HLA class ii-deficient cell capable of expressing HLA class ii protein, and HLA class i-deficient cells and uses thereof

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] The present invention relates to HLA class II-deficient cells capable of expressing HLA class II proteins, HLA class I-deficient cells, and uses thereof. [Background technology]

[0002] Human pluripotent stem cells have the potential to treat diseases affecting almost any organ system. However, a major limitation to the clinical use of human pluripotent stem cells and their derivatives is the rejection of transplanted cells by recipients due to differences in the major histocompatibility complex.

[0003] The major histocompatibility complex (MHC) is a multicomponent cell surface molecule found in all vertebrates that mediates the interaction of white blood cells with other white blood cells 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 exclusively on professional antigen-presenting cells (APCs), including macrophages, dendritic cells, and B cells. In addition, sometimes solid organs can express HLA class II genes that are involved in immune rejection. HLA class I (HLA-I) proteins are expressed on all nucleated cells and consist of HLA class I heavy chains (or α chains) and β-2 microglobulin (B2M). HLA class I proteins present peptides on the cell surface to CD8+ cytotoxic T cells. 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 α chain determines the peptide binding specificity on the peptide-binding groove of the HLA class I molecule. Cellular immunity is mediated by CD8+ T cell recognition of 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 a β2 microglobulin (B2M), whereas 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 those of extracellular pathogens, on the cell surface, whereas 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 activates B cells, leading to recruitment of phagocytes, local inflammation, and / or humoral responses. To date, several HLA class II loci have been identified, including HLA-DM (HLA-DMA and HLA-DMB, which encode the HLA-DM α and β chains, respectively), HLA-DO (HLA-DOA and HLA-DOB, which encode the HLA-DO α and β chains, respectively), HLA-DP (HLA-DPA and HLA-DPB, which encode the HLA-DP α and β chains, respectively), HLA-DQ (HLA-DQA and HLA-DQB, which encode the HLA-DQ α and HLA-DQ β chains, respectively), and HLA-DR (HLA-DRA and HLA-DRB, which encode the HLA-DR α and HLA-DR β chains, respectively).

[0006] In the context of transplantation, HLA class I and / or class II proteins from allogeneic sources represent foreign antigens, and recognition of non-self HLA class I and / or class II proteins is a significant obstacle in the use of pluripotent cells for transplantation or replacement therapy. Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, although personalized stem cell preparations or HLA-diverse stem cell banks could address current transplantation challenges, they require multiple cell lines to be characterized, differentiated into therapeutic cell products, and approved for human administration. This time-consuming, technically challenging, and expensive process is a major factor preventing stem cell-based therapies from moving into clinical trials. Thus, 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 aspect, the present invention provides an isolated cell comprising a genetically engineered disruption in a human leukocyte antigen (HLA) class II associated gene, wherein the cell is a primate cell. In one embodiment, the HLA class II associated gene is a regulatory factor X-associated ankyrin-containing protein (RFXANK). ankyrin-containing protein, regulatory factor 5 (RFX5), regulatory factor X-associated protein (RFXAP), class II transactivator (CIITA), 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 cell comprises a genetically engineered disruption in at least two, at least three, or all four of the HLA class II associated genes. In a further embodiment, the HLA class II associated gene is regulatory factor X-related ankyrin-containing protein (RFXANK). In yet another embodiment, the cell comprises a genetically engineered disruption in all copies of the HLA class II associated gene. In another embodiment, the cell further comprises one or more recombinant immunomodulatory genes, each capable of expressing an immunomodulatory polypeptide in a human cell. In a further embodiment, the one or more immunomodulatory genes comprise a polynucleotide capable of encoding an HLA II protein. In another embodiment, the one or more immunomodulatory genes comprise a polynucleotide capable of encoding a single-chain fusion HLA class II protein. In a further embodiment, the cell further comprises a genetically engineered disruption in the β2-microglobulin (B2M) gene.

[0009] In another aspect, the invention provides an isolated cell comprising: (a) a genetically engineered disruption in the beta2 microglobulin (B2M) gene; and (b) one or more polynucleotides capable of encoding an HLA class II protein, or a single chain fusion HLA class II protein; wherein the cell is a primate cell.

[0010] In one embodiment of any of these aspects, the cell comprises a genetically engineered disruption of all copies of the B2M gene, hi a further embodiment, the HLA II gene encodes an HLA protein selected from the group consisting of an HLA-DM α chain, an HLA-DM β chain, an HLA-DO α chain, an HLA-DO β chain, an HLA-DP α chain, an HLA-DP β chain, an HLA-DQ α chain, an HLA-DQ β chain, an HLA-DR α chain, and an HLA-DR β chain.

[0011] In another embodiment, the single chain fusion HLA class II protein comprises at least a portion of an HLA class II gene alpha chain covalently linked to at least a portion of an HLA class II gene beta 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 a further embodiment, the single chain fusion HLA class II protein comprises a plurality of different single chain fusion HLA class II proteins. In another embodiment, the single chain fusion HLA class II protein comprises at least a portion of an HLA-DQ alpha chain and at least a portion of an HLA-DQ beta chain. In yet another embodiment, the single chain fusion HLA class II protein comprises at least a portion of an HLA-DQ alpha chain allele HLA-DQA1. * 01 and the HLA-DQ beta chain allele HLA-DQB1 * and at least a portion of 02.

[0012] In further embodiments of any of the aspects of the cell of the invention, the HLA protein or single chain fusion HLA class II protein presents a first target peptide antigen on the cell surface, hi one such embodiment, the first target peptide antigen is covalently linked to the single chain fusion HLA class II protein.

[0013] In another embodiment, the cell further comprises a polynucleotide capable of encoding a single chain fusion HLA class I protein. In one such embodiment, the single chain fusion HLA class I protein comprises at least a portion of B2M covalently linked to at least a portion of an HLA class I α 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 chain fusion HLA class I protein comprises at least a portion of B2M covalently linked to at least a portion of HLA-A. In a further embodiment, the single chain fusion HLA class I protein comprises at least a portion of B2M covalently linked to at least a portion of HLA-A0201. In another embodiment, the cell further expresses a second target peptide antigen presented by the single chain fusion HLA class I protein on the cell surface. For example, the second target peptide antigen may be covalently linked to the single chain fusion HLA class I protein.

[0014] In another embodiment of any of the aspects of the cell of the invention, the cell further comprises one or more recombinant genes capable of encoding a suicide gene product. For example, the suicide gene product can comprise a protein selected from the group consisting of thymidine kinase and an apoptosis signaling protein.

[0015] The cells of any embodiment 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, pancreatic islet cells, hepatocytes, muscle cells, keratinocytes, nerve cells, hematopoietic cells, lymphocytes, red blood cells, platelets, skeletal muscle cells, eye cells, mesenchymal cells, fibroblasts, lung cells, gastrointestinal (GI) tract cells, vascular cells, endocrine cells, adipocytes, or cardiomyocytes. The cells may be human cells.

[0016] In another aspect, the invention provides a vaccine comprising a cell of any one of the embodiments or combination of embodiments of the cell of the invention, the cell comprising at least one target peptide antigen on the cell surface, wherein the vaccine is capable of inducing an immune response in a primate specific to the target peptide antigen.

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

[0018] [Figure 1] Diagram showing the structure 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 the vectors). Cells targeted by the TKNeo or HyTK vectors, respectively, can be isolated by selecting vector-infected cells with G418 or hygromycin (Hygro). Subsequent expression of Cre recombinase and selection with ganciclovir (GCV) then allows the isolation of clones in which the TKNeo or HyTK gene has been removed, leaving behind two inactivated RFXANK alleles with stop codons in all three reading frames, loxP sites, and polyadenylation sites (StopX3-loxP-pA). loxP is the recombination site for Cre recombinase. ITR is the vector inverted terminal repeat. Similar vectors can be designed to target other genes. [Diagram 2](A) is a schematic diagram of the targeting strategy for infecting human embryonic stem cells with the construct AAV-RFXANK-ETKNpA, and (B) is a photograph of a stained gel showing the polymerase chain reaction (PCR) products obtained after infection of human embryonic stem cells with AAV-RFXANK-ETKNpA and PCR, using a forward primer homologous to the neomycin sequence of the selection cassette and a reverse primer homologous to the RFXANK gene outside the targeting homology arms, as indicated by the arrows above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] All cited references are incorporated herein by reference in their entirety. In this application, unless otherwise specified, the techniques utilized are based on a number of well-known references, such as "Molecular Cloning: A Laboratory Manual" (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, CA), "Guide to Protein Purification", Methods in Enzymology (edited by MP Deutschcer, 1990, Academic Press, Inc.); "PCR Protocols: A Guide to Methods and Applications" (J.S.M. Deutschcer, ed., 1990, Academic Press, Inc.); (Innis et al., 1990, Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Techniques, 2nd ed. (RI Freshney, 1987, Liss, Inc., New York, NY), Gene Transfer and Expression Protocols ... For example, see "Medical Procedures for the Treatment of Cardiovascular Disease," pp. 109-128, edited by E. J. Murray, Humana Press Inc., Clifton, NJ, and the Ambion 1998 Catalog (Ambion, Austin, Tex.).

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

[0021] All embodiments of any aspect of the 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 associated gene, wherein the cell is a primate cell. HLA class II associated gene, as used herein, broadly refers to a gene encoding a protein involved in HLA class II mediated immune response. Thus, HLA class II associated gene includes genes encoding HLA class II molecules such as HLA-DM (SEQ ID NO: 48, 50), HLA-DO (SEQ ID NO: 52, 54), HLA-DP (SEQ ID NO: 36, 38), HLA-DQ (SEQ ID NO: 40, 42), and HLA-DR (SEQ ID NO: 44, 46). Exemplary HLA class II gene / protein sequences are listed in publicly available databases as follows: NM_033554.3 for HLA-DPA (SEQ ID NOs: 36, 37), HLA00514 for HLA-DPB (SEQ ID NOs: 38, 39), HLA00601 for HLA-DQA (SEQ ID NOs: 40, 41), HLA00622 for HLA-DQB (SEQ ID NOs: 42, 43), and NM_019111 for HLA-DRA (SEQ ID NOs: 44, 45). The HLA-DRB and HLA-DOB sequences can be referenced based on the GenBank or IMGT / HLA database numbers: HLA00664 (SEQ ID NOs: 46, 47), NM_006120 (SEQ ID NOs: 48, 49), HLA-DMA, NM_002118 (SEQ ID NOs: 50, 51), HLA-DMB, NM_002119 (SEQ ID NOs: 52, 53), and HLA-DOB, NM_002120 (SEQ ID NOs: 54, 55).

[0022] In addition, HLA class II-associated genes also include genes encoding HLA class II regulatory proteins that regulate the expression of HLA class II molecules, including, without limitation, regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-related protein (RFXAP), and class II transactivator (CIITA). For example, regulatory factor X-related ankyrin-containing protein (RFXANK) forms a complex together with regulatory factor X-related protein and regulatory factor 5 that binds to the X-box motif of the HLA class II gene promoter to activate the transcription of HLA class II genes. Exemplary sequences of HLA class II associated genes that regulate expression of HLA class II molecules can be found in publicly available databases under GenBank Accession Nos. NM_134440.1 (SEQ ID NOs: 24, 25) and NM_003721.2 (SEQ ID NOs: 26, 27) for RFXANK, NM_000449.3 (SEQ ID NOs: 28, 29) and NM_001025603.1 (SEQ ID NOs: 30, 31) for RFX5, NM_000538.3 (SEQ ID NOs: 32, 33) for RFXAP, and NM_000246.3 (SEQ ID NOs: 34, 35) for CIITA. All sequences disclosed under these GenBank Accession Nos. are incorporated herein by reference.

[0023] In certain embodiments, the present invention provides an isolated primate cell, preferably a human cell, comprising a genetic disruption in at least one HLA class II associated gene as defined herein.In certain detailed embodiments, the cell comprises a genetic disruption of all copies of at least one HLA class II associated gene.In certain other embodiments, the cell comprises multiple genetic disruptions in multiple HLA class II associated genes.

[0024] In certain embodiments, the HLA class II associated genes are selected from the group consisting of regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-related protein (RFXAP), and class II transactivator (CIITA). In certain detailed embodiments, the cells comprise at least one genetically engineered disruption in at least one, at least two, at least three, or all of the HLA class II associated genes selected from the group consisting of regulatory factor X-related ankyrin-containing protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-related protein (RFXAP), and class II transactivator (CIITA). Any combination of these four HLA class II associated genes as gene disruption targets for generating HLA class II-deficient cells is included within the scope of the present invention.

[0025] In certain other embodiments, the cells are immunocompetent for regulatory factor X-related ankyrin-containing protein (RFXANK) (SEQ ID NOs: 24-27), regulatory factor 5 (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 The invention comprises the disruption by genetic manipulation of at least one HLA class II associated gene selected from the group consisting of 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).

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

[0027] In another aspect, the invention provides HLA class I and HLA class II deficient cells. In a particular embodiment, the invention provides primate cells, preferably human cells, comprising a genetically engineered disruption in an HLA class II associated gene, and further comprising a genetically engineered disruption in the beta2 microglobulin (B2M) gene (SEQ ID NO:1). In other particular embodiments, the cells further comprise a genetically engineered disruption of all copies of the B2M gene (SEQ ID NO:1). In a particular embodiment, the genetic disruption in the B2M (SEQ ID NO:1) gene results in defective or no expression of the B2M protein (SEQ ID NO:2). In other particular embodiments, the cells further comprise a genetically engineered disruption of all copies of the B2M gene. In a particular embodiment, the genetic disruption in the B2M gene results in defective or no expression of the B2M protein. As 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 invention, HLA class I / class II deficient cells are provided. The B2M coding sequence is shown in SEQ ID NO: 1 (GenBank Accession No. NM_004048), and the B2M protein sequence is shown in SEQ ID NO: 2. There can be many single nucleotide polymorphisms (SNPs) in the gene; as one of skill in the art will appreciate, the human cells and methods of the invention are applicable to any such B2M genes and SNPs.

[0028] Any suitable technique can be used to introduce the disruption (in the HLA class II associated gene, the B2M gene, or any other suitable gene) by genetic engineering; exemplary gene disruption techniques are disclosed throughout this application and are within the level of skill of the art based on the teachings herein and known in the art. Other exemplary techniques can be found, for example, in U.S. Patent Application Publication No. 2008 / 0219956, published Sep. 11, 2008, which is incorporated herein by reference in its entirety. These techniques can optionally include removing non-human DNA sequences from the cells after disruption of the HLA class II associated gene and optionally disruption of the B2M gene.

[0029] One such technique utilizes an adeno-associated virus gene targeting vector, optionally including removal of the transgene used for targeting using techniques such as those described below, or by removing the transgene used for targeting by 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 its entirety. Exemplary targeting vectors and exemplary vector diagrams are also disclosed herein. It is within the level of skill of one of ordinary skill in the art to utilize various techniques for the generation of the HLA class II cells, preferably human cells, of the present invention, based on the teachings herein and known in the art.

[0030] In certain embodiments, the cellular genome of the HLA class II deficient cell may comprise 100 or less, 50 or less, or 30 or less non-human DNA sequence nucleotides. In certain other embodiments, the cellular genome may comprise 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 FIG. 1. The non-human DNA sequence may be removed by a second targeting round to delete the HyTK or TKNeo transgene in the initial vector, or by Cre-mediated loxP recombination.

[0031] In certain other embodiments, the HLA class II or HLA class I / class II deficient cells further comprise one or more recombinant immune modifier genes. Suitable immune modifier 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 term "single chain fusion HLA class II protein", "single chain fusion HLA class II molecule" or "single chain fusion HLA class II antigen" refers to a fusion protein comprising at least a portion of an HLA class II α chain covalently linked directly or via a linker sequence to at least a portion of an HLA class II β chain or a class II α chain or β chain linked to a peptide antigen, or a combined class II α chain and β chain that is also linked to a peptide antigen. On the other hand, the term "HLA class II protein", "HLA class II molecule" or "HLA class II antigen" refers to a non-covalently associated heterodimer of HLA class II α chain and HLA β chain expressed on the surface of wild-type cells. In embodiments where the gene encodes an HLA class II protein (as opposed to a single-chain fusion HLA class II protein), the gene is under the control of a promoter that is not involved in normal class II expression in the cell. In one embodiment, the gene is expressed episomally; in another embodiment, the gene is integrated into the genome of the cell. In either embodiment, the gene is operably linked to (i.e.: under transcriptional control of) a promoter that is not involved in normal class II expression in the cell. Any suitable promoter can be used, as can be determined by one of skill in the art based on the specific intended design and use of the construct and cell.

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

[0033] HLA class II deficient cells, HLA class I deficient cells, or HLA class I / class II deficient cells can be used as universal 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 in transplantation. In addition, solid organ cells can sometimes express HLA class II genes involved in immune rejection. Thus, 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 associated with solid organs.

[0034] In certain detailed embodiments of any of the cells of the invention, the HLA α 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 need not, be from the same HLA class II gene. For example, the HLA class II protein or single chain fusion HLA class II protein may comprise at least a portion of an HLA-DQ α chain and at least a portion of an HLA-DQ β chain (also referred to as a dimeric construct). HLA class II proteins and single chain fusion HLA class II proteins that comprise mismatched HLA class II alleles are also contemplated. In certain detailed embodiments, the HLA class II protein or single chain fusion HLA class II protein comprises at least a portion of the HLA-DQ α chain allele HLA-DQA1. * 01 (SEQ ID NO: 41) and at least a portion of the HLA-DQ beta chain allele HLA-DQB1 *02 (SEQ ID NO: 43). In certain preferred embodiments, the fusion construct has the leader sequence (or signal peptide) of the second portion of the fusion protein removed. For example, the HLA-DQB1 * HLA-DQA1 at the N-terminus covalently linked to at least a portion of 02 (SEQ ID NO: 43) * In a single chain fusion HLA class II protein comprising at least a portion of HLA-DQA1 (SEQ ID NO: 41), * The 01 (SEQ ID NO: 41) leader sequence was left in the construct, and the HLA-DQB1 * 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 chain fusion HLA class II proteins. In certain detailed embodiments, the HLA class II protein or single chain fusion HLA class II protein also comprises a first target peptide antigen that occupies the peptide binding site of the HLA class II protein or single chain fusion HLA class II protein, where the peptide antigen is covalently linked to the HLA class II protein or single chain fusion HLA class II protein (also referred to as a trimeric construct). In certain other embodiments, the covalently linked peptide antigen is cleaved at an incorporated protease cleavage site, and the cleaved peptide antigen can bind to the peptide binding site of the single chain fusion HLA-II protein for presentation.

[0035] Thus, HLA class II, HLA class I deficient cells, or HLA class I / class II deficient cells also encompass cells with a genetically engineered disruption in all copies of the HLA class II genes (e.g., a disruption in all copies of the HLA-DQ α and / or β chains), where one HLA class II allele is genetically engineered to express an HLA class II protein or single-chain fusion HLA class II protein of interest in place of the wild-type HLA class II protein (i.e., a gene-targeted knock-in in one HLA-II allele). Taking HLA-DQ as an example, in certain embodiments, HLA-DQ- / - The cells express HLA-DQ proteins only in the context of single-chain fusion HLA-DQ proteins from the HLA-DQ locus. In certain advantageous embodiments, 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 with a genetically engineered disruption in all copies of a particular HLA-II gene, where all alleles of that specific HLA-II gene are genetically engineered to express a single-chain fusion HLA class II protein in place of the wild-type HLA-II protein (i.e., a genetically targeted knock-in in all HLA-II alleles). HLA class II, HLA class I deficient cells, or HLA class I / class II deficient cells with such a genetic disruption express a particular HLA-II protein only in the context of a single-chain fusion HLA class II protein from all allele loci of that particular HLA-II gene.

[0037] HLA class II proteins and single chain fusion HLA class II proteins comprising sequence variants and fragments of HLA class II α and β chains are contemplated by the present invention, where such HLA class II proteins or single chain fusion constructs still have normal HLA class II functions, such as forming the proper secondary structure of the heterodimer on the cell surface, presenting peptides at the peptide binding site, interacting with CD4+ helper T cells, and eliciting an HLA class II mediated immune response. 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 a naturally occurring HLA class II α or β chain sequence, where the variants have normal HLA class II functions. In certain other embodiments, the variant shares at least 75%, 80%, 81%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence identity with an HLA class II α chain or β chain sequence as set forth in SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55.

[0038] Additionally, HLA class II, HLA class I deficient cells, or HLA class I / class II deficient cells can be engineered to recombinantly express a single chain fusion HLA class I protein in a B2M- / - genetic background. HLA class I deficient cells or HLA class I / class II deficient cells recombinantly expressing a single chain fusion HLA class I protein are nevertheless deficient in normal B2M function in that the wild-type B2M protein (SEQ ID NO:2), which can form non-covalently associated heterodimers with any HLA class I α chain on the cell surface, is not expressed by the cell.

[0039] The terms "single-chain fusion HLA class I protein", "single-chain fusion HLA class I molecule" or "single-chain fusion HLA class I antigen" refer to a fusion protein comprising at least a portion of a B2M protein covalently linked, either directly or via a linker sequence, to at least a portion of an HLA-Iα chain, whereas the terms "HLA class I protein", "HLA class I molecule" or "HLA class I antigen" refer to a non-covalently associated heterodimer of B2M and an HLAα chain expressed on the surface of a wild-type cell.

[0040] As used herein, the term "HLA class I alpha chain" or "HLA-I heavy chain" refers to the alpha chain of the HLA class I heterodimer. HLA class I heavy chains include, without limitation, the HLA class I alpha 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 No. K02883.1, SEQ ID NO: 3; UniProt No. 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, the term "HLA class I or II protein / molecule" is known to refer to MHC class I or II protein / molecule in humans, although the terms HLA and MHC are sometimes used synonymously throughout this application: for example, the terms HLA class I or HLA class II protein can also be used in primates to refer to the primate equivalents of HLA class I or HLA class II proteins, respectively. One of skill in the art will be able to interpret the meaning of this term based on the context.

[0042] Thus, HLA class I deficient cells or HLA class I / class II deficient cells also encompass cells with a genetically engineered disruption in all copies of the B2M gene, where one B2M allele is engineered to express a single-chain fusion HLA class I protein instead of the wild-type B2M protein (i.e., a gene-targeted knock-in in one B2M allele). B2M- / - cells with such a genetic background express B2M only in the context of the single-chain fusion HLA class I protein from the B2M locus. In a particular advantageous embodiment, expression of the single-chain fusion HLA class I protein is regulated by the 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 include cells with a genetically engineered disruption in all copies of the B2M gene, where all B2M alleles are engineered to express a single-chain fusion HLA class I protein in place of the wild-type B2M protein (i.e., a genetically targeted knock-in in all B2M alleles). HLA class I deficient cells or HLA class I / class II deficient cells with such a genetic disruption express B2M only in the context of a single-chain fusion HLA class I protein from all allelic loci of the B2M gene. In certain embodiments, the cells are engineered to express a homogeneous single-chain fusion HLA class I protein from all allelic loci of the B2M gene; while in other embodiments, the cells are engineered to express a heterologous single-chain fusion HLA class I protein from a different locus of the B2M gene.

[0044] In certain embodiments, the single chain 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 dimeric construct). In certain preferred embodiments, the HLA α chain contained in the single chain fusion HLA class I protein does not include the leader sequence (or signal sequence) of the HLA class I α chain (leaderless HLA α chain). In certain other embodiments, the single chain 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 chain 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 chain fusion HLA class I protein comprises a leader sequence (or signal peptide) covalently linked to at least a portion of B2M and at least a portion of the HLA α chain to ensure proper folding of the single chain fusion on the cell surface. The leader sequence may be the leader sequence of a B2M protein, the leader sequence of an HLA α chain protein or the leader sequence of another secretory protein. In certain detailed embodiments, the single chain fusion HLA class I protein comprises a B2M protein with its leader sequence removed. In certain other detailed embodiments, the single chain fusion HLA class I protein comprises an HLA α chain protein with its leader sequence removed. Certain HLA class I α chains are highly polymorphic. As one of skill in the art will appreciate, the human cells and methods of the present invention are applicable to any such HLA α chain and polymorphisms thereof.

[0045] Single chain fusion HLA class I proteins comprising sequence variants and fragments of B2M and / or HLA α chains are contemplated by the present invention, where such single chain fusion constructs still have normal HLA class I functions, e.g., forming the proper secondary structure of the heterodimer on the cell surface, presenting peptides in the peptide binding groove, 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, where the variants have normal HLA class I functions. In certain other embodiments, the variant shares at least 75%, 80%, 81%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence identity with a B2M or HLA heavy chain sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14.

[0046] In certain detailed embodiments, the HLA-A variants share 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 variants share 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 variants share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or complete sequence homology with SEQ ID NO: 8. In still other embodiments, the HLA-E variant shares at least 97%, 98%, 99%, or complete sequence homology with SEQ ID NO: 10. In certain particular 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 chain fusion HLA class I protein comprises full-length B2M (SEQ ID NO:2) (including its leader sequence) and an HLA alpha chain without a leader sequence (a leaderless HLA alpha chain); while in certain other embodiments, the single chain fusion HLA class I protein comprises a B2M (SEQ ID NO:2) protein without a leader sequence. It is understood that B2M- / - cells expressing two, three or more heterologous single chain fusion HLA class I proteins in any combination, such as, for example, expressing an SC fusion comprising HLA-A (SEQ ID NO: 4) (or leaderless HLA-A) and an SC fusion comprising HLA-C (SEQ ID NO: 8) (or leaderless HLA-C), expressing an SC fusion comprising HLA-A (SEQ ID NO: 4) (or leaderless HLA-A) and an SC fusion comprising HLA-E (SEQ ID NO: 10) (or leaderless HLA-E), or expressing an SC fusion comprising HLA-B (SEQ ID NO: 6) (or leaderless HLA-B), an SC fusion comprising HLA-E (SEQ ID NO: 10) (or leaderless HLA-E), and an SC fusion comprising HLA-G (SEQ ID NO: 14) (or leaderless HLA-G), are all contemplated by and within the scope of the present invention.

[0048] Natural killer (NK) cells are part of the innate immune response. Some pathogens can downregulate HLA class I protein expression in infected cells. NK cells monitor infection by recognizing and inducing apoptosis of cells that do not express HLA class I proteins. Inhibitory receptors on the surface of NK cells recognize HLA class I α-chain alleles, thereby preventing NK-mediated apoptosis in uninfected normal cells. Thus, in certain detailed embodiments, single-chain fusion HLA-I proteins inhibit NK-mediated killing of cells that do not express endogenous HLA class I proteins by binding to inhibitory receptors on NK cells. For example, HLA-E is a ligand for the CD94 / NKG2 receptor of NK cells, which inhibits NK cell-mediated apoptosis. Thus, in certain detailed embodiments, B2M- / - cells express single-chain fusion HLA class I proteins that include at least a portion of B2M and at least a portion of HLA-E. In addition, HLA-G is expressed on the surface of placental cytotrophoblasts, which do not normally express HLA-A, B, or C, and protects those cells from NK cell-mediated lysis by interacting with the inhibitory ILT2 (LIR1) receptor on NK cells (Pazmany et al., 1996, Science 274:792-795). Thus, in certain other preferred embodiments, B2M- / - cells express a single-chain 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 chain 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 percentage of the US population. Thus, in certain advantageous embodiments, the isolated cells express a single chain fusion HLA class I protein comprising at least a portion of B2M and at least a portion of HLA-A0201 in a B2M- / - genetic background, where the isolated cells are immunocompatible with a large percentage of the US population. Other suitable common alleles that can 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 chain fusion HLA class I protein also includes a second target peptide antigen that occupies the peptide binding groove of the single chain fusion HLA class I protein, where the peptide antigen is covalently linked to the single chain fusion HLA class I protein (also referred to as a trimeric construct). An example of a trimeric construct is shown in FIG. 2. The HLA-bGBE construct in FIG. 2 includes B2M and HLA-E covalently linked to a peptide antigen (such as, but not limited to, HLA-G signal peptide as illustrated in the figure) (SEQ ID NO: 23) designed to occupy the peptide binding groove of the single chain fusion HLA class I protein. In certain other embodiments, the covalently linked 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 chain fusion HLA-I protein for presentation.

[0051] In certain alternative embodiments, the peptide antigens occupying the peptide-binding groove of the single-chain fusion HLA class I protein are produced by the intracellular antigen processing pathway, where the peptide antigens are produced by the proteasome and transported to and loaded onto the single-chain fusion HLA class I protein in the endoplasmic reticulum. In certain detailed embodiments, the peptide antigens comprise peptides of tumor antigens. In certain other embodiments, the peptide antigens comprise peptides of proteins from pathogens, including, without limitation, bacteria, viruses, fungi, and parasites. In further embodiments, the peptide antigens comprise peptides of tumor antigens. In certain detailed embodiments, the HLA class I-deficient cells or HLA class I / class II-deficient cells express single-chain fusion HLA class I proteins covalently linked to peptides that do not contain autoantigens or neoantigens to the patient. It is within the capabilities of one of skill in the art to design single-chain fusion HLA class I proteins and peptide antigens presented thereon to tailor the immune response that can be induced in the recipient.

[0052] Isolated HLA class II deficient cells, HLA class I deficient cells, or HLA class I / class II deficient cells expressing HLA class II proteins or single chain fusion HLA class II proteins, optionally with a specific peptide antigen covalently or non-covalently linked to a single chain fusion protein, can be used, for example, to administer to a recipient to induce an immune response. Thus, in a related aspect, the invention provides a vaccine comprising the isolated cells of the invention, wherein the vaccine is capable of inducing an immune response in the recipient specific to the target peptide antigen. 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 a cytokine. 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 a human. Thus, in a further aspect, the present invention provides a kit comprising a vaccine of the invention and optionally an immune adjuvant.

[0053] Single-chain fusion HLA class I protein, HLA class II protein, or single-chain fusion HLA class II protein can be expressed from an expression vector that can express the protein transiently or, more preferably, stably in cells. Exemplary suitable expression vectors are known in the art. One such example is a retroviral vector, which has the ability to integrate into the cell genome and provide long-term stable expression of exogenous genes. In certain detailed embodiments, the viral vector is derived from human foamy virus, which is a type of retrovirus. Other suitable viral vectors include, without limitation, vectors derived from retrovirus, adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, vaccinia virus, and poxvirus.

[0054] In certain preferred embodiments, the polynucleotide capable of encoding the single-chain fusion HLA class I or class II protein or the HLA class II protein is integrated into the chromosome of the cell, preferably into the B2M or HLA class II locus, for stable expression. Thus, in certain preferred embodiments, the B2M locus is disrupted by inserting a polynucleotide capable of encoding the single-chain fusion HLA class I protein into the B2M locus to replace the expression of the endogenous wild-type B2M protein. Thus, in certain other preferred embodiments, a specific HLA-II locus is disrupted by inserting a polynucleotide capable of encoding the HLA class II protein or the single-chain fusion HLA class II protein into the HLA-II locus to replace 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 protein is not possible, but the selected given HLA class II protein or single-chain fusion HLA class I or class II protein can be expressed on the surface of cells that are naturally HLA class II deficient. Other expression vectors are also contemplated, and the selection of an appropriate expression vector is within the capabilities of one of ordinary skill in the art.

[0055] An "isolated cell" may be any suitable cell type for a given purpose. For example, the cell may be a pluripotent stem cell or a differentiated cell. "Stem cell" broadly encompasses any cell that has the potential for further differentiation. "Pluripotent stem cell" refers to a stem cell that has the potential to differentiate into any of the three germ layers, namely endoderm, mesoderm or ectoderm. "Adult stem cell" on the other hand has pluripotency in that it can only produce a limited number of cell types. "Embryonic stem (ES) cell" refers to a pluripotent stem cell derived from the inner cell mass of the blastocyst, an early embryo. "Induced pluripotent stem cell (iPS cell)" is a pluripotent stem cell artificially obtained by artificially inducing and expressing a specific gene from a non-pluripotent cell, typically an adult somatic cell.

[0056] In another aspect, the present invention provides a method of transplantation in a patient in need thereof, comprising administering to the patient an effective amount of cells of the present invention for transplantation. Because HLA class II-deficient cells and / or HLA class I-deficient cells do not express wild-type HLA class II proteins (and optionally also HLA class I proteins) on the cell surface, the cells, when administered to a patient, induce minimal or no immune response in the patient. Thus, transplantation using HLA class II-deficient cells and / or HLA class I-deficient cells obviates the need to undergo immunosuppressant drug therapy. Thus, in certain preferred embodiments, the patient is immunocompetent. In certain other embodiments, the cells are syngeneic cells; while in other embodiments, the cells are allogeneic cells.

[0057] In certain further embodiments, the cells of the invention are pluripotent stem cells; while in other embodiments, the cells of the 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 administering an effective amount of pluripotent stem cells or differentiated cells to a human. In certain preferred embodiments, the cells of the invention further express one or more engineered single-chain fusion HLA class II proteins, and optionally also single-chain fusion HLA class I proteins. In certain other embodiments, the cells are capable of avoiding NK cell-mediated killing and inducing minimal or no immune response in the recipient after transplantation.

[0058] Transplantation therapy, replacement therapy or regeneration therapy refers to the treatment of disease conditions by administering cells or tissues to a patient to supplement or replace missing cellular functions in a target organ. In certain detailed embodiments, the need for transplantation arises as a result of physical or pathological damage 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 transplantation of the cells of the invention supplements or replaces the missing cellular functions in the patient without the need to correct the patient's underlying genetic mutations by gene therapy. In certain further embodiments, transplantation includes, without limitation, hematopoietic stem cell transplantation or transplantation of cells incorporated into 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 solid organ.

[0059] In certain detailed embodiments, the cells administered to the recipient may or may not incorporate into the organ in need of such therapy. In certain embodiments, the cells of the invention differentiate into the desired cell type before or after transplantation and provide the required cellular function without themselves incorporating into the tissue at the transplant site. For example, in certain embodiments to treat diabetes, the cells of the invention are transplanted into the diabetic patient as either pluripotent stem cells or differentiated pancreatic beta islet cells. It is not necessary for the transplanted cells to reconstitute a functional pancreas: they merely need to secrete insulin in response to glucose levels. In certain detailed embodiments, the cells are transplanted into an ectopic site and do not fully incorporate into the pancreas. Transplantation of the pluripotent cells of the invention, the differentiated cells of the invention, or tissue differentiated and grown ex vivo from the cells of the invention are all contemplated 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 invention express one or more single-chain fusion HLA class II proteins, and optionally also single-chain fusion HLA class I proteins.

[0060] In a further aspect, the present invention provides a method of treating a disease condition in a patient in need thereof, comprising administering to the patient an effective amount of the cells of the present invention, thereby treating the disease condition, wherein the disease condition is diabetes, an autoimmune disease, cancer, an infectious disease, anemia, cytopenia, myocardial infarction, heart failure, skeletal or joint conditions, osteogenesis imperfecta, or burn. In certain detailed embodiments, the disease condition is caused by pathological or physical injury of a tissue or organ. In certain embodiments, the cells of the present invention are stem cells; while 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 tissues 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-chain fusion HLA class II proteins, and optionally also one or more single-chain fusion HLA class I proteins. In certain embodiments, the cells are syngeneic cells; while in other embodiments, the cells are allogeneic cells.

[0061] In certain detailed embodiments, the cells are differentiated cells, including, without limitation, dendritic cells, lymphocytes, erythrocytes, platelets, hematopoietic cells, pancreatic islet cells, hepatocytes, muscle cells, keratinocytes, cardiomyocytes, nerve cells, skeletal muscle cells, ocular cells, mesenchymal cells, fibroblasts, lung cells, GI tract cells, vascular cells, endocrine cells, and adipocytes. In certain other detailed embodiments, the invention provides methods for treating disease conditions in solid organs. In certain embodiments, the cells of the invention used to treat disease conditions express one or more single chain fusion HLA class I proteins, and optionally also express one or more single chain fusion HLA class I proteins.

[0062] "Treating" a patient with a disease or disorder means accomplishing one or more of the following: (a) reducing the severity of the disease; (b) arresting the progression of the disease or disorder; (c) preventing the worsening of the disease or disorder; (d) inhibiting or preventing the recurrence of the disease or disorder in a patient who has previously had the disease or disorder; (e) causing regression of the disease or disorder; (f) ameliorating or eliminating symptoms of the disease or disorder; and (f) improving survival. In certain preferred embodiments, the disease or disorder is one that can be treated by tissue or cell transplantation.

[0063] The effective amount of the isolated cells of the present invention for transplantation or for treating a disease condition depends on a number of factors, such as the type of tissue, the severity of the disease condition, the transplantation response, the reason for transplantation, and the age and general health of the patient. The effective amount can be determined by a skilled researcher or clinician through routine practice. Due to the reduced immunogenicity of the transplanted cells, a relatively large amount of cells can be tolerated by the patient to achieve the desired therapeutic effect. Alternatively, the 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 an injured joint, a broken bone, an infarct site, an ischemic site, or the surrounding area.

[0065] In certain detailed embodiments, the cells are administered using a delivery device, including, without limitation, a syringe. For example, the cells can be suspended in a solution or pharmaceutical composition contained in such a delivery device. A "solution" or "pharmaceutical composition" includes a physiologically compatible buffer and, optionally, a pharma- ceutical acceptable carrier or diluent that maintains the viability of the cells of the invention. The use of such carriers and diluents is well known in the art. Solutions include, without limitation, physiologically compatible buffers such as Hank's solution, Ringer's solution, or buffered saline. The cells can be kept in the solution or pharmaceutical composition for short-term storage without loss of viability. In certain detailed embodiments, the cells are frozen according to cryopreservation methods well known in the art for long-term storage without loss of viability.

[0066] Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran, but are still fluid in that they can be easily delivered by syringe injection. The solutions are preferably sterile, stable under the conditions of manufacture and storage, and free of microbial contamination, for example, by the use of 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 pharma- ceutically acceptable carrier or diluent, and optionally other components as noted above.

[0067] The cells may be administered systemically (e.g., intravenously) or locally (e.g., by addition directly to abnormal myocardium under echocardiographic guidance or to damaged tissue or organs accessible during open surgery). For injection, the cells may be in an injectable suspension formulation or in a biocompatible medium that becomes semi-solid at the site of damaged tissue for injection in liquid form. A syringe, controllable endoscopic delivery device, or other similar device may be used, so long as the needle lumen is of sufficient diameter (e.g., at least 30 gauge or larger) to avoid physical damage to the cells during delivery.

[0068] In certain other embodiments, the cells may be implanted using a solid support, such as a planar or three-dimensional matrix. The matrix or planar surface is surgically implanted into the patient at the appropriate site. For example, a patient in need of a pancreatic graft may have differentiated cells on a solid support surgically implanted into the pancreatic tissue. Exemplary solid supports include, without limitation, patches, gel matrices (such as GELFOAM® from Pharmacia-Upjohn), polyvinyl alcohol sponge (PVA)-collagen gel implants (such as IVALON, Unipoint Industries, High Point, NC), and other similar or equivalent devices. A variety of other encapsulation techniques may be used with the cells of the invention (e.g., WO 91 / 10470; WO 91 / 10425; U.S. Pat. No. 5,837,234; U.S. Pat. No. 5,011,472; U.S. Pat. No. 4,892,538).

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

[0070] In certain embodiments, the cells of the present invention can be differentiated into hematopoietic cells for the treatment of various hematopoietic disorders currently treated by bone marrow transplantation. Patients receiving blood transfusions can be refractory to platelet transfusions due to HLA incompatibility. Anemic or cytopenic patients can 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 dendritic cells derived from the stem cells of the invention are antigen presenting cells that can be used as cellular vaccines when appropriately engineered. In certain embodiments, the cells of the invention engineered to express HLA class II proteins or single chain fusion HLA class II proteins, and optionally also single chain fusion HLA class I proteins 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, first pluripotent cells were allowed to form embryoid bodies, and then non-adherent cells were cultured in the presence of hematopoietic cytokines to grow into specific cell lineages. Hematopoietic cell differentiation from the cells of the invention expressing HLA class II proteins or single-chain fusion HLA class II proteins, and optionally also single-chain fusion HLA class I proteins, can be analyzed by flow cytometry and colony assays. Different cell populations are selected based on their surface markers and used to monitor the expression of HLA genes and reactivity with human NK cells and T cells, as measured by Elispot, mixed lymphocyte reaction, and cytotoxicity assays. The effectiveness of single-chain fusion HLA constructs in suppressing NK cell-mediated killing can be examined at different differentiation and transplantation stages. See Bix et al., 1991, Nature 349:329-331. Hematopoietic stem cells can also be assayed using xenograft models, such as immunodeficient mice (SCID repopulating cells or SRC).

[0073] The cells of the invention may be differentiated into hematopoietic cells either before or after administration of the cells to a 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, Slukvin et al., 2006, J Immunol 176:2924-32 and Chang et al., 2006, Blood 108:1515-23.

[0074] In certain other embodiments, the cells of the invention are mesenchymal stem cells. In certain embodiments, the cells of the invention express one or more HLA class II proteins or single chain fusion HLA class II proteins, and optionally also one or more single chain fusion HLA class I proteins. MSCs have the potential to form several differentiated cell types, including bone marrow stromal cells, adipocytes, osteoblasts, and chondrocytes. Thus, inducing pluripotent stem cells to form MSCs (iMSCs) is useful in the treatment of skeletal and joint pathologies. iMSCs can further differentiate into osteoblasts and form bone in vivo. Deyle et al., 2012, Mol Ther. 20(1):204-13. T cell and NK cell cellular responses to ESCs, iMSCs, and their more terminally differentiated derivatives, such as osteoblasts, can be examined.

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

[0076] In yet other detailed embodiments, the cells of the invention may be differentiated into insulin-producing pancreatic islet cells. In a particular embodiment, the cells of the invention express one or more HLA class II proteins or single-chain fusion HLA class II proteins, and optionally also one or more single-chain fusion HLA class I proteins. The cells of the invention may 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, the treatment may be successful at different cell doses, with cells that are not fully differentiated into adult cell types, and also when the cells are transplanted at ectopic sites. Certain autoantigens, such as those derived from GAD65 or insulin, may cause autoimmune destruction of beta cells in diabetes (Di Lorenzo et al., 2007, Clin Exp Immunol 148:1-16). Thus, HLA class II-deficient, HLA class I-deficient, or HLA class I / class II-deficient cells expressing single-chain fusion HLA proteins presenting predetermined peptide antigens offer the added advantage that they do not present these self-antigens and may avoid autoimmune rejection after transplantation to prevent recurrence of diabetes.

[0077] The cells of the invention can be differentiated into pancreatic cells as previously described, using exposure of the cells to various cytokines and drugs to promote the sequential formation of mesendoderm, definitive endoderm, and pancreatic progenitor cells (Kroon et al., 2008, Nat Biotechnol 26:443-452). These cells can be further cultured in explants in immunodeficient mice. Wild-type cells and the cells of the invention expressing or not expressing one or more single-chain fusion HLA class II proteins or one or more single-chain fusion HLA class II proteins and optionally also one or more single-chain 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 invention are differentiated into pancreatic islet 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 performed according to established protocols. See, for example, Kroon et al., 2008, Nat Biotechnol 26:443-452.

[0079] In certain other detailed embodiments, the cells of the invention can be differentiated into cardiomyocytes. In certain embodiments, the cells of the invention further express one or more HLA class II proteins or single chain fusion HLA class II proteins, and optionally also one or more single chain fusion HLA class I proteins. Common clinical problems of myocardial infarction and congestive heart failure can be treated by transplanting and engrafting healthy stem cell derived cardiomyocytes to reconstitute a functional myocardium. Cardiomyocytes derived from the cells of the invention allow these treatments to proceed with prepackaged cells, avoiding the immunosuppression required for current allogeneic heart transplants. Physiologically relevant tests, such as electrical conduction and contraction tests, can be performed on cardiomyocytes derived from the cells of the 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 chain fusion HLA class I proteins can be tested to determine their immunological reactivity when expressing cardiomyocyte genes and establish which HLA modifications minimize such immune responses.

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

[0081] In yet other detailed embodiments, the cells of the invention may be differentiated into keratinocytes. In certain embodiments, the cells of the invention used for differentiation into keratinocytes express one or more single chain fusion HLA class II proteins, and optionally also one or more HLA class II proteins or single chain fusion HLA class I proteins. Severe burns and genetic skin conditions require treatment by skin grafting, which is currently done with various cell sources, such as pig skin grafts and cultured autologous human keratinocytes. Keratinocytes derived from the cells of the invention may provide a significant clinical advance, since burns may be first-aided with prepackaged cells, and genetic diseases such as epidermolysis bullosa may 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 genetic mutation. In many cases, the cells only need to remain engrafted long enough for adjacent host cells to repopulate the affected area. The cells of the invention may be embedded in a polyvinyl alcohol sponge (PVA)-collagen gel implant for transplantation into a recipient for in vivo differentiation. The cells of the invention may 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 aspect, the invention provides use of the cells of the invention to prepare a medicament for transplantation. In a related aspect, the invention provides use of the cells of the invention to prepare a medicament for the treatment of a disease condition.

[0083] Furthermore, the cells of the invention may serve as research tools, providing a system for studying the function of immunomodulatory proteins in HLA class II-deficient, HLA class I-deficient, or HLA class I / class II-deficient genetic backgrounds. In certain embodiments, the cells of the invention further express one or more HLA class II proteins or single-chain fusion HLA class II proteins, and optionally also one or more single-chain fusion HLA class I proteins. Thus, in a related aspect, the invention provides a method for determining the function of an immunomodulatory protein, comprising the steps of introducing one or more immunomodulatory genes into the cells of the invention and assaying the activity of the immunomodulatory genes. In certain preferred embodiments, the cells are human cells. For example, the cells of the invention can be used to study the function of immunomodulatory genes or to study immune responses in the absence of unwanted HLA class II antigens or HLA class I / class II antigens. In a further related aspect, the invention provides a method for identifying a compound or molecule that modulates the function of an immunomodulatory protein, the method comprising contacting an HLA class II deficient cell, an HLA class I deficient cell, or an HLA class I / class II deficient cell that contains one or more immunomodulatory genes with the compound or molecule of interest and assaying the activity of the immunomodulatory gene. In certain preferred embodiments, the cell is a human cell.

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

[0085] In yet another aspect, the invention provides an in vivo research tool in a mammal, particularly a non-human primate, to which the cells of the invention are administered, to study the function of immune-modulating genes in an HLA class II-deficient, HLA class I-deficient, or HLA class I / class II-deficient genetic background, or to identify compounds that modify the function of immune-modulating genes in the administered cells. In certain embodiments, the cells of the invention further express one or more HLA class II proteins or single-chain fusion HLA class II proteins, and optionally also one or more single-chain fusion HLA class I proteins.

[0086] Mice, especially immunodeficient mice, have been used as a model system for in vivo studies of human cells. Human stem cells may behave differently in mice. In addition, the mouse immune system differs from the human immune system in HLA class II genes, NK cell receptors, and non-classical MHC class I genes (e.g., HLA-E, F, and G). Thus, a Macaca nemestrina (Mn, pig-tailed macaque) model may be developed for the study of the cells of the present invention. The Macaca mulatta genome has been sequenced and has a high degree of homology with the nemestrina genome. Furthermore, the organization of the macaque MHC locus is similar to human HLA, including the non-classical genes. Homologs 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 deficient, HLA class I deficient, or HLA class I / class II deficient ESCs can be used for transplantation in macaques.

[0087] The same AAV-mediated gene targeting strategy described for human cells can be used to develop MHC class II deficiency in MHC class I / class II deficient macaque ESCs. Single chain fusion HLA class II proteins and optionally Mn versions of single chain 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 vector expressing GFP for subsequent identification of transplanted cells. Cells can be embedded in polyvinyl alcohol sponge (PVA)-collagen gel implants and placed subcutaneously in macaques. The implants can be harvested, sectioned and stained to determine the cell types present. Specific antibodies can be used to identify differentiated cell types formed by the transplanted cells.

[0089] Any embodiment described above applies to any aspect of the invention unless the context clearly dictates otherwise. All embodiments within and between the various aspects can be combined unless the context clearly dictates otherwise.

[0090] (Example) Example 1 Construction of human embryonic stem cells carrying a knockout mutation in the RFXANK gene FIG. 1 shows the structure 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 the vectors). By selecting vector-infected cells with G418 or hygromycin (Hygro), it is possible to isolate cells targeted by the TKNeo or HyTK vector, respectively. Subsequent expression of Cre recombinase and selection with ganciclovir (GCV) then allows the isolation of clones in which the TKNeo or HyTK gene has been removed, leaving behind two inactivated RFXANK alleles with stop codons in all three reading frames, loxP sites, and polyadenylation sites (StopX3-loxP-pA). loxP are recombination sites for Cre recombinase. ITR are vector inverted terminal repeats. Similar vectors can be designed to target 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 for targeting by PCR. The PCR used a forward primer that is homologous to the neomycin sequence of the selection cassette, as shown by the arrow above, and a reverse primer that is homologous to the RFXANK gene outside the targeting homology arm. As shown in Figure 2, five positive clones of the correct size were shown above among the 40 clones screened, resulting in a targeting frequency of 12.5%. (Additional Note) As a preferred embodiment, the technical concept that can be understood from the above embodiment will be described. [Item 1] An isolated cell that contains a genetically engineered disruption in a human leukocyte antigen (HLA) class II associated gene, the cell being a primate cell. [Item 2] The HLA class II-related gene is selected from the group consisting of regulatory factor X-related ankyrin-containing protein (RFXANK) (SEQ ID NOs: 24 to 27), regulatory factor 5 (RFX5) (SEQ ID NOs: 28 to 31), regulatory factor X-related protein (RFXAP) (SEQ ID NOs: 32 to 33), class II transactivator (CIITA) (SEQ ID NOs: 34 to 35), HLA-DPA (α chain) (SEQ ID NOs: 36 to 37), HLA-DPB (β chain) (SEQ ID NOs: 38 to 39), and HLA-DPA (α chain) (SEQ ID NOs: 39 to 40). The cell according to item 1, wherein the HLA-specific nucleotide sequence is selected from the group consisting of HLA-DQA (SEQ ID NOs: 40 to 41), HLA-DQB (SEQ ID NOs: 42 to 43), HLA-DRA (SEQ ID NOs: 44 to 45), HLA-DRB (SEQ ID NOs: 46 to 47), HLA-DMA (SEQ ID NOs: 48 to 49), HLA-DMB (SEQ ID NOs: 50 to 51), HLA-DOA (SEQ ID NOs: 52 to 53) and HLA-DOB (SEQ ID NOs: 54 to 55). [Item 3] 3. The cell of item 2, comprising a genetically engineered disruption in at least two, at least three, or all four of the HLA class II associated genes. [Item 4] 4. The cell according to any one of items 1 to 3, wherein the HLA class II-associated gene is regulatory factor X-related ankyrin-containing protein (RFXANK) (SEQ ID NOs: 24 to 27). [Item 5] 5. The cell according to any one of items 1 to 4, comprising a genetically engineered disruption in all copies of the HLA class II associated gene. [Item 6] 6. The cell according to any one of items 1 to 5, further comprising one or more recombinant immunomodulating genes, each capable of expressing an immunomodulating polypeptide in a human cell. [Item 7] 7. The cell according to any one of items 1 to 6, wherein the one or more immune modulating genes comprise a single chain fusion HLA class II protein, or a polynucleotide capable of encoding an HLA class II protein. [Item 8] 8. The cell according to any one of items 1 to 7, further comprising a genetically engineered disruption in the β2 microglobulin (B2M) gene (SEQ ID NO: 1). [Item 9] (a) a genetically engineered disruption in the β2 microglobulin (B2M) gene (SEQ ID NO:1); (b) one or more polynucleotides capable of encoding a single chain fusion HLA class II protein or an HLA class II protein; 1. An isolated cell comprising: The cells are primate cells. [Item 10] 10. The cell of any one of items 1 to 9, comprising a genetically engineered disruption of all copies of the B2M gene. [Item 11] 11. The cell according to any one of items 7 to 10, wherein the one or more immune modulating genes comprise one or more single-chain fusion HLA class II proteins, the one or more single-chain fusion HLA class II proteins comprising at least a portion of an HLA class II gene α chain covalently linked to at least a portion of an HLA class II gene β chain, and the HLA class II genes are selected from the group consisting of HLA-DP (SEQ ID NOs: 36 to 39), HLA-DQ (SEQ ID NOs: 40 to 43), HLA-DR (SEQ ID NOs: 44 to 47), HLA-DM (SEQ ID NOs: 48 to 51), and HLA-DO (SEQ ID NOs: 52 to 55). [Item 12] 12. The cell of claim 11, wherein the single chain fusion HLA class II protein comprises a plurality of different single chain fusion HLA class II proteins. [Item 13] 13. The cell according to item 11 or 12, wherein the single chain fusion HLA class II protein comprises at least a portion of an HLA-DQ alpha chain (SEQ ID NO: 41) and at least a portion of an HLA-DQ beta chain (SEQ ID NO: 43). [Item 14] The single chain fusion HLA class II protein is an HLA-DQ α chain allele HLA-DQA1 * 01 (SEQ ID NO: 41) and at least a portion of the HLA-DQ beta chain allele HLA-DQB1 *14. The cell according to any one of items 11 to 13, comprising at least a portion of sequence ID No. 02 (SEQ ID NO: 43). [Item 15] 15. The cell according to any one of items 11 to 14, wherein the single-chain fusion HLA class II protein presents a first target peptide antigen on the cell surface. [Item 16] 16. The cell of claim 15, wherein the first target peptide antigen is covalently linked to the single chain fusion HLA class II protein. [Item 17] 11. The cell according to any one of items 1 to 10, wherein the one or more immune modulating genes comprise one or more HLA class II proteins, and the one or more HLA class II proteins are selected from the group consisting of HLA-DM α chain, HLA-DM β chain, HLA-DO α chain, HLA-DO β chain, HLA-DP α chain, HLA-DP β chain, HLA-DQ α chain, HLA-DQ β chain, HLA-DR α chain and HLA-DR β chain. [Item 18] 17. The cell according to any one of items 8 to 16, further comprising a polynucleotide capable of encoding a single-chain fusion HLA class I protein. [Item 19] 19. The cell of item 18, wherein the single chain fusion HLA class I protein comprises at least a portion of B2M (SEQ ID NO: 2) covalently linked to at least a portion of an HLA class I alpha chain, the HLA class I alpha chain being 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] 20. The cell of item 18 or 19, wherein the single chain fusion HLA class I protein comprises at least a portion of B2M (SEQ ID NO: 2) covalently linked to at least a portion of HLA-A (SEQ ID NO: 3). [Item 21] 21. The cell according to any one of items 18 to 20, wherein the single chain fusion HLA class I protein comprises at least a portion of B2M covalently linked to at least a portion of HLA-A0201 (SEQ ID NO: 4). [Item 22] 22. The cell according to any one of items 18 to 21, further expressing a second target peptide antigen presented by the single-chain fusion HLA class I protein on the cell surface. [Item 23] 23. The cell of claim 22, wherein the second target peptide antigen is covalently linked to the single chain fusion HLA class I protein. [Item 24] 24. The cell of any one of items 1 to 23, further comprising one or more recombinant genes capable of encoding a suicide gene product. [Item 25] 25. The cell of item 24, wherein the suicide gene product comprises a protein selected from the group consisting of thymidine kinase and apoptosis signaling protein. [Item 26] 26. The cell according to any one of items 1 to 25, having a normal karyotype. [Item 27] 27. The cell according to any one of items 1 to 26, which is a non-transformed cell. [Item 28] 28. The cell according to any one of items 1 to 27, which is a stem cell. [Item 29] 29. The cell according to item 28, wherein the stem cell is 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] 30. The cell according to item 28 or 29, wherein the stem cell is a pluripotent stem cell. [Item 31] 31. The cell according to any one of items 28 to 30, wherein the stem cell is differentiated. [Item 32] 32. The cell according to item 31, wherein the differentiated cell is selected from the group consisting of dendritic cells, pancreatic islet cells, hepatic cells, muscle cells, keratinocytes, nerve cells, hematopoietic cells, lymphocytes, red blood cells, platelets, skeletal muscle cells, eye cells, mesenchymal cells, fibroblasts, lung cells, GI tract cells, vascular cells, endocrine cells, adipocytes and cardiomyocytes. [Item 33] 32. The cell according to any one of items 1 to 31, which is a human cell. [Item 34] A vaccine comprising the cell according to any one of items 16 to 33, A vaccine capable of inducing an immune response in a primate specific to at least one of a first target peptide antigen and a second target peptide antigen. [Item 35] 35. The vaccine of item 34, wherein the primate is a human and the cells are human cells. [Item 36] 1. A method of transplantation in a patient in need thereof, comprising: A method comprising the step of administering an effective amount of the cell or vaccine according to any one of items 1 to 35 to the patient. [Item 37] 37. The method of claim 36, wherein the patient is immunocompetent. [Item 38] 38. The method of item 36 or 37, wherein the cells or vaccine comprise differentiated cells. [Item 39] 39. The method of any one of items 36 to 38, wherein the patient is a human and the cells are human cells.

Claims

1. An isolated cell comprising a genetically engineered disruption in a human leukocyte antigen (HLA) class II associated gene, (a) the HLA class II-associated gene is a gene encoding an HLA class II-associated protein selected from the group consisting of regulatory factor X-related ankyrin-containing proteins (RFXANK) of SEQ ID NOs: 24 to 27, regulatory factor 5 (RFX5) of SEQ ID NOs: 28 to 31, regulatory factor X-related proteins (RFXAP) of SEQ ID NOs: 32 to 33, and class II transactivator (CIITA) of SEQ ID NOs: 34 to 35; (b) the cell further comprises a genetically engineered disruption in the beta 2 microglobulin (B2M) gene of SEQ ID NO:1; (c) the cell further comprises a polynucleotide encoding a single chain fusion HLA class I protein, the single chain fusion HLA class I protein comprising at least a portion of B2M of SEQ ID NO:2 covalently linked to at least a portion of an HLA class I α chain; said single chain fusion HLA class I protein is capable of forming the proper secondary structure of a heterodimer on the cell surface, presenting peptides in the peptide binding groove, and exerting normal HLA class I functions with respect to binding to inhibitory receptors on the surface of NK cells; (d) the cells optionally further comprise one or more recombinant immunomodulating genes each capable of expressing an immunomodulating polypeptide, the one or more immunomodulating polypeptides being single-chain fusion HLA class II proteins; said single chain fusion HLA class II protein, if present, comprises at least a portion of an HLA class II gene alpha chain covalently linked to at least a portion of an HLA class II gene beta chain, said HLA class II genes being selected from the group consisting of HLA-DP of SEQ ID NOs: 36-39, HLA-DQ of SEQ ID NOs: 40-43, HLA-DR of SEQ ID NOs: 44-47, HLA-DM of SEQ ID NOs: 48-51, and HLA-DO of SEQ ID NOs: 52-55; (e) the single chain fusion HLA class I protein and / or the single chain fusion HLA class II protein present one or more peptide antigens on the cell surface, the peptides comprising a target peptide antigen; cell.

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

3. A cell described in claim 1 or 2, wherein the HLA class I α chain is selected from the group consisting of HLA-A of sequence number 4, HLA-B of sequence number 6, and HLA-C of sequence number 8.

4. A cell described in claim 1 or 2, wherein the HLA class I α 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. A cell described in claim 1 or 2, wherein the HLA class I α chain is HLA-E of sequence number 10.

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

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

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

9. A 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 kinase and apoptosis signalling protein.

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

11. A cell described in any one of claims 1 to 10, wherein the cell is a non-transformed cell.

12. The cell described in 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 described in claim 12, wherein the stem cell is a pluripotent stem cell.

14. A cell described in any one of claims 1 to 12, wherein the cell is a differentiated cell.

15. The cell of claim 14, wherein the differentiated cell is selected from the group consisting of dendritic cells, pancreatic islet cells, hepatic cells, muscle cells, keratinocytes, nerve cells, hematopoietic cells, lymphocytes, red blood cells, platelets, skeletal muscle cells, eye cells, mesenchymal cells, fibroblasts, lung cells, GI tract cells, vascular cells, endocrine cells, adipocytes, and cardiomyocytes.

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

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

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

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

20. A kit comprising a cell described in any one of claims 1 to 15.

21. The cells described in any one of claims 1 to 15, wherein the cells are for use in transplantation in a patient in need of a transplant, and the use comprises administering an effective amount of the cells to the patient.

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

23. A cell for use as described in claim 21 or 22, wherein the cell is a differentiated cell.

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

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