β2 microglobulin-deficient cells

Abstract

To provide β2 microglobulin-deficient cells.SOLUTION: The invention provides isolated cells having a genetic modification through disruptive recombination in β2 microglobulin (B2M) gene, and the cells further comprising one or more recombinant immunomodulatory genes comprising a polynucleotide capable of encoding single chain fusion human leukocyte antigen (HLA) class I protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to β2 microglobulin-deficient cells. [Background technology]

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

[0003] The major histocompatibility complex (MHC) is a cell surface multicomponent molecule found in all vertebrates that mediates the interaction of leukocytes with other leukocytes or other cells. The MHC gene family is divided into three groups: class I, class II, and class III. In humans, MHC is called human leukocyte antigens (HLA). HLA class I (HLA-I) proteins are expressed in all nucleated cells and consist of HLA class I heavy chains (or α chains) and β2 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 (HLA-A, HLA-B, and HLA-C) α chains and three nonclassical (HLA-E, HLA-F, and HLA-G) α chains. The specificity of peptides binding to the HLA class I molecule peptide-binding cleft is determined by the α chain. Recognition of peptides presented by HLA class I molecules by CD8+ T cells mediates cell-mediated immunity.

[0004] HLA class I proteins from allogeneic sources themselves constitute foreign antigens in the context of transplantation. Recognition of non-self HLA class I proteins is a major obstacle to using pluripotent cells for transplantation or replacement therapy. The first two clinical trials of human embryonic stem cells (ESCs) were conducted, delivering ESCs to immune-privileged sites (e.g., spinal cord and eye) where xenogeneic cells may remain. However, most potential clinical applications do not involve immune-privileged sites, because even these immune-privileged sites will eventually reject the xenogeneic cells. Alternatively, HLA-matched or partially matched cells from HLA-type stem cell banks, or pluripotent stem cell (iPSC) lines derived from each patient can be generated for transplantation. However, the generation of an individually matched cell line requires significant costs, months of cell culture, highly trained staff, and thorough validation of the final product, all of which must be done after regulatory approval. Moreover, each cell line is likely to behave somewhat differently in gene expression patterns, culture characteristics, differentiation potential, and genetic mutations. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, although individualized stem cell preparations or HLA-diverse stem cell banks could address the current transplantation problem, they require multiple cell lines to be characterized, differentiated into therapeutic cell products, and approved for administration to humans. This time-consuming, technically challenging, and costly process is the main reason that stem cell-based therapies have not entered clinical trials. Thus, there is a need for more effective, low-cost cell-based therapies that are not hindered by rejection. [Means for solving the problem]

[0006] According to the invention, in one aspect, the invention provides an isolated primate cell comprising a genetically engineered disruption in the β2 microglobulin (B2M) gene. In certain embodiments, the cell comprises a genetically engineered disruption of all copies of the B2M gene.

[0007] In certain other embodiments, the cell further comprises one or more recombinant immunomodulatory genes. Suitable immunomodulatory genes include, but are not limited to, genes encoding viral proteins that inhibit antigen presentation, microRNA genes, and genes encoding single-chain (SC) fusion human leukocyte antigen (HLA) class I proteins, as described below. In certain preferred embodiments, the primate cell is a human cell.

[0008] In certain preferred embodiments, the one or more immune-modulating genes comprise a polynucleotide capable of encoding a single-chain fusion HLA class I protein. In certain embodiments, 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 certain preferred embodiments, the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of an HLA class I α chain selected from the group consisting of HLA-C, HLA-E, and HLA-G. In certain other preferred embodiments, the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of an HLA class I α chain selected from the group consisting of HLA-A, HLA-E, and HLA-G. In certain embodiments, the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A0201 (e.g., SEQ ID NO: 16). In certain other specific embodiments, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-E (eg, SEQ ID NOs: 18 and 20).

[0009] In yet other specific embodiments, the cell has a normal karyotype. In other certain embodiments, the cell is a non-transformed cell. In particular, the cell is a stem cell 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. In further certain embodiments, the cell further comprises one or more recombinant genes that can code for a suicide gene product. In certain embodiments, the suicide gene product comprises a protein selected from the group consisting of thymidine kinase and apoptosis signaling protein.

[0010] In certain preferred embodiments, the stem cells are pluripotent stem cells that express a single-chain fusion HLA class I protein comprising at least a portion of B2M and at least a portion of an HLA class I alpha chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In certain embodiments, the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A0201.

[0011] In certain other embodiments, the stem cell is a differentiated cell. In certain embodiments, the differentiated cell is selected from the group consisting of 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 cells, vascular cells, endocrine cells, adipocytes, and cardiomyocytes. In certain preferred embodiments, the differentiated cell is a human cell that expresses a single-chain fusion HLA class I protein comprising at least a portion of B2M and at least a portion of an HLA class I alpha chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In certain embodiments, the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A0201.

[0012] In certain other embodiments, the cell further expresses a target peptide antigen that is presented on the surface of the cell by a single-chain fusion HLA class I protein. In certain embodiments, the target peptide antigen is covalently linked to the single-chain fusion HLA class I protein. In certain preferred embodiments, the target peptide antigen is derived from a protein of a pathogen or a cancer cell. Thus, in a related aspect, the invention provides a vaccine comprising a B2M- / - cell of the invention, which is capable of eliciting an immune response in a primate specific for the target peptide antigen. In certain embodiments, the vaccine comprises a cell of the invention that is a differentiated dendritic cell. In certain other embodiments, the cell is a human cell of the invention, which expresses a cytokine that further enhances the immune response. In certain preferred embodiments, the cytokine is IL2. In certain other preferred embodiments, the cytokine is IFN-γ. In certain embodiments, the immune response comprises a humoral immune response; however, in other embodiments, the immune response comprises a cellular immune response. In a further related aspect, the invention provides a kit comprising a vaccine comprising the isolated cell of the invention and an immune adjuvant. In certain embodiments, the cell is a human cell.

[0013] In yet another aspect, the present invention provides a method of transplantation to a patient in need of transplantation, comprising administering to the patient an effective amount of the isolated cell of the present invention.In some embodiments, the patient is immunocompetent.In certain embodiments, the patient is a primate, preferably a human.In some preferred embodiments, the patient is a human, and the cell is a human cell.In further embodiments, the cell is a stem cell or a differentiated cell, optionally expressing a single-chain fusion HLA class I protein.

[0014] In yet a further aspect, the present invention provides a method for treating a condition in a patient in need of such treatment, comprising administering to the patient an effective amount of the B2M- / - cells of the present invention, the condition including, but not limited to, endocrine disorders, diabetes, autoimmune diseases, cancer, infectious diseases, anemia, platelet disorders, immunodeficiency, leukopenia, myocardial infarction, heart failure, liver failure, skeletal or joint conditions, neurological diseases, stroke, paralysis, blindness or other visual impairments, muscular dystrophy, osteogenesis imperfecta, lung diseases, skin diseases, or burns. In certain embodiments, the patient is immunocompetent. In certain embodiments, the patient is a primate, preferably a human. In certain preferred embodiments, the patient is a human, and the cell is a human cell. In further embodiments, the cell is a stem cell or a differentiated cell, optionally expressing a single-chain fusion HLA class I protein. In certain embodiments, the condition is diabetes, and the cell is a differentiated pancreatic islet cell. In a further embodiment, the differentiated pancreatic islet cells express a single chain fusion HLA class I protein.

[0015] In another aspect, the invention provides a kit comprising the isolated primate cells, preferably human cells, of the invention. In certain embodiments, the kit is used for transplantation or for use in treating a medical condition. In other certain embodiments, the kit comprises a graft comprising the isolated primate cells, preferably human cells, of the invention.

[0016] Specific embodiments of the present invention will become apparent from the following more detailed description of certain preferred embodiments and the appended claims. [Brief description of the drawings]

[0017] [Figure 1]FIG. 1 shows the generation of B2M- / -ESCs. FIG. 1A is an illustration of an AAV B2M targeting vector with exons shown in large boxes. FIG. 1B shows Southern blots showing the results of gene targeting and Cre-mediated transgene excision (Cre-out). FIG. 1C shows the results of flow cytometry (using isotype controls) showing the lack of HLA class I expression after gene targeting. [Diagram 2] Histological sections of teratomas developed from B2M- / -Cre-out human ESCs transplanted into immunodeficient mice were stained with DAPI, hematoxylin, and eosin, or lineage-specific markers MAP-2 (microtubule-associated protein 2) for ectoderm, α-SAM (α-smooth muscle actin) for mesoderm, or FoxA2 (forkhead box protein A2) for endoderm. Scale bar = 100 microns. [Diagram 3] Figure 3 shows single chain fusion HLA class I constructs. Figure 3A shows the design of foamy virus vectors for expressing single chain fusion HLA class I proteins. Figure 3B illustrates the linear protein structure of single chain fusion HLA class I proteins. Exemplary single chain fusion HLA class I protein sequences are shown for HLA-bGBE (SEQ ID NOs: 19 and 20, DNA and protein sequences, respectively), HLA-gBE (SEQ ID NOs: 17 and 18), and HLA-bBA0201 (SEQ ID NOs: 15 and 16). Figure 3C shows the results of flow cytometry (isotype control) showing single chain fusion HLA-E expression in B2M- / -ESCs. Figure 3D shows the results of flow cytometry (isotype control) showing single chain fusion HLA-A0201 expression in B2M- / -ESCs. [Figure 4] The experimental design for each differentiated cell type is outlined. [Diagram 5] 1 shows differentiation of B2M− / − ESC-derived keratinocytes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference for all purposes. In this application, unless otherwise indicated, the techniques utilized are generally described in detail in a number of well-known references, such as "Molecular Cloning: A Laboratory Manual" (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) and "PCR Protocols: A Guide to Methods and Applications." The literature can be found in either "Innis et al., Academic Press, San Diego, CA, 1990" or "Theoretical Modeling for Quantum Computational Biology: A Guide to Methods and Applications."

[0019] As used herein, the singular forms "a," "an," and "the" include plural referents unless specifically stated otherwise. For example, "an isolated cell" means one or more isolated cells.

[0020] All embodiments disclosed herein can be combined unless otherwise stated. In one aspect, the invention provides a B2M-deficient cell. In particular, the invention provides an isolated primate cell, preferably a human cell, comprising a genetic modification in the B2M gene. In certain preferred embodiments, the cell is a human cell comprising a genetic modification in the B2M gene. In a related aspect, the cell comprises a genetic modification in all copies of the B2M gene. In certain embodiments, the genetic modification of the B2M gene results in a lack of B2M protein or in the absence of expression of B2M protein. Since B2M is a common element of all HLA class I proteins, the genetic modification prevents the expression of all native HLA class I proteins at the cell surface. The sequence encoding B2M 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 may be a large number of single nucleotide polymorphisms (SNPs) in this gene; as will be appreciated by those skilled in the art, the human cells and methods of the invention are applicable to any such B2M genes and SNPs.

[0021] The cells of these embodiments of the present invention can be used, for example, as donor cells for transplantation of recipients in need of transplantation. B2M-deficient cells include cells that contain a B2M- / - genetic background (B2M- / - cells). The term "B2M- / - cells" refers to primate cells, preferably human cells, that contain genetically engineered modifications in all copies of the B2M gene. B2M- / - cells can function as "universal donor cells" in that they are immunologically compatible with all or most of the recipients in a population. A recipient or patient as used herein refers to a primate, preferably a human. In certain embodiments, the cells are human cells and the patient is a human.

[0022] The cells of the present invention can be genetically engineered to modify the B2M gene so that no functional endogenous B2M protein is produced from the modified locus. In certain embodiments, the modification results in the expression of a non-functional B2M protein, including but not limited to truncations, deletions, point mutations, and insertions. In other embodiments, the modification results in no protein being expressed from the B2M gene at all.

[0023] Cells that lack B2M expression cannot express HLA class I protein on cell surface.The lack of HLA class I provides additional advantages; for example, cells that do not express HLA class I cannot present self-antigens that would otherwise prevent the success of cell therapy for autoimmune diseases, such as diabetes and rheumatoid arthritis.Similarly, therapeutic gene products (e.g., dystrophin) that are introduced by the cell therapy of the present invention, which are missing in patients with certain genetic diseases (e.g., muscular dystrophy), are also not presented and are not recognized as neo-antigens in replacement therapy.

[0024] Any suitable technique for modifying one, two, or all copies of the B2M gene can be used; exemplary techniques are disclosed throughout this application and are within the level of ordinary skill in the art based on the teachings herein and known in the art. Other exemplary techniques can be found, for example, in U.S. Provisional Patent Application No. 2008 / 0219956, published Sep. 11, 2008, which is incorporated herein by reference in its entirety. These techniques can optionally include a step of removing non-human DNA sequences from cells after modification of the B2M gene.

[0025] An exemplary embodiment of this method includes using an adeno-associated virus gene targeting vector as disclosed throughout this application, and optionally removing the transgene used for targeting by techniques such as those described below, or by Cre-mediated loxP recombination or other suitable recombination techniques. See Khan et al., Protocol, 2011, vol. 6, pp. 482-501, which is incorporated herein by reference in its entirety. Exemplary targeting vectors and diagrams of exemplary vectors are also disclosed herein. Utilizing various techniques to generate the B2M- / - cells, preferably human cells, of the present invention is within the level of skill of one of ordinary skill in the art based on the teachings herein and known in the art.

[0026] In certain embodiments, the cellular genome of the B2M- / - cell may contain less than 100, less than 50, or less than 30 nucleotides of non-human DNA sequence. In other certain embodiments, the cellular genome may contain 6, 5, 4, 3, 2, 1, or 0 nucleotides of non-human DNA sequence. An exemplary technique for removing any non-human DNA introduced in the modification of the B2M gene is shown in FIG. 1A. The non-human DNA sequence may be removed by a second round of targeting to remove the HyTK or TKNeo transgene in the first vector, or by Cre-mediated loxP recombination.

[0027] In other embodiments, the cells can instead be engineered to recombinantly express single-chain fusion HLA class I proteins in a B2M- / - genetic background. Thus, B2M- / - cells as used herein also encompass primate cells, preferably human cells, that express one or more single-chain fusion HLA class I proteins in a B2M- / - genetic background. B2M- / - cells that recombinantly express single-chain fusion HLA class I proteins are nevertheless deficient in normal B2M function, such that the cells do not express wild-type B2M proteins that form heterodimers non-covalently bound to any HLA class I α chain on the cell surface.

[0028] 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 that includes 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 linked heterodimer of B2M and an HLAα chain that is expressed on the surface of wild-type cells.

[0029] 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, but are not limited to, 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 shown 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_00507, 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).

[0030] In addition, the term "HLA class I protein / molecule" is known to refer to human MHC class I proteins / molecules, and the terms HLA and MHC are sometimes used interchangeably throughout this application: for example, the term HLA class I protein can also be used to refer to the primate equivalents of primate HLA class I proteins. A person skilled in the art will be able to understand the meaning of the terms based on the context.

[0031] The term B2M- / - cell as used herein also encompasses cells with genetic modification in all copies of the B2M gene, where one B2M allele is genetically modified to express single-chain fusion HLA class I protein instead of wild-type B2M protein (i.e., gene-targeted knock-in in one B2M allele). B2M- / - cells with such a genetic background express B2M from the B2M locus only in the context of single-chain fusion HLA class I protein. In certain advantageous embodiments, the expression of single-chain fusion HLA class I protein is regulated by the endogenous B2M regulatory sequence located in the B2M locus.

[0032] In related embodiments, B2M- / - cells further encompass cells with genetic modifications in all copies of the B2M gene, where all B2M alleles have been genetically modified to express a single-chain fusion HLA class I protein instead of wild-type B2M protein (i.e., gene-targeted knock-in in all B2M alleles). B2M- / - cells with such genetic modifications express B2M from all allelic loci of the B2M gene only in the context of a single-chain fusion HLA class I protein. In certain embodiments, the cells are genetically modified to express the same type of single-chain fusion HLA class I protein from all allelic loci of the B2M gene; however, in other embodiments, the cells are genetically modified to express different types of single-chain fusion HLA class I proteins from different allelic loci of the B2M gene.

[0033] Throughout this application, "cells of the invention", "isolated cells of the invention", "B2M- / - cells", "B2M- / - cells of the invention", or "stem or differentiated cells of the invention" are sometimes used interchangeably and may encompass all B2M- / - cells described herein. In certain embodiments, the B2M- / - cells of the invention express a single chain fusion HLA class I protein as defined herein in a B2M- / - background. The B2M- / - cells can be genetically engineered to express a single chain HLA class I protein from the B2M locus or other locations in the genome. In certain embodiments, the cells of the invention contain a genetically engineered modification in an allele of the B2M gene that prevents expression of wild-type B2M protein, but still expresses a single chain fusion HLA class I protein from the B2M locus. In other certain embodiments, the cells of the invention contain a genetically engineered modification in all alleles of the B2M gene that prevents expression of wild-type B2M protein, but still expresses a single chain fusion HLA class I protein from all B2M loci. The terms "gene," "allele," and "locus" may be used interchangeably throughout this application.

[0034] An "isolated cell" can be any cell type appropriate for a given purpose. For example, the cell can be a pluripotent stem cell or a differentiated cell. "Stem cell" broadly encompasses all cells that can be further differentiated. "Pluripotent stem cells" are stem cells that have the ability to differentiate into any of the three germ layers: endoderm, mesoderm, or ectoderm. "Adult stem cells," on the other hand, are pluripotent in that they can only produce a limited number of cell types. "Embryonic stem (ES) cells" are pluripotent stem cells derived from the blastocyst, the inner cell mass of an early embryo. "Induced pluripotent stem cells (iPS cells)" are pluripotent stem cells artificially created from non-pluripotent cells, typically adult somatic cells, by artificially inducing the expression of certain genes.

[0035] In certain embodiments, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G (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 comprise the leader sequence (or signal sequence) of the HLA class I α chain (leaderless HLA α chain). In other certain embodiments, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-C, HLA-E, or HLA-G. In further certain embodiments, the single chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A, HLA-E, or HLA-G. In certain preferred embodiments, the single-chain fusion HLA class I protein comprises a leader sequence (or signal sequence) covalently linked to at least a portion of B2M and at least a portion of an HLA alpha chain that ensures proper folding of the single-chain fusion HLA class I protein at the cell surface. The leader sequence can be the leader sequence of a B2M protein, the leader sequence of an HLA alpha chain protein, or the leader sequence of another secreted protein. In certain embodiments, the single-chain fusion HLA class I protein comprises a B2M protein with the leader sequence removed. In other certain embodiments, the single-chain fusion HLA class I protein comprises an HLA alpha chain protein with the leader sequence removed. Certain HLA class I alpha chains are highly polymorphic. As will be appreciated by those skilled in the art, the human cells and methods of the present invention are applicable to any such HLA alpha chain and polymorphisms thereof.

[0036] Single chain fusion HLA class I proteins comprising sequence variants and fragments of B2M and / or HLA alpha chains are encompassed by the present invention, and such single chain fusion constructs still have normal HLA class I functions, such as forming the proper secondary structure of the heterodimer on the cell surface, presenting peptides to peptide binding clefts, and binding to inhibitory receptors on the surface of NK cells. In certain embodiments, the sequence variants have 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, and the sequence variants have normal HLA class I functions. In certain other embodiments, the sequence variant has at least 75%, 80%, 81%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence identity to the B2M or HLA heavy chain sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, or 14.

[0037] In certain embodiments, the HLA-A variants have at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or complete sequence homology with SEQ ID NO: 4. In other certain embodiments, the HLA-B variants have 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 further certain embodiments, the HLA-C variants have 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 has at least 97%, 98%, 99%, or complete sequence identity to SEQ ID NO: 10. In certain embodiments, the HLA-F variant has at least 99% or complete sequence identity to SEQ ID NO: 12. In certain other embodiments, the HLA-G variant has at least 98%, 99%, or complete sequence identity to SEQ ID NO: 14.

[0038] In certain other embodiments, the single chain fusion HLA class I protein comprises full-length B2M (including its leader sequence) and an HLA alpha chain without a leader sequence (leaderless HLA alpha chain); while in certain other embodiments, the single chain fusion HLA class I protein comprises a B2M protein without a leader sequence. It is understood that B2M- / - cells expressing two, three or more different types of single chain fusion HLA class I proteins in any combination are all encompassed by the present invention, for example B2M- / - cells expressing an SC fusion comprising HLA-A (or leaderless HLA-A) and an SC fusion comprising HLA-C (or leaderless HLA-C), B2M- / - cells expressing an SC fusion comprising HLA-A (or leaderless HLA-A) and an SC fusion comprising HLA-E (or leaderless HLA-E), or B2M- / - cells expressing an SC fusion comprising HLA-B (or leaderless HLA-B), an SC fusion comprising HLA-E (or leaderless HLA-E), and an SC fusion comprising HLA-G (or leaderless HLA-G).

[0039] Natural killer (NK) cells are part of the innate immune response. Some pathogens can downregulate the expression of HLA class I proteins in infected cells. NK cells monitor infection by recognizing cells that do not express HLA class I proteins and inducing apoptosis. Inhibitory receptors on the surface of NK cells recognize HLA class I α-chain alleles, thereby preventing NK-induced apoptosis of uninfected normal cells. Thus, in certain embodiments, single-chain fusion HLA-I proteins inhibit NK cell-induced death 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-induced apoptosis. Thus, in certain 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 normally expressed on the surface of placental cytotrophoblasts, which do not express HLA-A, B, or C, and protects these cells from NK cell-induced lysis by interacting with the inhibitory ILT2 (LIR1) receptor on NK cells (Pazmany et al., Science 274:792-795, 1996). Thus, in certain other preferred embodiments, B2M- / - cells express a single-chain fusion HLA class I protein that comprises at least a portion of B2M and at least a portion of HLA-G.

[0040] In certain embodiments, the single-chain fusion HLA class I protein comprises at least a portion of B2M and at least a portion of HLA-A0201, an HLA-A allele. HLA-A0201 (SEQ ID NO: 4) is a common HLA class I allele found in the majority of the population of the United States. Thus, in certain advantageous embodiments, the isolated cell expresses 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, and the isolated cell is immunocompatible with the majority of the population of the United States. Other suitable common alleles that can be used include, but are not limited to, HLA-A0101, HLA-A0301, HLA-B0702, HLA-B0801, HLA-C0401, HLA-C0701, and HLA-C0702. In certain preferred embodiments, the HLA allele comprises HLA-A0201 (SEQ ID NO: 4), HLA-B0702 (SEQ ID NO: 6), or HLA-C0401 (SEQ ID NO: 8).

[0041] In further certain embodiments, the single chain fusion HLA class I protein also contains a specific peptide antigen that occludes its peptide binding cleft, which is covalently linked to the single chain fusion HLA class I protein (also called a trimeric construct). An example of a trimeric construct is shown in FIG. 3B. The HLA-bGBE construct in FIG. 3B contains HLA-E and B2M covalently linked to a peptide antigen (such as, but not limited to, the HLA-G peptide antigen exemplified in this figure) (SEQ ID NO: 23) designed to occlude the peptide binding cleft of the single chain fusion HLA class I protein. In other certain embodiments, the covalently linked peptide antigen is cleaved by an inbuilt protease cleavage site, and the cleaved peptide antigen can be bound to a peptide that binds to the cleft of the single chain fusion HLA-I protein for presentation. In certain alternative embodiments, the peptide antigen occupying the peptide-binding cleft of the single-chain fusion HLA class I protein is produced by an intracellular antigen processing pathway in which the peptide antigen is produced by the proteasome and transported to and attached to the single-chain fusion HLA class I protein in the endoplasmic reticulum. In certain embodiments, the peptide antigen comprises a peptide of a tumor antigen. In certain other embodiments, the peptide antigen comprises a peptide of a protein from a pathogen, including but not limited to bacteria, viruses, fungi, and parasites. In further embodiments, the peptide antigen comprises a peptide of a tumor antigen. In certain embodiments, B2M- / - cells express a single-chain fusion HLA class I protein covalently linked to a peptide that does not contain the patient's self- or neo-antigens. It is within the ability of one skilled in the art to design the single-chain fusion HLA class I protein and the peptide antigen presented on the single-chain fusion HLA class I protein to modulate the immune response that may be elicited in the recipient.

[0042] Isolated B2M- / - cells expressing a single-chain fusion HLA class I protein comprising a specific peptide antigen covalently or non-covalently linked to the single-chain fusion HLA class I protein can be used, for example, to administer to a recipient to elicit an immune response. Thus, in a related aspect, the invention provides a vaccine comprising the isolated cells of the invention, which can elicit an immune response in the recipient specific for the target peptide antigen. The immune response includes, but is not limited to, a cellular immune response and / or a humoral immune response. The vaccine may comprise stem cells or differentiated cells; in certain embodiments, such cells are differentiated dendritic cells. In certain other embodiments, the cells further express a cytokine. Any suitable cytokine may be used; in certain embodiments, such cytokine is IL2 or IFN-γ. In certain preferred embodiments, the cells are human cells and the recipient is human.

[0043] Single-chain fusion HLA class I protein can be expressed from an expression vector that allows for transient expression of protein in B2M- / - cells, more preferably stable expression. Exemplary suitable expression vectors are known in the art. One such example is a retroviral vector, which is integrated into the genome of a cell to stably express exogenous genes for a long period of time. In certain embodiments, the viral vector is derived from a retrovirus type of human foamy virus. Other suitable viral vectors include, but are not limited to, retrovirus, adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, vaccinia virus, and poxvirus.

[0044] In certain preferred embodiments, a polynucleotide capable of encoding a single-chain fusion HLA class I protein is integrated into a chromosome of a cell, preferably into the B2M or HLA locus, for stable expression. Thus, in certain preferred embodiments, the B2M locus is modified by inserting a polynucleotide capable of encoding a single-chain fusion HLA class I protein into the B2M locus to replace the expression of endogenous wild-type B2M protein. As a result of such gene targeting, the expression of normal B2M is modified, preventing the formation of wild-type HLA class I protein, but allowing the expression of the optimal selected single-chain fusion HLA class I protein on the surface of cells that are otherwise deficient in B2M. Other expression vectors are also contemplated, and the selection of an appropriate expression vector is within the capabilities of one skilled in the art.

[0045] Depending on the vector design, a polynucleotide capable of expressing a single-chain fusion HLA class I protein is delivered to cells by viral infection (if a viral vector is used) or by other delivery methods including, but not limited to, transfection, electroporation, gene targeting, or liposome-mediated DNA delivery.

[0046] Any immune effects of B2M- / - cells expressing single chain fusion HLA class I proteins can be tested by various means. For example, B2M- / - cells expressing SC fusion HLA class I proteins can be differentiated into antigen-presenting dendritic cells (iDCs). Inhibition of NK cell-induced lysis can be measured by chromium release assay after incubating iDCs with normal human NK cells and NKL cell lines. Various controls (untransduced B2M- / - iDCs, B2M+ / + iDCs, 721.221 class I negative cell line, as well as anti-receptor antigens and anti-HLA antibodies) can be used to establish the specificity of the interaction. Further characterization can be performed in Elispot assays by incubating cells with T cells.

[0047] In a related aspect, the invention provides an HLA class I type B2M- / - cell bank, the cells of which comprise a B2M- / - genetic background and have been engineered to express one or more types of single chain fusion HLA class I proteins in which the HLA alpha chain is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In certain embodiments, the cell bank comprises a cell population expressing a single chain fusion HLA class I protein in which the HLA alpha chain comprises HLA-A. In other certain embodiments, the cell bank comprises a cell population expressing a single chain fusion HLA class I protein in which the HLA alpha chain comprises HLA-B. In further certain embodiments, the cell bank comprises a cell population expressing a single chain fusion HLA class I protein in which the HLA alpha chain comprises HLA-C. In yet other embodiments, the cell bank comprises a cell population expressing a single chain fusion HLA class I protein in which the HLA alpha chain comprises HLA-E. In certain other embodiments, the cell bank comprises a cell population expressing a single-chain fusion HLA class I protein whose HLA alpha chain comprises HLA-F. In certain embodiments, the cell bank comprises a cell population expressing a single-chain fusion HLA class I protein whose HLA alpha chain comprises HLA-G. In certain embodiments, the cell bank comprises one or more, preferably all, of the cell populations described above.

[0048] The cells of the cell bank can be pluripotent stem cells or differentiated cells. In certain embodiments, the cell bank contains different types of differentiated cells, such as skin cells, pancreatic islet β cells, etc., that express the same single-chain fusion HLA class I protein. Meanwhile, in other embodiments, the cell bank contains different types of differentiated cells, such as skin cells, pancreatic islet β cells, etc., that each express a different single-chain fusion HLA class I protein. The selection of suitable donor cells from the cell bank for a given patient can be determined by a skilled researcher or clinician. In certain other embodiments, some cells of the cell bank express HLA class I alleles that match the HLA class I alleles of the patient to whom the cells are administered. In certain preferred embodiments, the cells are human cells and the patient is human. In certain embodiments, the cells express single-chain fusion HLA class I proteins that include HLA-A0201 and B2M that match the HLA alleles of the majority of the U.S. population.

[0049] In another aspect, the invention provides a method of transplantation to a patient in need of transplantation, comprising administering to the patient an effective amount of the cells of the invention for transplantation. Because B2M- / - cells do not express wild-type HLA class I proteins on the cell surface, the cells induce little or no immune response in the patient when administered to the patient. Thus, transplantation with B2M- / - cells limits the need for immunosuppressive therapy. Thus, in certain preferred embodiments, the patient is immunocompetent. In certain other embodiments, the cells are isogeneic cells; while in other embodiments, the cells are allogeneic cells.

[0050] In further certain embodiments, the cell of the present invention is a pluripotent stem cell; in other embodiments, the cell of the present invention is a differentiated cell. In certain preferred embodiments, the cell is a human cell, and the patient is a human patient. In certain embodiments, the method of transplantation comprises administering an effective amount of the pluripotent stem cell or differentiated cell to a human. In certain preferred embodiments, the cell of the present invention further expresses one or more recombinant single-chain fusion HLA class I proteins. In other certain embodiments, the cell can avoid NK cell-induced death after transplantation and induce little or no immune response in the recipient.

[0051] Transplantation therapy, replacement therapy, or regeneration therapy refers to the treatment of a disease condition by administering cells or tissue to a patient to supplement or replace the dysfunction of cells in a target organ. In certain embodiments, the need for transplantation arises as a result of the physical or physical disorder of tissue or organ. In other certain embodiments, the need for transplantation arises as a result of one or more genetic defects or mutations in a patient, and transplantation of the cells of the present invention supplements or replaces the dysfunction of cells in the patient without the need for gene therapy to correct the patient's underlying genetic mutation. In further certain embodiments, transplantation includes, but is not limited to, hematopoietic stem cell transplantation, or transplantation of cells that are incorporated into an organ, such as the liver, kidney, pancreas, lung, brain, muscle, heart, digestive tract, nervous system, skin, bone, bone marrow, fat, connective tissue, immune system, or blood vessels. In certain embodiments, the target organ is a solid organ.

[0052] In certain embodiments, the cells administered to the recipient may or may not incorporate into the organ requiring such therapy. In certain embodiments, the cells of the invention differentiate into the desired cell type before or after transplantation and perform the required cellular function without incorporating into the tissue at the transplantation site. For example, in certain embodiments to treat diabetes, the cells of the invention are transplanted into the diabetic patient as pluripotent stem cells or differentiated pancreatic islet β cells. The transplanted cells do not have to constitute a functioning pancreas: they only need to secrete insulin in response to blood glucose levels. In certain embodiments, the cells are transplanted into an ectopic site and do not fully incorporate into the pancreas. The cell populations of the invention, the differentiated cells of the invention, or tissues developed from the cells of the invention by in vitro differentiation 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 I proteins.

[0053] In a further aspect, the invention provides a method for treating a condition in a patient in need of treatment, comprising administering to the patient an effective amount of a cell of the invention to treat the condition, the condition being diabetes, an autoimmune disease, cancer, an infectious disease, anemia, leukopenia, myocardial infarction, heart failure, skeletal or joint conditions, osteogenesis imperfecta, or burns. In certain embodiments, the condition results from physiological or physical damage to a tissue or organ. In certain embodiments, the cell of the invention is a stem cell; in other embodiments, the cell of the invention is a differentiated cell. In certain preferred embodiments, the cell is a human cell and the patient is a human patient. In certain embodiments, the human cell is a differentiated cell. Transplantation of tissues developed from the cell of the invention ex vivo is also contemplated by the invention. In certain preferred embodiments, the cell of the invention further expresses one or more single chain fusion HLA class I proteins. In certain embodiments, the cell is an isogenic cell; in other embodiments, the cell is an allogeneic cell.

[0054] In certain embodiments, the cell is a differentiated cell, including but not limited to 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, gastrointestinal cells, vascular cells, endocrine cells, and adipocytes.In other certain embodiments, the present invention provides a method for treating a pathology of a solid organ.In certain embodiments, the cell of the present invention used for treating a pathology expresses one or more single-chain fusion HLA class I proteins.

[0055] "Treating" a patient with a disease or disorder means achieving one or more of the following: (a) reducing the severity of the disease; (b) halting the progression of the disease or disorder; (c) inhibiting the worsening of the disease or disorder; (d) limiting or preventing the recurrence of the disease or disorder in a patient previously affected with the disease or disorder; (e) regressing 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.

[0056] The effective amount of the isolated cells of the present invention for transplantation or treatment of a disease state varies depending on many factors, such as tissue type, disease state severity, transplantation response, reason for transplantation, and age and general health of the patient.The effective amount can be determined by a skilled researcher or clinician in a routine manner.The immunogenicity of transplanted cells is reduced, so that a patient can tolerate a relatively large amount of cells to achieve the desired therapeutic effect.Alternatively, the cells can be transplanted repeatedly in a periodic manner until the desired therapeutic effect is achieved.

[0057] The administration route of the cells of the present invention is not limited to a specific route.Exemplary administration routes include, but are not limited to, intravenous, intramuscular, subcutaneous, intraperitoneal, transdermal, intradermal, and subcutaneous routes.The cells of the present invention can also be administered locally by injection.For example, the cells can be injected into or around an injured joint, a fractured bone, an infarcted site, an ischemic site.

[0058] In certain embodiments, the cells are administered by a delivery device, including, but not limited to, a syringe. For example, the cells can be suspended in a solution or pharmaceutical composition contained within such a delivery device. A "solution" or "pharmaceutical composition" includes a physiologically compatible buffer, and optionally a pharma- ceutical acceptable carrier or diluent in which the cells of the invention survive. The use of such carriers and diluents is well known in the art. Such solutions include, but are not limited to, physiologically compatible buffers, such as Hank's solution, Ringer's solution, or physiologically buffered saline. The cells can be stored in the solution or pharmaceutical composition for short periods without loss of viability. In certain embodiments, the cells are stored for long periods without loss of viability according to cryopreservation methods well known in the art.

[0059] Aqueous injection suspensions may contain substances that increase their viscosity, such as sodium carboxymethylcellulose, sorbitol, or dextran, but are still liquid enough to be easily delivered by syringe injection.The solution is preferably sterile, stable under the conditions of manufacture and storage, and free of microbial contamination, such as by using parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal.The cells contained in the solution can be stem cells or differentiated cells as described herein, in a pharmaceutically acceptable carrier or diluent, and other components as necessary.

[0060] The cells can be administered systemically (e.g., intravenously) or locally (e.g., directly into a myocardial defect under echographic guidance, or by direct application to damaged tissue or organs accessible during open surgery). For injection, the cells can be included in an injectable liquid suspension preparation, or in a biocompatible medium that is in an injectable liquid form and becomes semi-solid at the site of damaged tissue. A syringe, controllable endoscopic delivery device, or other similar device can be used, provided that the lumen of the needle is of sufficient diameter (e.g., at least 30 gauge or larger) to avoid physical damage to the cells during delivery.

[0061] In certain other embodiments, the cells can be implanted by a solid support, such as a planar surface or a three-dimensional matrix. The matrix or surface is surgically implanted into the appropriate site of the patient. For example, in a patient requiring a pancreas transplant, the solid support provided with differentiated cells can be surgically implanted into the pancreatic tissue. Exemplary solid supports include, but are not limited to, patches, gel matrices (e.g., GELFOAM® from Pharmacia-Upjohn), polyvinyl alcohol sponge (PVA)-collagen gel implants (e.g., IVALON, Unipoint Industries, High Point, NC), and other similar or equivalent devices. A variety of other encapsulation techniques can be used with the cells of the present invention. See, e.g., WO 91 / 10470, WO 91 / 10425, U.S. Pat. Nos. 5,837,234; 5,011,472; and 4,892,538.

[0062] The cells of the present invention can be differentiated into various cell types of all three lineages, including but not limited to hematopoietic cells, mesenchymal cells, pancreatic endoderm cells, cardiac cells, and keratinocyte cells.In certain embodiments, such differentiated cells further express single-chain fusion HLA class I proteins.Generally, each cell type can be analyzed for HLA class I protein expression, reactivity with human T cells and NK cells, appropriate differentiation markers, and xenografting in immunodeficient mice to test in vivo developmental potential.See Figure 4.A brief description of each differentiated cell type is provided below.

[0063] In certain embodiments, the cells of the present invention can be differentiated into hematopoietic cells for the treatment of various hematopoietic disorders that are currently treated by bone marrow transplantation. Transfusion recipients may be unable to receive platelet transfusions due to HLA mismatch. Patients with anemia or thrombocytopenia can be treated by delivering red blood cells, platelets, or neutrophils derived from the cells of the present invention to treat bleeding or infection.

[0064] Furthermore, the dendritic cells derived from stem cells of the present invention are antigen-presenting cells that can be used as cellular vaccines when appropriately engineered. In certain embodiments, the cells of the present invention engineered to express single-chain fusion HLA class I proteins and unique peptide antigens are used to vaccinate against specific pathogens or tumor antigens. In other certain embodiments, differentiated B2M- / - cytotoxic lymphocytes with HLA-restricted reactivity to specific antigens are used to eliminate infected or tumor cells.

[0065] To obtain hematopoietic cells, pluripotent cells were first allowed to form 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 invention expressing single-chain fusion HLA class I proteins can be analyzed by flow cytometry and colony assays. Differentiated cell populations are stored based on their surface markers and used to monitor the expression of HLA genes and reactivity with human NK cells and T cells measured by Elispot, mixed lymphocyte reaction, and cytotoxicity assays. The effectiveness of single-chain fusion HLA constructs in suppressing NK cell-induced death can be tested at different stages of differentiation and transplantation. See Bix et al., Nature, 1991, 349:329-331. Hematopoietic stem cells can also be assayed using xenograft models, for example in immunodeficient mice (SCID repopulating cells or SRC).

[0066] The cells of the present invention can be differentiated into hematopoietic cells before or after the cells are administered 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., J Immunol, 2006, 176:2924-32, and Chang et al., Blood, 2006, 108:1515-23.

[0067] In certain other embodiments, the cells of the invention can differentiate into mesenchymal stem cells. In certain embodiments, the cells of the invention express one or more single chain fusion HLA class I proteins. MSCs have the ability 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 for treating skeletal and joint conditions. iMSCs can further differentiate into osteoblasts to form bone in vivo. Deyle et al., Mol Ther. 2012, 20(1), 204-13. The cellular responses of T cells and NK cells to ESCs, iMSCs, and their more terminally differentiated derivatives, such as osteoblasts, can be examined.

[0068] In certain embodiments, mesenchymal stem cells can differentiate into cell types, including but not limited to bone marrow stromal cells, adipocytes, osteoblasts, osteocytes, and chondrocytes. The cells of the present invention are differentiated into mesenchymal stem cells 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 performed according to established protocols. See, for example, Deyle et al., supra.

[0069] In yet other specific embodiments, the cells of the invention can be differentiated into insulin-producing pancreatic islet cells. In certain embodiments, the cells of the invention express one or more single-chain fusion HLA class I proteins. The cells of the invention can be used to treat insulin-dependent diabetes. Advantageously, the transplanted cells do not need to reconstitute a functioning pancreas: they only need to secrete insulin in response to blood glucose levels. Thus, this treatment can be successful with different cell doses, with cells that are not fully differentiated into mature cell types, and when cells are transplanted into ectopic sites. Certain autoantigens, such as those derived from GAD65 or insulin, can trigger autoimmune destruction of beta cells in diabetes (Di Lorenzo et al., Clin Exp Immunol, 2007, vol. 148, pp. 1-16). Thus, B2M- / - cells, or B2M- / - cells expressing single-chain fusion HLA class I proteins presenting defined peptide antigens, offer the added advantage of not presenting these self-antigens and avoiding autoimmune rejection to prevent recurrence of diabetes after transplantation.

[0070] The cells of the invention can be differentiated into pancreatic cells as previously described, utilizing exposure of the cells to various cytokines and drugs to promote the sequential formation of mesoendoderm, definitive endoderm, and pancreatic precursors (Kroon et al., Nat Biotechnol 2008, 26:443-452). These cells can be further cultured in explants in immunodeficient mice. The cells of the invention and wild-type cell lines expressing or not expressing single-chain fusion HLA class I proteins can be analyzed for reactivity with T cells and NK cells at various stages of development.

[0071] The cells of the present invention are differentiated into pancreatic islet cells before or after administration to a patient. In certain preferred embodiments, the cells are human cells and the patient is a human patient. In vitro hematopoietic differentiation can be performed according to established protocols. See, for example, Kroon et al., Nat Biotechnol, 2008, vol. 26, pp. 443-452.

[0072] In other certain embodiments, the cells of the present invention can be differentiated into cardiomyocytes. In certain embodiments, the cells of the present invention further express one or more single-chain fusion HLA class I proteins. The common clinical problems of myocardial infarction and ischemic heart failure can be addressed by transplanting healthy stem cell-derived cardiomyocytes that engraft and reconstruct functional myocardium. Cardiomyocytes derived from the cells of the present invention allow these treatments to be performed with pre-packaged cells, avoiding the immunosuppression currently required for allogeneic heart transplantation. Physiologically relevant tests, such as electrical conduction and contraction tests, can be performed on cardiomyocytes derived from the cells of the present invention. B2M- / - stem cells or differentiated cardiomyocytes that express or do not express single-chain fusion HLA class I proteins are tested to determine their immunological reactivity when expressing cardiomyocyte genes to establish which HLA modification minimizes their immune response.

[0073] The cells of the present invention can be differentiated into cardiomyocytes 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 are differentiated into cardiomyocytes to treat diseases including, but not limited to, myocardial infarction and ischemic heart failure. In vitro cardiomyocyte differentiation can be performed according to established protocols. See, for example, Lafiamme et al., Nat Biotechnol, 2007, vol. 25, pp. 1015-1024.

[0074] In yet other specific embodiments, the cells of the present invention can be differentiated into keratinocytes. In certain embodiments, the cells of the present invention that can be used to differentiate into keratinocytes express one or more single chain fusion HLA class I proteins. Severe burns and genetic skin diseases require treatment with skin grafts, which are currently performed using various cell sources, such as pig skin grafts and cultured autologous human keratinocytes. Keratinocytes derived from the cells of the present invention provide a major clinical advancement, as burns can be treated as an emergency with prepackaged cells, and genetic diseases, such as epidermolysis bullosa, can be treated with normal cells (even with a B2M- / - background of the cell chromosomes) that do not require correction of the causative genetic mutation. In many cases, the cells only need to be transplanted long enough for nearby host cells to repopulate the affected area. Figure 5 shows the in vitro differentiation of keratin 5+ and keratin 14+ keratinocyte colonies derived from the cells of the present invention. The cells of the invention are cultured in Matrigel culture and then expanded in serum-free keratinocyte medium containing all-trans retinoic acid and BMP4 as previously described (Itoh et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS USA), 2011, vol. 108, pp. 8797-8802). For in vivo differentiation, the cells of the invention can be embedded in a polyvinyl alcohol sponge (PVA)-collagen gel implant for transplantation into a recipient. The cells of the invention can be differentiated into keratinocytes before or after transplantation. In certain preferred embodiments, the cells are human cells and the patient is human.

[0075] In yet another aspect, the invention provides the use of the cells of the invention in the preparation of a medicament for transplantation. In a related aspect, the invention provides the use of the cells of the invention in the preparation of a medicament for treating a medical condition.

[0076] Furthermore, the cells of the present invention can serve as a research tool, providing a system for testing the function of immunomodulatory proteins in a B2M- / - genetic background. In certain embodiments, the cells of the present invention further express one or more single-chain fusion HLA class I proteins. Thus, in a related aspect, the present invention provides a method for determining the function of an immunomodulatory protein, comprising introducing one or more immunomodulatory genes into the cells of the present invention of the present invention and assaying for 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 test the function of an immunomodulatory gene or test an immune response in the absence of unwanted class I antigens. In certain embodiments, the cells of the present invention express HLA-F, or a single-chain fusion HLA class I protein comprising B2M and HLA-F, and the function of HLA-F can be tested in a B2M- / - background. In a further related aspect, the invention provides a method for identifying a compound or molecule that modulates the function of an immunomodulatory protein, comprising contacting a B2M- / - cell containing one or more immunomodulatory genes with the compound or molecule of interest and assaying for the activity of the immunomodulatory gene. In certain preferred embodiments, the cell is a human cell.

[0077] In yet another related aspect, the invention provides an in vivo research tool in a mammal, particularly a non-human primate, which administers the cells of the invention to test the function of immune-modulating genes or to identify compounds that modulate the function of immune-modulating genes of the administered cells in a B2M- / - genetic background. In certain embodiments, the cells of the invention further express one or more single-chain fusion HLA class I proteins.

[0078] Mice, especially immunodeficient mice, were used as a model system to test human cells in vitro. Human stem cells may behave differently in mice. In addition, mouse and human immune systems have different NK cell receptors and non-classical HLA class I genes (e.g., HLA-E, F, and G). Therefore, a pigtailed macaque (Mn) model can be created to test the cells of the present invention. The genome of rhesus macaques has been sequenced and is highly homologous to that of pigtailed macaques. Furthermore, the organization of the macaque MHC locus is similar to human HLA, including non-classical genes. Homologs of 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 B2M- / -ESCs and Mn B2M- / -ESCs can be used for transplantation of macaques.

[0079] MHC class I-deficient (B2M- / -) macaque ESCs can be generated using the same AAV-mediated gene targeting strategy as described for human cells. Mn versions of single-chain fusion HLA class I proteins are expressed in B2M- / - macaque ESCs using similar viral vectors as described above.

[0080] Cells can be grown in vitro and labeled with a vector expressing GFP for later 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 removed, sectioned, and stained to determine the cell types present. Specific antibodies can be used to identify differentiated cell types formed by the transplanted cells.

[0081] Any embodiment described above applies to any aspect of the invention unless otherwise stated. All embodiments within and between the various aspects can be combined unless otherwise stated.

[0082] The following examples are illustrative of specific embodiments of the invention and various uses thereof. These examples are presented for illustrative purposes only and should not be construed as limitations of the invention. (Example) Example 1: Generation of human pluripotent stem cells carrying a knockout mutation in the B2M gene Human pluripotent stem cells were generated with knockout mutations of both alleles of the β2 microglobulin (B2M) gene, which encodes a common subunit required for surface expression of all HLA class I heterodimers (HLA-A, B, C, D, E, F, and G). B2M- / - (class I negative) H1 human ESCs were generated using adeno-associated virus (AAV) gene targeting vectors (University of Wisconsin). Human pluripotent stem cells were infected with the AAV gene targeting vector to inactivate the B2M gene by homologous recombination. AAV-mediated gene targeting methods have been previously described, for example, in Khan et al., Protocol, 2011, 482:482-501, and Khan et al., Science, 1990, 248:1227-30. These references are incorporated herein by reference in their entirety.

[0083] Figure 1 shows the generation of HLA class I negative human H1 ESC cells using adeno-associated virus (AAV) gene targeting vectors. The two AAV vectors used contain homology arms surrounding exon 1 of the human B2M gene and are designed to insert either TKNeo or HyTK fusion genes into exon 1, encoding G418 resistance or hygromycin resistance, respectively (Figure 1A). H1 ESCs were infected with the AAV-B2M-ETKNpA vector, and 30% of the G418-resistant cells were targeted at one B2M allele based on Southern blot analysis. One of these clones was then infected with the AAV-B2M-EHyTKpA vector, and 10% of the hygromycin-resistant cells were targeted at B2M. Southern blot analysis of a representative clone with a deletion in both B2M alleles (B2M- / -) is shown in Figure 1B (HyTK / TKN). None of the targeted clones analyzed contained random integrants. The sequences of the targeting vector plasmids pA2-B2METKMpA and pA2-B2MEHuTKpA are shown in SEQ ID NO:21 and SEQ ID NO:22, respectively.

[0084] Cre recombinase was then used to remove the loxP-floxed TKNeo and HyTK transgenes from the B2M locus. Cre was delivered transiently by a non-integrating foamy virus vector, a type of previously described retroviral vector that efficiently infects human ESCs. See Deyle et al., J. Virol. 84:9341-9 (2010) and Gharwan et al., Mol Ther. 15:1827-1833 (2007). Four clones in which the TKNeo and HyTK transgenes were deleted were selected with a moiety of ganciclovir, which kills cells expressing thymidine kinase (TK). The results shown by Southern blot analysis demonstrated a transgene-free double knockout (FIG. 1B).

[0085] The karyotype of two of these clones was checked and found to be normal (data not shown), and teratoma assays performed in immunodeficient mice demonstrated that these cells had trilineage developmental potential (Figure 2). Flow cytometry using antibodies against B2M (anti-B2M-01-PE, SantaCruz Biotechnology) and pan-HLA class I antigens (W6 / 32, Sigma-Aldrich) confirmed that these cells did not express HLA class I proteins on their surface (Figure 1C).

[0086] Example 2 Expression of single-chain fusion HLA class I proteins in B2M knockout cells In mice, HLA class I negative cells can be destroyed through a "missing self" mechanism by natural killer (NK) cells. Bix et al., Nature 349:329-331, 1991. Although human NK cells have different receptors, a similar inhibition of NK cytotoxicity is mediated by the interaction of HLA-C, E, and G with the NK cell receptor. The "missing self" phenomenon has been described in detail for class I deficient hematopoietic cells. Previous mouse transplantation data showing that many types of B2M- / - organs survive in B2M+ / + hosts suggests that the "missing self" phenomenon may not be as important when transplanted cells form solid organs. However, given that the "missing self" phenomenon may significantly affect donor cell survival in some circumstances, specific HLA class I genes that inhibit NK cytotoxicity were introduced into B2M- / - cells as single-chain fusion proteins.

[0087] The strategy to express a specific HLA class I gene in a B2M- / - background is shown in Figure 3. The B2M chain was fused to a specific HLA class I heavy chain, allowing surface expression of this class I chain even in B2M- / - cells. These single-chain fusion proteins were expressed using integrative foamy virus vectors. Foamy virus vectors are a type of retroviral vector with a large packaging capacity that can fully infect human pluripotent stem cells (Gharwan et al., supra). One such representative single-chain fusion HLA class I protein foamy virus construct is shown in Figure 3A. The ΔΦ-EGP-PHLA-SC foamy vector contains a GFP-Puro fusion protein gene driven by the EF1α promoter and a separate expression cassette with the ubiquitously expressed PGK promoter driving the HLA single-chain fusion (HLA-SC) construct (Figure 3A), where the GFP-Puro fusion protein gene allows puromycin selection and GFP expression in transduced cells. This vector design resulted in constitutive expression of both transgenes, although many other vector designs and internal promoters can be used. For example, the GFP-Pur gene can be driven by the pGK promoter, and the EF1α promoter controls expression of the SC HLA genes.

[0088] As shown in FIG. 3B, the HLA-bGBE trimer single chain fusion construct contained the HLA-G peptide (SEQ ID NO: 23) covalently linked to the HLA-E peptide binding cleft, while the HLA-gBE dimer construct contained the HLA-G signal peptide truncated but still non-covalently linked to the HLA-E molecule. See Crew et al., Mol Immunol 42:1205-1214 (2005). B2M- / - cells were transduced with these vectors. Puromycin-resistant clones were selected and HLA-E surface expression was analyzed by flow cytometry in pluripotent cells expressing these constructs (FIG. 3C).

[0089] Additionally, specific classical HLA class I allele single chain fusion proteins were generated and expressed in B2M- / -H1 ESCs to generate "quasi-universal" donor cells for improved compatibility with the recipient. For example, in the United States, HLA-A0201 alleles are present in 48% of Caucasians, 46% of Hispanics, and 24% of African Americans, all of which should tolerate HLA-A0201+ stem cells. See www.allelfrequencies.net and Storkus et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS USA), 1989, 86:2361-2364. HLA-bBA0201 dimeric single chain fusion constructs were introduced into B2M- / -H1 ESCs by foamy virus vectors (Figure 3B), and expression of single chain fusion proteins was analyzed by flow cytometry (Figure 3D).

[0090] It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention, and that all modifications and alternatives equivalent to those embodiments are within the concept and scope of the invention as set forth in the appended claims. (Additional Note) As a preferred embodiment, the technical concept that can be understood from the above embodiment will be described. [Item 1] 1. An isolated cell comprising a genetic modification in all copies of the beta 2 microglobulin (B2M) gene, said cell being a human cell; The cells further comprise one or more recombinant immunomodulatory genes comprising a polynucleotide capable of encoding a single-chain fusion human leukocyte antigen (HLA) class I protein; the single chain fusion HLA class I protein comprises an exogenous B2M protein covalently linked, either directly or via a linker sequence, to at least one HLA class-I α chain; the HLA class-I α chain may or may not contain a signal sequence of an HLA class-I chain protein, and the exogenous B2M protein may or may not contain a signal sequence of a B2M protein; An isolated cell, wherein the cell has a normal karyotype and is a non-transformed cell. [Item 2] 2. The cell of claim 1, wherein the single chain fusion HLA class I protein comprises the B2M protein covalently linked to an HLA class I alpha chain selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, and the selected HLA class I alpha chain may or may not contain a signal sequence of the HLA class-I chain protein. [Item 3] 2. The cell of claim 1, wherein the single chain fusion HLA class I protein comprises the B2M protein and an HLA class I alpha chain selected from the group consisting of HLA-E, HLA-G, and HLA-A, and the selected HLA class I alpha chain includes or does not include a signal sequence of the HLA class-I chain protein. [Item 4] 4. The cell according to any one of items 1 to 3, wherein the single-chain fusion HLA class I protein comprises the B2M protein and HLA-A with or without a signal sequence of the HLA-A protein. [Item 5] 5. The cell according to any one of items 1 to 4, wherein the single-chain fusion HLA class I protein comprises the B2M protein and HLA-A0201 with or without a signal sequence of HLA-A0201 protein. [Item 6] 4. The cell according to any one of items 1 to 3, wherein the single-chain fusion HLA class I protein comprises the B2M protein and HLA-E with or without a signal sequence of the HLA-E protein. [Item 7] 4. The cell according to any one of items 1 to 3, wherein the single-chain fusion HLA class I protein comprises the B2M protein and HLA-G with or without a signal sequence of HLA-G protein. [Item 8] 3. The cell according to item 1 or 2, wherein the single-chain fusion HLA class I protein comprises the B2M protein and HLA-B with or without the signal sequence of the HLA-B protein. [Item 9] 3. The cell according to item 1 or 2, wherein the single-chain fusion HLA class I protein comprises the B2M protein and HLA-C with or without the signal sequence of the HLA-C protein. [Item 10] 3. The cell according to item 1 or 2, wherein the single-chain fusion HLA class I protein comprises the B2M protein and HLA-F with or without the signal sequence of the HLA-F protein. [Item 11] 11. The cell according to any one of items 1 to 10, wherein the cell further expresses a target peptide antigen that is presented on the surface of the cell by the single-chain fusion HLA class I protein. [Item 12] 12. The cell of claim 11, wherein the target peptide antigen is covalently linked to the single chain fusion HLA class I protein. [Item 13] 13. The cell according to any one of items 1 to 12, wherein the cell further comprises one or more recombinant genes capable of encoding a suicide gene product. [Item 14] 14. The cell of item 13, wherein the suicide gene product comprises a protein selected from the group consisting of thymidine kinase and an apoptosis signaling protein. [Item 15] 15. The cell according to any one of items 1 to 14, wherein the cell is a stem cell. [Item 16] 16. The cell of item 15, 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 17] 17. The cell of item 15 or 16, wherein the stem cell is a pluripotent stem cell. [Item 18] 18. The cell according to any one of items 15 to 17, wherein the stem cell is differentiated. [Item 19] 20. The cell of item 18, 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, gastrointestinal cells, vascular cells, endocrine cells, adipocytes, and cardiomyocytes. [Item 20] 13. A vaccine comprising the cells according to item 11 or 12, capable of inducing an immune response in a primate specific to the target peptide antigen. [Item 21] 21. The vaccine of item 20, wherein the primate is a human. [Item 22] A kit comprising the cell according to any one of items 1 to 19. [Item 23] 23. The kit according to item 22, further comprising a graft, the graft comprising the cells according to any one of items 1 to 19. [Item 24] 21. A kit comprising the vaccine of item 20, further comprising an immune adjuvant.

Claims

**Claim 1**: An isolated eye cell comprising a genetically engineered disruption in the β2-microglobulin (B2M) gene, which is a primate cell, wherein the cell further comprises one or more polynucleotides capable of encoding a single-chain fusion non-classical human leukocyte antigen (HLA) class I protein, wherein the single-chain fusion non-classical HLA class I protein comprises at least a portion of B2M covalently bound to at least a portion of an HLA class I α-chain selected from the group consisting of HLA-E, HLA-F, and HLA-G, wherein the single-chain fusion non-classical HLA class I protein is capable of having a normal function for binding to inhibitory receptors on the surface of NK cells, a cell. **Claim 2**: The cell according to claim 1, wherein the non-classical HLA class I protein is HLA-E. **Claim 3**: The cell according to claim 1, wherein the non-classical HLA class I protein is HLA-F. **Claim 4**: The cell according to claim 1, wherein the non-classical HLA class I protein is HLA-G. **Claim 5**: The cell according to claim 1, comprising a plurality of different single-chain fusion non-classical HLA class I proteins. **Claim 6**: The plurality of different single-chain fusion non-classical HLA class I proteins are (i) a non-classical HLA class I protein that is HLA-E, and (ii) a non-classical HLA class I protein selected from the group consisting of HLA-F and HLA-G The cell according to claim 5, comprising. **Claim 7**: The cell according to any one of claims 1 to 6, wherein the cell comprises a genetically engineered disruption in all copies of the B2M gene. **Claim 8**: The cell according to any one of claims 1 to 7, wherein the cell further comprises a peptide presented on the surface of the cell by the single-chain fusion non-classical HLA class I protein. **Claim 9**: The cell according to claim 8, wherein the peptide is covalently bound to the single-chain fusion non-classical HLA class I protein. **Claim 10**: The cell according to any one of claims 1 to 9, wherein the B2M protein of the single-chain fusion non-classical human leukocyte antigen (HLA) class I protein is a full-length B2M protein.

11. The cell according to any one of claims 1 to 10, wherein the B2M protein and the non-classical HLA class-I α chain of the single-chain fusion non-classical human leukocyte antigen (HLA) class I protein are linked via a linker sequence.

12. The cell according to any one of claims 1 to 11, wherein the B2M protein of the single-chain fusion non-classical human leukocyte antigen (HLA) class I protein does not contain a leader sequence.

13. The cell according to any one of claims 1 to 12, wherein the HLA class-I α chain does not contain a leader sequence.

14. The cell according to any one of claims 1 to 13, further comprising one or more recombinant genes capable of encoding a suicide gene product.

15. The cell according to claim 14, wherein the suicide gene product comprises a protein selected from the group consisting of thymidine kinase and apoptosis signaling protein.

16. The cell according to any one of claims 1 to 15, having a normal karyotype.

17. The cell according to any one of claims 1 to 16, wherein the cell is a human cell.

18. A pharmaceutical composition comprising the cell according to any one of claims 1 to 17 and a physiologically compatible buffer.

19. A pharmaceutical composition for use in transplantation in a patient in need of transplantation, wherein an effective amount of the cell is administered to the patient, according to claim 18.

20. The pharmaceutical composition according to claim 19, wherein the patient is immunocompetent.

21. The pharmaceutical composition according to any one of claims 18 to 20, wherein the patient is human and the cell is a human cell.

22. A pharmaceutical composition for use in the treatment of a medical condition in a patient in need of treatment of the medical condition, wherein an effective amount of the cell is administered to the patient and the medical condition is an eye disease, according to claim 18.

23. A kit comprising the cell according to any one of claims 1 to 17.

24. The kit according to claim 23, further comprising a graft, wherein the graft comprises the cell according to any one of claims 1 to 17.

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