficient, or HLA class I / class II-deficient cytotoxic lymphocytes with limited HLA reactivity to specific antigens are used for the elimination of infected or tumor cells.

[0072] To obtain hematopoietic cells, pluripotent cells were first made capable of forming embryoid bodies, and then non-adherent cells were cultured in the presence of hematopoietic cytokines to grow into specific cell lineages. Differentiation of hematopoietic cells from the cells of the present invention expressing HLA class II protein or single-strand fusion HLA class II protein, and optionally single-strand fusion HLA class I protein, can be analyzed by flow cytometry and colony assays. Various cell populations are selected based on their surface markers, and their HLA gene expression and reactivity with human NK cells and T cells are monitored by measurements using Elispot, mixed lymphocyte assay, and cytotoxicity assays. The effectiveness of single-strand fusion HLA constructs in suppressing NK cell-mediated death can be investigated at various differentiation and transplantation stages. See Bix et al., 1991, Nature, Vol. 349, pp. 329-331. Hematopoietic stem cells can also be assayed using xenograft models, such as immunodeficient mice (SCID regrowth cells, i.e., SRCs).

[0073] The cells of the present invention can differentiate into hematopoietic cells either before or after administration to a patient. In certain preferred embodiments, the cells are human cells, and the patient is human. In vitro hematopoietic differentiation can be carried out according to established protocols. See, for example, Slukvin et al., 2006, Journal of Immunology (J Immunol), Vol. 176, pp. 2924-32, and Chang et al., 2006, Blood, Vol. 108, pp. 1515-23.

[0074] In certain other embodiments, the cells of the present invention are mesenchymal stem cells. They can differentiate into stem cells. In certain embodiments, the cells of the present invention express one or more HLA class II proteins or single-strand fusion HLA class II proteins, and optionally also express one or more single-strand fusion HLA class I proteins. MSCs have the potential to form several differentiated cell types, including bone marrow stromal cells, adipocytes, osteoblasts, and chondrocytes. Therefore, inducing pluripotent stem cells to form MSCs (iMSCs) is useful in the treatment of skeletal and joint diseases. iMSCs can further differentiate into osteoblasts and form bone in vivo. Deyle et al., 2012, Molecular Therapy (Mol Ther.), Vol. 20(1), pp. 204-2013. The cellular responses of T cells and NK cells to ESCs, iMSCs, and their more terminally differentiated derivatives such as osteoblasts can be investigated.

[0075] In certain detailed embodiments, mesenchymal stem cells have the ability to differentiate into non-limiting exemplary cell types such as bone marrow stromal cells, adipocytes, osteoblasts, osteocytes, and chondrocytes. The cells of the present invention differentiate into mesenchymal stem cells either before or after administration to a patient. In certain preferred embodiments, the cells are human cells, and the patient is human. In vitro mesenchymal differentiation can be carried out according to established protocols. See, for example, Deyle et al., cited above.

[0076] In further detailed embodiments, the cells of the present invention may differentiate into insulin-producing pancreatic islet cells. In certain embodiments, the cells of the present invention express one or more HLA class II proteins or single-strand fusion HLA class II proteins, and optionally also express one or more single-strand fusion HLA class I proteins. The cells of the present invention can be used to treat insulin-dependent diabetes mellitus. Advantageously, the transplanted cells do not need to reconstitute a functional pancreas: they simply need to secrete insulin in response to glucose levels. Thus, this treatment can be effective at various cell doses, with cells that are not fully differentiated into adult cell types, and even when the cells are transplanted to ectopic sites. Certain autoantigens, such as GAD65 or those derived from insulin, can cause autoimmune destruction of β-cells in diabetes (Di Lorenzo et al., 2007, Clinical and Experimental Immunology (Clin Exp Immunol), Vol. 148, pp. 1-16). Therefore, HLA class II-deficient, HLA class I-deficient, or HLA class I / class II-deficient cells expressing single-strand fusion HLA proteins that present specific peptide antigens offer further advantages in that they do not present these autoantigens and can prevent relapse of diabetes by avoiding autoimmune rejection after transplantation.

[0077] The cells of the present invention can differentiate into pancreatic cells as described above, by exposing the cells to various cytokines and drugs to promote the sequential formation of mesoendoderm, final endoderm, and pancreatic progenitor cells (Kroon et al., 2008, Nature Biotechnol, Vol. 26, pp. 443-452). These cells can be further cultured in grafts in immunodeficient mice. Wild-type cells and cells of the present invention expressing or not expressing one or more single-strand fusion HLA class II proteins or one or more single-strand fusion HLA class II proteins and optionally one or more single-strand HLA class I proteins can be analyzed for their reactivity with T cells and NK cells at various developmental stages.

[0078] The cells of the present invention differentiate into pancreatic islet cells either before or after administration to the patient. In certain preferred embodiments, the cells are human cells, and the patient is human. In vitro hematopoietic differentiation can be performed according to established protocols. See, for example, Kroon et al., 2008, Nature Biotechnology, Vol. 26, pp. 443-452.

[0079] In certain other detailed embodiments, the cells of the present invention can differentiate into cardiomyocytes. In certain embodiments, the cells of the present invention further express one or more HLA class II proteins or single-strand fusion HLA class II proteins, and optionally also express one or more single-strand fusion HLA class I proteins. Common clinical problems of myocardial infarction and congestive heart failure can be treated by transplanting cardiomyocytes derived from healthy stem cells and allowing them to engraft to reconstruct a functional cardiomyocyte layer. Cardiomyocytes derived from the cells of the present invention make it possible to carry out these treatments with pre-packaged cells, avoiding the immunosuppression required for current allogeneic heart transplantation. Physiologically relevant tests, such as electrical conductivity tests and contraction tests, can be performed on cardiomyocytes derived from the cells of the present invention. HLA class II-deficient, HLA class I-deficient, or HLA class I / class II-deficient stem cells or differentiated cardiomyocytes expressing or not expressing single-strand fusion HLA class I proteins can be tested to determine their immunological reactivity when cardiomyocyte genes are expressed and to establish which HLA modifications minimize such immune responses.

[0080] The cells of the present invention can differentiate into cardiomyocytes either before or after administration to a patient. In certain preferred embodiments, the cells are human cells, and the patient is human. In certain embodiments, the cells of the present invention differentiate into cardiomyocytes for the treatment of diseases including myocardial infarction and congestive heart failure, without limitation. In vitro cardiomyocyte differentiation can be carried out according to established protocols. See, for example, Laflamme et al., 2007, Nature Biotechnology, Vol. 25, pp. 1015-1024.

[0081] In further detailed embodiments, the cells of the present invention can differentiate into keratinocytes. In certain embodiments, the cells of the present invention used for differentiation into keratinocytes express one or more single-strand fusion HLA class II proteins, and optionally also express one or more HLA class II proteins or single-strand fusion HLA class I proteins. Severe burns and genetic skin conditions require treatment with skin grafts, which are currently performed using various cell sources such as porcine skin grafts and cultured autologous human keratinocytes. Keratinocytes derived from the cells of the present invention can provide significant clinical advances because burns can be treated with emergency cells in a pre-packaged state, and genetic diseases such as epidermolysis bullosa can be treated with otherwise normal HLA class II-deficient, HLA class I-deficient, or HLA class I / class II-deficient cells without the need to correct the causative gene mutation. In many cases, the cells only need to engraft for a sufficient time for adjacent host cells to regrow in the affected area. The cells of the present invention may be embedded in a polyvinyl alcohol sponge (PVA)-collagen gel implant for transplantation into a recipient, in order to differentiate them in vivo. The cells of the present invention can differentiate into keratinocytes either before or after transplantation. In certain preferred embodiments, the cells are human cells, and the patient is human.

[0082] In yet another embodiment, the present invention provides the use of the cells of the present invention for preparing a drug for transplantation. In a related embodiment, the present invention provides the use of the cells of the present invention for preparing a drug for treating a disease condition.

[0083] Furthermore, the cells of the present invention can serve as a research tool providing a system for studying the function of immunomodulatory proteins in a genetic background of HLA class II deficiency, HLA class I deficiency, or HLA class I / class II deficiency. In certain embodiments, the cells of the present invention further express one or more HLA class II proteins or single-strand fusion HLA class II proteins, and optionally also express one or more single-strand fusion HLA class I proteins. Accordingly, in relevant embodiments, the present invention provides a method for determining the function of immunomodulatory proteins, the method comprising the steps of introducing one or more immunomodulatory genes into the cells of the present invention and assaying the activity of the immunomodulatory genes. In certain preferred embodiments, the cells are human cells. For example, the cells of the present invention can be used to study the function of immunomodulatory genes or immune responses in the absence of undesirable HLA class II antigens or HLA class I / class II antigens. In a further relevant embodiment, the present invention provides a method for identifying compounds or molecules that modify the function of immunomodulatory proteins, the method comprising the steps of contacting HLA class II-deficient cells, HLA class I-deficient cells, or HLA class I / class II-deficient cells containing one or more immunomodulatory genes with the compound or molecule of interest, and assaying the activity of the immunomodulatory genes. In certain preferred embodiments, the cells are human cells.

[0084] In a related embodiment, the present invention provides a kit comprising HLA class II-deficient, HLA class I-deficient, or HLA class I / class II-deficient cells of the present invention and an implant, wherein the implant comprises the cells of the present invention.

[0085] In yet another embodiment, the present invention provides in vivo research tools in mammals, particularly non-human primates, to which cells of the present invention are administered, for studying the function of immunomodulatory genes in a genetic background of HLA class II deficiency, HLA class I deficiency, or HLA class I / class II deficiency, or for identifying compounds that modify the function of immunomodulatory genes in administered cells. In certain embodiments, cells of the present invention further express one or more HLA class II proteins or single-strand fusion HLA class II proteins, and optionally also express one or more single-strand fusion HLA class I proteins.

[0086] Mice, particularly immunodeficient mice, are used as model systems for in vivo studies of human cells. Human stem cells may behave differently in mice. In addition, the mouse immune system and the human immune system differ in HLA class II genes, NK cell receptors, and non-classical MHC class I genes (e.g., HLA-E, F, and G). Therefore, a Macaca nemestrina (Mn, pig-tailed macaque) model can be developed for cell studies of the present invention. The Macaca mulatta genome has been sequenced and has a high degree of homology to the Nemestrina genome. Furthermore, the composition of the macaque MHC locus, including non-classical genes, is similar to human HLA. Homologous forms of the human HLA-E and HLA-G genes have been identified in macaques. The macaque MHC locus also contains homologs of many human NK cell receptors. Human and Mn HLA class II deficiency, HLA class I deficiency, or HLA class I / class II deficiency ESCs can be used for transplantation in macaques.

[0087] MHC class II deficiency can be developed in MHC class I / class II deficient macaque ESCs using the same AAV-mediated gene targeting strategies described for human cells. Single-strand fusion HLA class II proteins and, optionally, Mn versions of single-strand fusion HLA class I proteins are expressed in HLA class II or HLA class II / class I deficient macaque ESCs using viral vectors similar to those described above.

[0088] Cells can be grown in vitro and labeled with a GFP-expressing vector for subsequent identification of transplanted cells. The cells may also be embedded in a polyvinyl alcohol sponge (PVA)-collagen gel implant and subcutaneously implanted in a macaque. The implant can be retrieved, sectioned, and stained to determine the cell types present. Specific antibodies can be used to identify the differentiated cell types formed by the transplanted cells.

[0089] Any embodiment described above applies to any aspect of the present invention unless otherwise specifically indicated in the context. All embodiments within the range of various embodiments and between them can be combined unless otherwise specifically indicated in the context.

[0090] (Examples) Example 1: Construction of human embryonic stem cells with a knockout mutation in the RFXANK gene. Figure 1 shows the structures of two exemplary adeno-associated virus (AAV) gene targeting vectors, designed to insert either the TKNeo gene (AAV-RFXANK-ETKNpA) or the HyTK gene (AAV-RFXANK-HyTK), controlled by the EF1α promoter (EF), into exon 3 of the RFXANK gene (also shown below their vectors). By selecting vector-infected cells with G418 or hygromycin (Hygro), cells targeted by the TKNeo or HyTK vectors, respectively, can be isolated. Subsequently, by expression of Cre recombinase and selection with ganciclovir (GCV), clones from which the TKNeo or HyTK gene has been removed can be isolated, leaving behind two inactivated RFXANK alleles with stop codons, loxP sites, and polyadenylation sites (StopX3-loxP-pA) in all three reading frames. loxP is the recombination site for Cre recombinase. ITR is the reverse terminal repeat of the vector. Similar vectors can be designed targeting other genes.

[0091] A knockout mutation was created in the first allele of the RFXANK gene using the AAV-RFXANK-ETKNpA vector (SEQ ID NO: 56). Human embryonic stem cells were infected with AAV-RFXANK-ETKNpA and screened by PCR for targeting. In this PCR, as indicated by the arrows above, a forward primer homologous to the neomycin sequence of the selection cassette and a reverse primer homologous to the RFXANK gene outside the target homologous arm were used. As shown in Figure 2, five positive clones of the correct size were shown above out of the 40 clones screened, resulting in a targeting frequency of 12.5%. (Note) As a preferred embodiment, the technical concept that can be understood from the above embodiment is described below. [Item 1] Isolated cells that contain genetically engineered disruption of human leukocyte antigen (HLA) class II-related genes, and which are primate cells. [Item 2] The aforementioned HLA class II-related genes include regulatory factor X-related ankyrin-containing protein (RFXANK) (SEQ ID NOs. 24-27), regulatory factor V (RFX5) (SEQ ID NOs. 28-31), regulatory factor X-related protein (RFXAP) (SEQ ID NOs. 32-33), class II transactivator (CIITA) (SEQ ID NOs. 34-35), HLA-DPA (α chain) (SEQ ID NOs. 36-37), HLA-DPB (β chain) ( Cells as described in item 1, selected from the group consisting of HLA-DQA (sequence numbers 38-39), HLA-DQB (sequence numbers 42-43), HLA-DRA (sequence numbers 44-45), HLA-DRB (sequence numbers 46-47), HLA-DMA (sequence numbers 48-49), HLA-DMB (sequence numbers 50-51), HLA-DOA (sequence numbers 52-53), and HLA-DOB (sequence numbers 54-55). [Item 3] Cells as described in item 2, comprising genetically engineered disruption of at least two, at least three, or all four HLA class II-related genes. [Item 4] The cells described in any one of items 1 to 3, wherein the HLA class II-related gene is regulatory factor X-related ankyrin-containing protein (RFXANK) (SEQ ID NOs. 24-27). [Item 5] A cell as described in any one of items 1 to 4, wherein all copies of the aforementioned HLA class II-related gene have been genetically modified and destroyed. [Item 6] A cell according to any one of items 1 to 5, further comprising one or more recombinant immunomodulatory genes, each having the ability to express immunomodulatory polypeptides in human cells. [Item 7] A cell as described in any one of items 1 to 6, wherein one or more immunomodulatory genes contain a single-strand fusion HLA class II protein or a polynucleotide capable of encoding an HLA class II protein. [Item 8] Cells described in any one of items 1 to 7, further comprising genetically engineered disruption of the β2 microglobulin (B2M) gene (Sequence ID 1). [Item 9] (a) Genetic disruption of the β2 microglobulin (B2M) gene (Sequence ID 1) and; (b) a single-strand fusion HLA class II protein or one or more polynucleotides capable of encoding an HLA class II protein; Including isolated cells, A cell that is a primate cell. [Item 10] Cells described in any one of items 1-9, including genetically engineered destruction of all copies of the B2M gene. [Item 11] A cell according to any one of items 7 to 10, wherein one or more immunomodulatory genes contain one or more single-stranded fusion HLA class II proteins, and the one or more single-stranded fusion HLA class II proteins contain at least a portion of the HLA class II gene α chain covalently bonded to at least a portion of the HLA class II gene β chain, and the HLA class II gene is selected from the group consisting of HLA-DP (SEQ ID NOs. 36-39), HLA-DQ (SEQ ID NOs. 40-43), HLA-DR (SEQ ID NOs. 44-47), HLA-DM (SEQ ID NOs. 48-51), and HLA-DO (SEQ ID NOs. 52-55). [Item 12] The cells described in item 11, wherein the single-stranded fusion HLA class II protein comprises multiple different single-stranded fusion HLA class II proteins. [Item 13] The cell according to item 11 or 12, wherein the single-stranded fusion HLA class II protein comprises at least a portion of an HLA-DQα chain (SEQ ID NO: 41) and at least a portion of an HLA-DQβ chain (SEQ ID NO: 43). [Item 14] The aforementioned single-strand fusion HLA class II protein is HLA-DQα chain allele HLA-DQA1 * At least a portion of 01 (sequence number 41) and the HLA-DQβ chain allele HLA-DQB1 *A cell according to any one of items 11 to 13, comprising at least a portion of 02 (Sequence ID 43). [Item 15] The cell according to any one of items 11 to 14, wherein the single-strand fusion HLA class II protein presents a first target peptide antigen on the cell surface. [Item 16] The cell described in item 15, wherein the first target peptide antigen is covalently bound to the single-strand fusion HLA class II protein. [Item 17] A cell as described in any one of items 1 to 10, wherein one or more immunomodulatory genes contain one or more HLA class II proteins, and the one or more HLA class II proteins are selected from the group consisting of HLA-DMα chain, HLA-DMβ chain, HLA-DOα chain, HLA-DOβ chain, HLA-DPα chain, HLA-DPβ chain, HLA-DQα chain, HLA-DQβ chain, HLA-DRα chain, and HLA-DRβ chain. [Item 18] A cell according to any one of items 8 to 16, further comprising a polynucleotide capable of encoding a single-stranded fusion HLA class I protein. [Item 19] The cell according to item 18, wherein the single-stranded fusion HLA class I protein comprises at least a portion of B2M (SEQ ID NO: 2) covalently bonded to at least a portion of the HLA class Iα chain, and the HLA class Iα chain is selected from the group consisting of HLA-A (SEQ ID NO: 4), HLA-B (SEQ ID NO: 6), HLA-C (SEQ ID NO: 8), HLA-E (SEQ ID NO: 10), HLA-F (SEQ ID NO: 12), and HLA-G (SEQ ID NO: 14). [Item 20] The cell according to item 18 or 19, wherein the single-stranded fusion HLA class I protein comprises at least a portion of B2M (SEQ ID NO: 2) covalently bound to at least a portion of HLA-A (SEQ ID NO: 3). [Item 21] The cell according to any one of items 18 to 20, wherein the single-strand fusion HLA class I protein comprises at least a portion of B2M covalently bound to at least a portion of HLA-A0201 (SEQ ID NO: 4). [Item 22] The cell according to any one of items 18 to 21, further expressing a second target peptide antigen presented by the single-strand fusion HLA class I protein on the cell surface. [Item 23] The cell described in item 22, wherein the second target peptide antigen is covalently bound to the single-strand fusion HLA class I protein. [Item 24] A cell as described in any one of items 1 to 23, further comprising one or more recombinant genes capable of encoding a suicide gene product. [Item 25] The cell according to item 24, wherein the suicide gene product comprises a protein selected from the group consisting of thymidine kinases and apoptosis signaling proteins. [Item 26] Cells having a normal karyotype, as described in any one of items 1 to 25. [Item 27] Non-transformed cells, as described in any one of items 1 to 26. [Item 28] A stem cell, as described in any one of items 1 through 27. [Item 29] The cells described in item 28, wherein the aforementioned stem cells are selected from the group consisting of hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, hepatic stem cells, neural stem cells, pancreatic stem cells, and mesenchymal stem cells. [Item 30] The cells described in item 28 or 29, wherein the aforementioned stem cells are pluripotent stem cells. [Item 31] The cells described in any one of items 28 to 30, wherein the aforementioned stem cells are of the differentiated type. [Item 32] The differentiated cells described in item 31 are selected from the group consisting of dendritic cells, islet cells, hepatocytes, muscle cells, keratinocytes, nerve cells, hematopoietic cells, lymphocytes, erythrocytes, platelets, skeletal muscle cells, ophthalmic cells, mesenchymal cells, fibroblasts, lung cells, GI duct cells, vascular cells, endocrine cells, adipocytes, and cardiomyocytes. [Item 33] Human cells, as described in any one of items 1 to 31. [Item 34] A vaccine containing cells described in any one of items 16 to 33, A vaccine capable of inducing an immune response in primates that is specific to at least one of a first target peptide antigen and a second target peptide antigen. [Item 35] The vaccine described in item 34, wherein the primate is a human and the cells are human cells. [Item 36] A transplantation method for patients who require it, A method comprising the step of administering an effective amount of cells or vaccine described in any one of items 1 to 35 to the patient. [Item 37] The method according to item 36, wherein the patient is immunocompetent. [Item 38] The method according to item 36 or 37, wherein the cells or vaccine include differentiated cells. [Item 39] The method according to any one of items 36 to 38, wherein the patient is human and the cells are human cells.

Claims

1. Isolated human cells comprising genetically engineered disruption of endogenous human leukocyte antigen (HLA) class II related genes, (a) The HLA class II related gene is a gene encoding an HLA class II related protein selected from the group consisting of regulatory factor X-related ankyrin-containing protein (RFXANK) of SEQ ID NOs. 24-27, regulatory factor V (RFX5) of SEQ ID NOs. 28-31, regulatory factor X-related protein (RFXAP) of SEQ ID NOs. 32-33, and class II transactivator (CIITA) of SEQ ID NOs. 34-35. (b) The cells further include genetically engineered disruption of the endogenous β2 microglobulin (B2M) gene of Sequence ID No. 1, (c) The cell further comprises a polynucleotide encoding a single-strand fusion HLA class I protein, wherein the single-strand fusion HLA class I protein comprises at least a portion of exogenous SEQ ID NO: 2 B2M covalently bonded to at least a portion of the HLA class I α chain, The aforementioned single-strand fusion HLA class I protein can exert normal HLA class I function by forming an appropriate secondary structure of heterodimers on the cell surface, presenting peptides in the peptide binding grooves, and binding to inhibitory receptors on the surface of NK cells. (d) The cells optionally further comprise one or more exogenous recombinant immunomodulatory genes, each having the ability to express an immunomodulatory polypeptide, wherein the one or more immunomodulatory polypeptides are single-strand fusion HLA class II proteins. If the single-stranded fusion HLA class II protein is present, it includes at least a portion of the HLA class II gene α chain covalently bonded to at least a portion of the HLA class II gene β chain, and the HLA class II gene is selected from the group consisting of HLA-DP (SEQ ID NOs. 36-39), HLA-DQ (SEQ ID NOs. 40-43), HLA-DR (SEQ ID NOs. 44-47), HLA-DM (SEQ ID NOs. 48-51), and HLA-DO (SEQ ID NOs. 52-55). (e) The single-strand fusion HLA class I protein and / or single-strand fusion HLA class II protein present one or more peptide antigens on the cell surface, wherein the peptides include a target peptide antigen. cell.

2. The cell according to claim 1, wherein one or more peptides are covalently bound to the HLA class I protein and / or the HLA class II protein.

3. The cell according to claim 1 or 2, wherein the HLA class Iα chain is selected from the group consisting of HLA-A of SEQ ID NO: 4, HLA-B of SEQ ID NO: 6, and HLA-C of SEQ ID NO:

8.

4. The cell according to claim 1 or 2, wherein the HLA class Iα chain is selected from the group consisting of HLA-E of SEQ ID NO: 10, HLA-F of SEQ ID NO: 12, and HLA-G of SEQ ID NO:

14.

5. The cell according to claim 1 or 2, wherein the HLA class Iα chain is HLA-E of SEQ ID NO:

10.

6. The cell according to any one of claims 1 to 5, wherein the HLA class II-related gene encodes regulatory factor X-related ankyrin-containing protein (RFXANK) of sequence numbers 24 to 27.

7. The cell according to any one of claims 1 to 6, wherein the immunomodulatory polypeptide comprises a plurality of different single-strand fusion HLA class II proteins.

8. The single-stranded fusion HLA class II protein is (a) comprising at least a portion of the HLA-DQα chain of SEQ ID NO: 41 and at least a portion of the HLA-DQβ chain of SEQ ID NO: 43; or (b) comprising at least a portion of the HLA-DQα chain allele HLA-DQA1*01 of SEQ ID NO: 41 and at least a portion of the HLA-DQβ chain allele HLA-DQB1*02 of SEQ ID NO: 43 The cell according to any one of claims 1 to 7.

9. The cell according to any one of claims 1 to 8, wherein the cell further comprises a recombinant gene encoding a suicide gene product selected from the group consisting of thymidine kinases and apoptosis signaling proteins.

10. The cell according to any one of claims 1 to 9, wherein the cell has a normal karyotype.

11. The cell according to any one of claims 1 to 10, wherein the cell is a non-transformed cell.

12. The cell according to any one of claims 1 to 11, wherein the cell is a stem cell selected from the group consisting of pluripotent stem cells, hematopoietic stem cells, embryonic stem cells, adult stem cells, induced pluripotent stem cells, hepatic stem cells, neural stem cells, pancreatic stem cells, and mesenchymal stem cells.

13. The cell according to claim 12, wherein the stem cell is a pluripotent stem cell.

14. The cell according to any one of claims 1 to 12, wherein the cell is a differentiated cell.

15. The cell according to claim 14, wherein the differentiated cell is selected from the group consisting of dendritic cells, islet cells, hepatocytes, muscle cells, keratinocytes, nerve cells, hematopoietic cells, lymphocytes, erythrocytes, platelets, skeletal muscle cells, ophthalmic cells, mesenchymal cells, fibroblasts, lung cells, GI duct cells, vascular cells, endocrine cells, adipocytes, and cardiomyocytes.

16. A pharmaceutical composition comprising cells according to any one of claims 1 to 15 and a physiologically compatible buffer.

17. A vaccine for use in inducing a target peptide antigen-specific immune response in a patient, comprising the cells described in any one of claims 1 to 15.

18. The vaccine according to claim 17, wherein the patient is a human.

19. The vaccine according to claim 17 or 18, wherein the cells include differentiated cells.

20. A kit comprising the cells according to any one of claims 1 to 15.

21. The cell according to any one of claims 1 to 15, wherein the cell is a cell for use in transplantation in a patient in need of transplantation, and the use comprises administering an effective amount of the cell to the patient.

22. The cells for use according to claim 21, wherein the patient is immunocompetent.

23. The cell for use according to claim 21 or 22, wherein the cell is a differentiated cell.

24. The cells for use according to any one of claims 21 to 23, wherein the patient is a human.

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