RU2795302C2 - Universal donor cells and related methods - Google Patents

Abstract

FIELD: cell biology; medicine.

SUBSTANCE: invention relates in particular to a cell composition for transplantation containing a population of human pancreatic cells in vitro, its use for transplantation and for the production of insulin in a subject, as well as a method for reducing transplant rejection of pancreatic endoderm cells in a human and a method for obtaining insulin in an immunocompromised or immunocompromised person. The said cell composition contains a population of human pancreatic cells in vitro containing CHGA^-, NKX6.1⁺, PDX1⁺ pancreatic cell precursor cells, and CHGA⁺ pancreatic endocrine cells, in which the function of at least one gene of the major histocompatibility complex (MCHC) class I and at least one natural killer (NK) cell activating ligand is disrupted or inhibited, resulting in reduced binding of the NK activating ligand to the NK activating receptor in the said human pancreatic cell population in vitro.

EFFECT: obtaining target cells that lack some or all of the classical expression of human leukocyte antigen cells, but are not attacked by NK cells and are not subsequently lysed.

26 cl, 21 dwg, 5 tbl, 17 ex

Description

RELATED APPLICATIONS

[001] The present application claims priority to U.S. Application No. 15/648,337 entitled "Universal Donor Cells and Related Methods", filed July 12, 2107, incorporated herein by reference in its entirety.

FIELD OF TECHNOLOGY

[002] The present invention relates to the field of gene expression, genetic engineering and gene/cell therapy.

BACKGROUND OF THE INVENTION

[003] Human pluripotent stem cells (hPSCs) are a useful tool for obtaining mature cells of any type for transplantation to patients. In principle, hPSC cell based therapies have the potential to treat most if not all degenerative diseases, however the success of such therapies may be limited by the subject's immune response. The immune system protects the body from infections with multi-level protection of increasing specificity. Simply put, physical barriers prevent pathogens such as bacteria and viruses from entering the body. If a pathogen breaches these barriers, the innate immune system provides an immediate but non-specific response. If pathogens successfully evade the innate response, vertebrates have a second layer of defense, an adaptive immune system that is activated by the innate response. The adaptive immune system generates a much more specific response. In this case, the immune system adapts its response during infection to improve recognition of the pathogen. This improved response is then stored after the pathogen is eliminated in the form of an immunological memory, allowing the adaptive immune system to speed up and escalate attacks each time that pathogen is detected. The adaptive immune response is antigen-specific and requires the recognition of specific "foreign" antigens during a process called antigen presentation. Antigenic specificity allows the generation of responses that are tailored to specific pathogens or pathogen-infected cells. Interferon gamma (IFN-γ) plays an important role in the fight against infectious and non-communicable diseases. T cells are the main source of IFN-γ in the human immune response. NK cells, macrophages, and interferons play an important role in both innate and adaptive immunity.

[004] The major histocompatibility complex (MHC) is a set of cell surface proteins essential for the regulation of the immune system. The main function of MHC molecules is to bind antigens derived from pathogens and expose them to the cell surface for recognition by the corresponding T cells. The MHC gene family is divided into three subgroups: class I, class II, and class III. Human MHC is also called the HLA complex (Human Leukocyte Antigens, English - HLA). Natural killer cells (NK cells) are lymphocytes that function at the interface between innate and adaptive immunity. NK cells directly contribute to immune defense through their effector functions such as cytotoxicity and cytokine secretion, as well as through the regulation of innate and adaptive immune responses. When a target or host cell encounters NK cells, several outcomes are possible. The degree of response of NK cells is determined by the number and type of activating and inhibitory receptors on NK cells, as well as the number and type of activating and inhibitory ligands on the target cell. See Fig. 1. In scenario A, when target cells do not have class I human leukocyte antigens (HLA) and do not activate NK cell ligands, NK cells expressing inhibitory class I MHC receptors and ligand-activating receptors do not attack the target cells ( no response or response not allowed). In scenario B, when target cells express class I LAS but lack activating ligands, NK cells expressing inhibitory receptors and activating receptors cannot attack the targets. In scenario C, when target cells have reduced levels of class I LAS or do not have class I LH and express NK cell activating ligands, NK cells expressing inhibitory receptors and activating receptors attack the target cells. In scenario D, when target cells express both ligands that activate their own class I LAS proteins and ligands that activate NK cells, the level of responses of NK cells expressing inhibitory receptors and activating receptors is determined by the balance of inhibitory and activating signals for NK -cells. Haynes et al., THE IMMUNE SYSTEM IN HEALTH AND DISEASE, PART 15: Immune-Mediated, Inflammatory, and Rheumatotogic Disorders, 372e, Introduction to the Immune System.

[005] Historically, research efforts to overcome the host's immune response to allogeneic cells have focused on the adaptive immune response, that is, interfering with the adhesion between T cells and MHC class I antigens presented on foreign cells. Thus, the CRISPR and TALEN systems have been used to generate loss-of-function genetic modifications and, consequently, to obtain stem cells that do not express one or more of the classic MHC/LAH genes. However, these cells and cells derived from them are still vulnerable to the host's innate immune response (NK cells). See, for example, Parham et al. (2005) Nat Rev Immunol. 5(3):201-214. To overcome the host's innate immune response, other researchers have tried to introduce tolerogenic factors back into the target cell; the focus was on "losing one's own". See WO 2016183041 A2, which is hereby incorporated by reference in its entirety. The present inventors surprisingly found that the key to evading the host's NK-mediated immune response is not "losing one's self" but the expression and level of expression of NK cell activating ligands on target cells.

[006] Thus, there remains a need for compositions and methods for developing target cells that lack some or all of the classical LAS expression, but that are not attacked by NK cells and subsequently lysed.

BRIEF DESCRIPTION OF THE PRESENT INVENTION

[007] Strategies for overcoming transplant rejection, in particular allogeneic immune transplant rejection in cell transplantation therapy, are disclosed herein by providing universal donor cell lines. In one embodiment of the present invention, human pluripotent stem cells are provided that lack some or all of the classical expression of the class I LAS protein on the cell surface and the expression of an NK cell activating ligand. In one embodiment, the present invention provides a cell derived from a human pluripotent stem cell, such as a pancreatic cell, that lacks some or all of the classical cell surface expression of LAH class I protein and NK-activating ligand expression. In one embodiment, the present invention provides a method for preventing cell transplant rejection by providing transplanted pancreatic cells in which at least one MHC gene, such as beta-2-microglobulin (B2M), and at least one NK activating ligand gene, such as Intercellular adhesion molecule 1 (ICAM-1), destroyed, removed, modified or inhibited. In another embodiment, the present invention provides a method for preventing cell transplant rejection by providing transplanted pancreatic cells in which the expression of at least one MHC protein, such as B2M, and at least one NK cell activating protein, such as ICAM-1 , destroyed, removed, modified or inhibited. Destruction, removal, modification, or inhibition of B2M leads to a deficiency in the expression and function of class I LAH on the cell surface.

BRIEF DESCRIPTION OF THE FIGURES

[008] FIG. 1 is a reproduction from an article by Haynes et al., supra, Figure 372e-4 (which is incorporated herein in its entirety), showing various scenarios for an NK cell mediated response to target cells. In the absence of MHC class I and in the absence of NK-activating ligands on the target cell, inhibitory and activating receptors on NK cells are not activated and NK cells remain inactive (Scenario A). In the presence of MHC class I, but in the absence of NK-activating ligands on the target cell, inhibitory receptors on NK cells are activated, but activating receptors on NK cells are not activated and NK cells remain inactive (Scenario B). In the absence of native MHC class I, but in the presence of NK-activating ligands on the target cell, inhibitory receptors on NK cells are not activated, but activating receptors on NK cells are activated and NK cells attack (Scenario C). In the presence of MHC class I and NK-activating ligands on the target cell, inhibitory and activating receptors on NK cells are activated and the result is determined by the signal balance.

[009] In FIG. 2A shows a representative flow cytometry analysis of cell surface B2M protein expression in wild-type hES cells without IFN-γ (Line B) and after treatment with IFN-γ (Line A). The shaded area denotes baseline expression without antibody staining. IFN-γ treatment increases cell surface B2M protein expression in wild-type hES cells.

[0010] In FIG. 2B shows a representative flow cytometry analysis of cell surface B2M protein expression in B2M knockout (B2M -/-) hES cells without IFN-γ (Line B) and after treatment with IFN-γ (Line A). B2M -/- hES cells have very little cell surface B2M protein expression, which does not change significantly after treatment with IFN-γ.

[0011] In FIG. 3A shows a representative flow cytometry analysis of cell surface LAH-ABC protein expression in wild-type hES cells without IFN-γ (Line B) and after treatment with IFN-γ (Line A) using pan-LAH class I antibodies. The shaded area denotes baseline expression. without staining with antibodies.

[0012] In FIG. 3B shows a representative flow cytometry analysis of cell surface LAH-ABC protein expression in B2M knockout hES cells without IFN-γ (Line B) and after treatment with IFN-γ (Line A). hES B2M -/- cells do not express the LAH-ABC cell surface protein.

[0013] In FIG. 4A shows a representative flow cytometry analysis of cell surface B2M protein expression in wild-type pancreatic endoderm (PEC) cells without IFN-γ (Line B) and after treatment with IFN-γ (Line A). The shaded area denotes baseline expression without antibody staining. IFN-γ treatment upregulates cell surface B2M protein expression in wild-type PEC cells.

[0014] In FIG. 4B shows a representative flow cytometry analysis of cell surface B2M protein expression in B2M knockout PEC cells without IFN-γ (Line B) and after treatment with IFN-γ (Line A). B2M -/- PEC cells do not express the B2M cell surface protein.

[0015] In FIG. 5A shows a representative flow cytometry analysis of cell surface LAH-ABC protein expression in wild-type PEC cells without IFN-γ (Line B) and after treatment with IFN-γ (Line A). The shaded area denotes baseline expression without antibody staining. IFN-γ treatment upregulates cell surface LAH-ABC protein expression in wild-type RES cells.

[0016] In FIG. 5B shows a representative flow cytometry analysis of cell surface LAH-ABC protein expression in B2M knockout PEC cells without IFN-γ (Line B) and after treatment with IFN-γ (Line A). B2M -/- RES cells do not express the LAH-ABC cell surface protein.

[0017] In FIG. 6A shows a representative flow cytometry analysis of cell surface ICAM-1 protein expression in wild-type hES cells without IFN-γ (Line B) and after treatment with IFN-γ (Line A). The shaded area denotes baseline expression without antibody staining. IFN-γ treatment increases cell surface protein expression of ICAM-1 in wild-type hES cells.

[0018] In FIG. 6B shows a representative flow cytometry analysis of cell surface ICAM-1 protein expression in B2M knockout hES cells without IFN-γ (Line B) and after IFN-γ treatment (Line A). B2M -/- hES cells have cell surface protein ICAM-1 expression similar to that in wild-type hES cells before and after IFN-γ treatment.

[0019] In FIG. 7A shows a representative flow cytometry analysis of cell surface ICAM-1 protein expression in wild-type PEC cells without IFN-γ (Line B) and after treatment with IFN-γ (Line A). The shaded area denotes baseline expression without antibody staining. IFN-γ treatment upregulates cell surface protein ICAM-1 expression in wild-type RES cells.

[0020] In FIG. 7B shows a representative flow cytometry analysis of cell surface ICAM-1 protein expression in B2M knockout PEC cells without IFN-γ (Line B) and after treatment with IFN-γ (Line A). After treatment with IFN-γ, B2M -/- PEC cells have cell surface protein ICAM-1 expression similar to that in wild-type PEC cells, which is higher than baseline expression (shaded area).

[0021] In FIG. 8 is a bar graph showing ICAM-1 mRNA expression data (Asymetrix Biochip Expression Assay) in wild-type hES cells, B2M (-/-) hES cells, wild-type PEC cells, and B2M (-/-) PEC cells, each of which not treated with IFN-γ (control) or treated with IFN-γ. ICAM-1 expression was also determined in cells known to have low cell surface ICAM protein expression: cancer cells (K562 and SKBR3), transplanted PEC cells that have matured into cells that produce insulin in vivo, islets human cells and two different samples of peripheral blood mononuclear cells (PBMC) (without IFN-γ treatment). Expression of ICAM-1 mRNA was increased after treatment of hES (wild-type or B2M-/-) or PEC (wild-type or B2M-/-) cells with IFN-γ.

[0022] In FIG. 9 shows a representative flow cytometry analysis of cell surface expression of CD58 proteins (designated: LFA-3) in wild-type PEC cells without IFN-γ treatment (Line B) and after IFN-γ treatment (Line A). Line C indicates baseline expression without antibody staining. IFN-γ treatment slightly increased the expression of cell surface CD58 proteins in wild-type PEC cells compared to the untreated control. We used antibodies from BioLegend, cat. No. 330909.

[0023] In FIG. 10 shows a representative flow cytometry analysis of cell surface CD155 protein expression on wild-type PEC cells without IFN-γ treatment (Line B) and after IFN-γ treatment (Line A). Line C indicates baseline expression without antibody staining. After treatment with IFN-γ, wild-type RES cells had cell surface CD155 protein expression similar to that in untreated control wild-type RES cells. PVR gene designation (designated as: CD 155, NECL-5, HVED). Antibodies from Milteneyi Biotech Inc., cat. No. 130-105-905.

[0024] In FIG. 11 shows a representative flow cytometry analysis of cell surface expression of CEACAM1 proteins (designated: CD66a, BGP, BGP1) in wild-type PEC cells without IFN-γ treatment (Line B) and after IFN-γ treatment (Line A). Line C indicates baseline expression without antibody staining. IFN-γ treatment slightly increased the expression of cell surface CEACAM1 proteins in wild-type PEC cells compared to untreated controls. Antibodies from Milteneyi Biotech Inc., cat. No. 130-098-858.

[0025] In FIG. 12 shows a representative flow cytometry analysis of cell surface BAT3 protein expression in wild-type PEC cells without IFN-γ treatment (Line B) and after IFN-γ treatment (Line A). Line C indicates baseline expression without antibody staining. Wild-type RES cells in the untreated control have cell surface BAT3 protein expression similar to that in wild-type RES treated with IFN-γ. Designation of the BAG6 gene (designated as: BAT3, transcript 3 associated with LAH-B). Used antibodies company Abeam, Inc., cat. No. ab210838.

[0026] In FIG. 13 shows a representative flow cytometry analysis of cell surface expression of CADM1 proteins (designated as: NECL2, TSLC1, IGSF4, RA175) in wild-type PEC cells without IFN-γ treatment (Line B) and after IFN-γ treatment (Line A). Line C indicates baseline expression without antibody staining. After treatment with IFN-γ, wild-type PEC cells have cell surface CADM1 protein expression similar to that of the untreated control. Antibodies from MBL International Corp. were used. cat. No. CM004-4.

[0027] In FIG. 14 shows a representative flow cytometry analysis of cell surface CD112 protein expression in wild-type PEC cells without IFN-γ treatment (Line B) and after IFN-γ treatment (Line A). Line C indicates baseline expression without antibody staining. After treatment with IFN-γ, wild-type RES cells have expression of cell surface CD 112 proteins similar to that of the untreated control. PVRL2 gene designation (designated as; CD112, Nectin-2, PVRR2, HVEB). Antibodies from Milteneyi Biotech Inc., cat. No. 130-109-056.

[0028] In FIG. 15 shows a histogram demonstrating the reduction in NK target cell lysis after blocking ICAM-1 expression in target cells with anti-ICAM1 antibodies (left to right, wild-type hESC cells, IFN-γ treated hESC cells, B2M -/-hESC cells , B2M -/-hES cells treated with IFN-γ, wild-type RES cells, wild-type RES cells treated with IFN-γ, B2M -/- PEC cells, B2M -/-PEC cells treated with IFN-γ, control cell line K562 for NK cytotoxicity assay). From left to right, each set of three columns represents: NK-062216; NK-062216 + ICAM1 AB (5 µg/ml); NK-062216+ICAM1 AB (10 µg/ml).

SEQUENCE LIST

[0029] The nucleic acid and amino acid sequences given in the accompanying sequence listing are given using the standard letter abbreviations for nucleic acid bases and the three letter codes for amino acids established by 37 C.F.R. 1.822. Only one strand of each of the nucleic acids is shown, but it should be understood that the corresponding complementary strand is included in addition to the strand shown. This sequence listing is provided via an ASCII text file called Sequence_Listing, dated July 3, 2018, 11 KB in size, which is incorporated herein by reference.

DETAILED DESCRIPTION

[0030] Class I MHC molecules are one of two main classes of major histocompatibility complex (MCHC) molecules (the second class being class II MHC molecules). Their function is to expose peptide fragments of foreign proteins from cells to cytotoxic T cells; this elicits an immediate immune system response against a specific foreign antigen exposed by the MHC class I protein. F and LACH-G. Human LAHS-E, LAHS-F and LAHS-G molecules are non-classical MHC class I molecules characterized by limited polymorphism and low expression on the cell surface compared to classical paralogs (LAH-A, -B and -C). All MHC class I proteins must be associated with p2-microglobulin (B2M) to obtain a functional heterodimeric MHC class I protein complex prior to functional cell surface expression. Class I MHC molecules can also act as inhibitory ligands for NK cells. A decrease in normal levels of MHC class I molecules on the cell surface activates the killing of NK cells.

[0031] Historically, target cells containing MHC class I inhibitory ligands have been thought to avoid attack when NK cells are treated due to the presumed dominant nature of the MHC class I inhibitory signal (see Figure 1 Scenario B, reproduced from "Harrison's Principles of Internal Medicine 19 E" (vol. 1 and vol. 2) A Major Histocompatibility Complex, part 15, figs. 372e-4, in this document). But the applicants unexpectedly found that it was quite the opposite.

[0032] Exposure of cells to IFN-γ has been shown to increase mRNA expression of MHC class I molecules as well as expression of the MHC class I protein complex on the cell surface. This increase in MHC-1 class expression was expected to inhibit NK cells. Applicants found that wild-type (WT) hES cells, when treated with IFN-γ (which has been shown to increase the number of MHC class I molecules on the surface of hES cells, in Figures 2A and 3A), increased NK cell-mediated cytotoxicity. See Fig. 15 showing NK cell-mediated toxicity (lysis) increasing from 58% to 79% (compare the first bars under conditions 1 and 2 in FIG. 15). The same was true when wild-type RES cells were treated with IFN-γ, these cells also overexpressing B2M and LAH-ABC, see FIG. 4A and 5A where HNK-mediated toxicity (lysis) increased from 33% to 53% (compare the first bars under conditions 5 and 6 in FIG. 15). These data suggest that the key factor in overcoming the immune response of host NK cells is not the overexpression of MHC class I inhibitory signals, but the blocking of NK-activating ligand signals.

[0033] To further test this hypothesis in the context of the pronounced cytotoxicity mediated by NK cells, Applicants created hES B2M -/- (knockout) cells (similar to the cells in Fig. 1, Scenario C). As expected, B2M -/- cells abolished cell surface expression of MHC class I molecules on hES (FIGS. 2B and 3B) and PEC (FIGS. 4B and 5B) cells. Also, as expected, B2M -/- cells showed an increase in NK-mediated lysis relative to WT cells, from 58% to 71% for hES cells and from 33% to 54% for PEC cells (compare the first columns under conditions 1 vs. 3 or 5 versus 7 in Fig. 15). Applicants found that exposure of B2M -/- hES or PEC cells to IFN-γ further increased the percentage of NK cell-mediated toxicity (lysis) (compare the first columns under conditions 2 vs. 4 and 6 vs. 8 in Fig. 15). Accordingly, Applicants have found that cell surface expression of NK cell activating ligand (FIGS. 6B and 7B) and mRNA expression (FIG. 8) are increased by treatment with IFN-γ. These data suggested that NK cell activating ligands on the target cell play a critical role in NK cell cytotoxicity and led to the hypothesis that inhibition of NK cell activating ligand expression may protect against NK cell-mediated cytotoxicity in in the context of reduced MCH class I expression, for example in the context of B2M -/-.

[0034] In order to determine whether the toxicity of NK cells can be reduced by inhibiting the action of NK activating ligands on target cells, Applicants blocked the expression of NK activating ligand in WT and hES B2M -/- and PEC cells, for example, using blocking antibody ICAM1 to block the ICAM1 protein on the surface of target cells. Applicants surprisingly found that target cell lysis was reduced (compare the first columns with the second and third columns for conditions 2, 4, 6 and 8 in FIG. 15). Thus, Applicants have found that NK cell lysis can be reduced by blocking the NK activating ligand. Blocking ICAM1 expression using an anti-NK activating ligand antibody in B2M −/− cells is a proof of concept for obtaining a double knockout cell (class I LAS gene knockout and NK activating ligand gene knockout). In doing so, Applicants may transfer target cells (eg, hES cells and/or pancreatic cells) from Scenario C to Scenario A in FIG. 1. Specifically, the cells, tissues, and organs of the present invention inhibited or did not inhibit the expression of LH class I cell surface proteins (B2M -/-) and inhibited or did not inhibit the expression of NK activating ligand, the expression of cell surface proteins (e.g., ICAM1 - /-). Inhibition of cell surface protein expression can be achieved by gene knockout or by blocking protein expression using antibodies. Other strategies to interfere with cell surface protein expression include the use of antisense RNAs, RNA decoys, ribozymes, RNA aptamers, miRNAs, shRNA/miRNAs, transdominant negative proteins (TNPs), chimeric/fusion proteins, nucleases, chemokine ligands, anti-infective cellular proteins , intracellular antibodies (sFv), nucleoside analogs (NRT1), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides and chemical agents) and protease inhibitors. Double or multiple gene knockout effectively prevents both cytotoxic T cell (CTL) and NK cell mediated toxicity because class I LAS are present in low levels or are virtually absent and NK activating protein ligands that could be contact is practically not expressed on the surface of CTL or NK cells. In addition, to completely eliminate NK activation, Applicants expect that the expression of multiple NK activating ligands should be abolished/reduced either by gene knockout in the target cell (e.g. hES cell based cell therapy) or by blocking antibodies or other strategies currently known or developed in the future.

Agents that block ligands that activate NK cells

[0035] According to one aspect, the present invention provides a method for suppressing NK cell function treatment. According to another aspect, the present invention provides a method for suppressing at least one immune response. Each of the methods includes administering to a subject in need of treatment an agent that inhibits NK cell function. In some embodiments, said agent is an antibody. In some embodiments, said antibody selectively binds to ligands on target cells that activate NK cells.

[0036] It is contemplated that various types of reagents, including antibodies and blocking proteins, can be used to prevent adhesion between NK cells and ligands on target cells that activate NK cells.

[0037] In some embodiments, such NK activating ligands may be selected from Table 1.

NK-activating ligands are also described in Pegram et al., Activating and inhibiting receptors of NK-cells, Immunology and Cell Biology (2011) 89, 216-224, which is incorporated herein by reference in its entirety.

[0038] Hypoimmunogenic hES cells and their derivatives

[0039] LAHs are cell surface molecules that are encoded by a large family of genes and that can be divided into class I and class II molecules. Class 1 LAS molecules are found on the surface of every nuclear cell and are targeted by the present invention. Mismatch of LAH molecules between donor cells (target cells) and recipient immune cells (eg, T cells) during transplantation often results in immune or graft rejection. Class I LAS complexes are structurally composed of polymorphic heavy chains composed of LH class I peptides (eg, LH-A, LH-B, and LH-C) and beta-2-microglobulin light chains (β2m or B2M). In the absence of B2M, class I LAS complexes cannot assemble correctly and are also not expressed on the cell surface or cell membrane. In the invention described herein, Applicants have created cell lines of hES and cells derived from them by disrupting (adding and removing several base pairs, creating a frameshift in the mRNA encoding the protein, and introducing mutations that cause loss of activity) of the B2M gene, and thus a strong reduction in the expression of class 1 LAS on the surface of hESC cells.

[0040] The above methodology can also be used to obtain or generate hES cell lines and their derivatives by further disrupting genes that encode NK-activating ligands such as ICAM1. Thus, in one embodiment, the present invention provides compositions and methods for producing a target cell that lacks at least one LAT class I antigen and at least one NK activating ligand, thereby creating a hypoimmunogenic cell. It is expected that such a hypoimmunogenic cell will be less susceptible to immune rejection by the subject into which such cells are transplanted. In transplantation, this hypoimmunogenic cell must engraft (and must not be rejected). In one embodiment, such a target cell is able to engraft and survive with little or no immune suppression required from the recipient.

[0041] In one embodiment, inhibition, reduction, and/or deletion of both class I LAS expression and NK activating ligand expression (or class I LAS deficiency and NK activating ligand deficiency) in hESC cells and derived cells may serve as a universal source of donor cells for transplantation therapy. These double knockouts (class I LAS deficiency and NK activating ligand deficiency) can be transplanted ubiquitously without minor histocompatibility complex (MiHC) match, human leukocyte antigen (HLA) match, or immune suppression.

[0042] This document discloses new in vitro derived hypoimmunogenic compositions and cells. In particular, in certain embodiments, the present inventions disclosed herein relate to a stem cell whose genome has been altered (modified) to reduce or remove critical components of both the MHC class I gene(s) and the ligand gene(s), activating NK. In certain embodiments, the present inventions disclosed herein relate to pancreatic cell lines such as pancreatic endoderm cells, pancreatic epithelial cells, pancreatic progenitor cells, pancreatic endocrine progenitor cells, pancreatic endocrine cells, pre-beta pancreatic cells or pancreatic beta cells whose genome has been altered (modified) to reduce or remove critical components of both the MHC class I gene(s) and the NK activating ligand gene(s), thereby generating hypoimmunogenic cell lines pancreatic type. Natural killer activating ligands include, but are not limited to, the ligands listed in Table 1 from Category 1, 2, 3, or combinations thereof. Natural killer activating ligands include, for example, ICAM-1, CEACAM1, CADM1, MICA, and MICB. Class I MHC genes include LACH-A, LACH-B, LACH-S, LACH-E, LACH-F, LACH-G, and V2M. In certain aspects, such downregulation or knockout of MHC class I and/or MHC class II genes is accomplished by direct and/or indirect targeting of the NLRC5, B2M, and CIITA genes and other components of the MHC enhancersome (the enhancer is a higher order protein complex assembled on an enhancer and regulating the expression of the target gene (for example, MHC class I or MHC class II transcription regulators).

[0043] Methods for producing hypoimmunogenic cells are also disclosed herein, which method includes modulating the expression of one or more NK activating ligands expressed by the cell, and modulating the expression of one or more MHC class I and/or MHC class II by said cell, thereby thereby obtaining a hypoimmunogenic cell. In certain aspects, modulating the expression of cell surface proteins of one or more MHC class I and/or MHC class II complexes includes reducing, inhibiting and/or interfering with the expression of one or more MHC class I and/or MHC class II genes or proteins. In some embodiments, modulating the expression of one or more MHC class I and/or MHC class II complexes comprises removing one or more genes encoding one or more MHC class I or MHC class II transcriptional regulators from at least one allele of said cell. For example, in certain embodiments, such methods include removing one or more genes encoding one or more MHC class I or MHC class II transcriptional regulators selected from the group consisting of LRC5, CIITA, B2M, and combinations thereof. In certain aspects, modulating the expression of one or more NK activating ligands includes removing, inhibiting, or reducing the expression of one or more genes encoding NK activating ligands. In certain embodiments, such NK activating ligands are selected from Table 1. In certain embodiments, such NK activating ligands are selected from Table 1, category 1, 2, 3, or combinations thereof. In some embodiments, such NK activating ligands are selected from Table 1, categories 1, 2, and 3. In some embodiments, such NK activating ligands are selected from Table 1, categories 1 and 3. In some embodiments, such NK activating ligands are selected from Table 1 categories 1 and 2. In certain embodiments, such NK activating ligands are selected from Table 1, categories 2 and 3. In certain embodiments, such NK activating ligands are selected from the group consisting of ICAM-1, CEACAM1, CADM1 MICA, MICB and their combinations.

[0044] In some embodiments, said hypoimmunogenic cells to be implanted are in an animal product free environment, such as "XenoFree" products.

[0045] The present invention contemplates making changes to the target polynucleotide sequences by any means that are available to a person skilled in the art, for example, using any meganucleases or endodeoxyribocleases, nucleases with zinc finger protein domains (ZFN or ZNF), an effector nuclease like transcription activators (TALEN) or systems based on short palindromic repeats regularly arranged in groups (CRISPR/Cas or CRISPR/Cas9) or traditional homologous recombination techniques. Such CRISPR/Cas systems can use a wide variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005; 1(6)e60). In some embodiments, said CRISPR/Cas system is a type I CRISPR system. In some embodiments, said CRISPR/Cas system is a type II CRISPR system. In some embodiments, said CRISPR/Cas system is a type V CRISPR system. The NEXTGEN™ CRISPR system (Transposagen Inc., Lexington, KY), which includes a bidirectional RNA and a catalytically inactive Cas9 protein, can also be used to make changes to target polynucleotide sequences. , fused with FokI nuclease. To create these hypoimmunogenic cells described herein, other methods of changing the sequences of polynucleotides to reduce or suppress their expression in target cells, known to a person skilled in the art or discovered later, can be used.

[0046] In some embodiments, these changes result in reduced expression of the target polynucleotide sequence. In some embodiments, these changes are homozygous changes. In some embodiments, these changes are heterozygous changes.

[0047] In some embodiments, the target polynucleotide sequence is a genome sequence. In some embodiments, said target polynucleotide sequence is a human genome sequence. In some embodiments, said target polynucleotide sequence is a mammalian genome sequence. In some embodiments, said target polynucleotide sequence is a vertebrate genome sequence.

[0048] In some embodiments, said hypoimmunogenic cells are embryonic stem cells. In some embodiments, said hypoimmunogenic cells are pluripotent stem cells. In some embodiments, said hypoimmunogenic cells are induced pluripotent stem cells, reprogrammed cells, dedifferentiated or transdifferentiated cells. In some embodiments, said hypoimmunogenic cells are multipotent pancreatic progenitor cells. In some embodiments, said hypoimmunogenic cells are monohormonal or polyhormonal cells. In some embodiments, said hypoimmunogenic cells are mesendodermal cells, definitive endoderm cells, PDX1 negative foregut endoderm cells, PDX1 positive foregut endodermal cells, pancreatic endoderm cells, endocrine progenitor cells, endocrine cells, correctly identified endocrine cells, immature endocrine cells or functional beta cells. In some embodiments, said hypoimmunogenic cells may be homogeneous or heterogeneous populations of cells. In some embodiments, said hypoimmunogenic cells are cells that produce one or more biologically active substances of interest. Hypoimmunogenic cells may initially be therapeutically inactive upon transplantation, for example, they may be pancreatic progenitor cells or PDX1-positive pancreatic endoderm cells, but after transplantation they further develop, mature and exert a therapeutic effect.

[0049] In some embodiments, said hypoimmunogenic cells can be any cell that can be derived from human pluripotent stem cells, including any cell, tissue, or organ, and can include skin cells, beta cells (i.e., pancreatic cells located in the islets of Langerhans), parathyroid cells, intestinal cells, endocrine cells, heart cells, brain cells, kidney cells, liver cells, cells of the gastrointestinal tract and auxiliary organs of digestion, salivary gland cells, cells of the adrenal glands, prostate cells, lung cells, pancreas cells, bone cells, immune system cells, hematopoietic cells, vascular cells, eye cells, connective tissue cells, skeletal muscle cells, bone tissue, musculoskeletal tissue, corneal tissue, skin tissue, valves but limited to the heart, blood vessels, immune cells, connective tissue, lung tissue, skin, cornea, kidney, liver, lung, pancreas, heart, and intestines.

[0050] In some embodiments, said hypoimmunogenic cells may be individual (single) cells in suspension or in aggregates of cells. In some embodiments, said hypoimmunogenic cells include totipotent cells. In one embodiment, said hypoimmunogenic cells include multipotent cells. In one embodiment, said hypoimmunogenic cells include unipotent cells.

[0051] In some embodiments, said hypoimmunogenic cells are derived from a population of pluripotent cells lacking functional expression of class I LAS and expression of NK-activating ligands. Said derived cells may be selected from the group consisting of: any cell, tissue, or organ, and may include skin cells, beta cells (i.e. pancreatic cells located in the islets of Langerhans), parathyroid cells, intestinal cells, endocrine cells of the heart, brain cells, kidney cells, liver cells, cells of the digestive tract and auxiliary organs of digestion, salivary gland cells, adrenal gland cells, prostate cells, lung cells, pancreatic cells, bone cells, immune cells, hematopoietic cells, vascular cells, eye cells, connective tissue cells, musculoskeletal cells, bone tissue, musculoskeletal tissue, corneal tissue, skin tissue, heart valves, blood vessels, immune cells, connective tissue, lung tissue, skin, cornea, kidney, liver , lung, pancreas, heart and intestines.

[0052] The hypoimmunogenic cell, tissue, and/or organ to be transplanted may be syngeneic or allogeneic to the subject receiving the transplant.

[0053] In one embodiment, the hypoimmunogenic cell is a human pluripotent cell. In one embodiment, the hypoimmunogenic cell is a human pancreatic cell. In one embodiment, the hypoimmunogenic cell is a human pancreatic endoderm cell. In one embodiment, the hypoimmunogenic cell is a human pancreatic progenitor cell. In one embodiment, the hypoimmunogenic cell is a human pancreatic progenitor cell. In one embodiment, the hypoimmunogenic cell is a human pancreatic endocrine cell. In one embodiment, the hypoimmunogenic cell is a human pancreatic endocrine progenitor cell. In one embodiment, the hypoimmunogenic cell is a human pancreatic endocrine pre-beta cell. In one embodiment, the hypoimmunogenic cell is a human pancreatic beta cell. In one embodiment, the hypoimmunogenic cell is a single-hormonal or multi-hormonal human pancreatic cell. In one embodiment, the hypoimmunogenic cell is a cell that expresses human insulin.

[0054] In one embodiment, the hypoimmunogenic cells are well known, publicly available pluripotent cell lines. The invention described here is useful for all hES cells and iPSC lines, and at least for hESCs such as CyT49, CyT25, CyT203 and CyT212. The pluripotent cell line includes commercially available cells available from WiCell on the Internet at: wicell.org/home/stem-cell-lines/order-stem-cell-lines/obtain-stem-cell-lines.cmsx and, in specifically include BG01, BG02 and BG03.

[0055] In one embodiment, the hypoimmunogenic cells are substantially similar to those described in D'Amour et al. "Production of Pancreatic Hormone-Expressing Endocrine Cells From Human Embryonic Stem Cells" (Nov. 1, 2006) Mature Biotechnology 24, 1392-1401, which is incorporated herein by reference in its entirety. An article by D'Amour et al. describes a 5-step differentiation protocol: stage 1 (results mainly in endoderm production), stage 2 (results mainly in production of PDX1-negative foregut endoderm), stage 3 (results mainly in PDX1 production -positive foregut endoderm), stage 4 (results mainly in the production of pancreatic endoderm, also called multipotent pancreatic progenitors or production of pancreatic endocrine progenitors), and stage 5 (results mainly in the production of hormone-expressing endocrine cells). In one embodiment, said hypoimmunogenic cells are substantially the same as those described in US Pat.

[0056] In one embodiment, said hypoimmunogenic cells are substantially similar to those described in Schulz et al. A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells, PLoS One 7:5 1-17 (2012), which is incorporated herein by reference in its entirety. The article by Schulz et al. describes methods for growing and preserving hESCs and a suspension-based differentiation system. In particular, undifferentiated pluripotent cells were aggregated into clusters in a dynamic rotary suspension culture followed by total differentiation for two weeks according to a four-stage protocol. Briefly, from hES cell aggregate suspensions, hESC monolayers are dissociated from Accutase (Innovative Cell Technologies), harvested and resuspended at 1×10 ⁶ cells/mL in StemPro hESC SFM (Life Technologies; DMEM/F12 combo media containing Glutamax, StemPro hESC supplement , BSA, and 1% (v/v) penicillin/streptomycin; without FGF-2 and 2-mercaptoethanol). Single cell suspensions were dispensed into 6-well plates untreated with TC (5.5 ml/well) and rotated at 95 rpm on an Innova 2000 rotor (New Brunswick Scientific) or dispensed into 500 ml filtered Nalgene storage vials ( 150 ml/bottle) and rotated at 65 rpm on a Sartorius Certomat RM-50 rotor (5 cm rotation axis). Cells were shaken overnight in an incubator at 37°C/8% CO ₂ and formed aggregates with a size of approximately 100-200 μm. For aggregates with a diameter of 100 to 200 µm, rotation speeds from 60 to 140 rpm can be used for a 6-well plate; for 500 ml bottles, a rotation speed of 5-20 rpm can be used. The suspension aggregate differentiation included only a few modifications from D'Amour. The TGF-βRI kinase inhibitor IV was added during step 2, and retinoic acid was replaced with a more stable retinoid analog, TTNPB (3 nM), in step 3. Growth factors KGF (50 ng/mL) and EGF (50 ng/mL) were added at stage 4 to preserve cell mass. Noggin (50 ng/ml) was also added in step 4. In one embodiment, the hypoimmunogenic cells are essentially the same as those described in US Pat.

[0057] In one embodiment, the hypoimmunogenic cells are substantially similar to those described in Agulnick et al. Insulin-Producing Endocrine Cells Differentiated In Vitro From Human Embryonic Stem Cells Function in Macroencapsulation Devices In Vivo Stem Cells. Translational Medicine, 4: 1-9 (2015), which is incorporated herein by reference in its entirety. An article by Agulnick et al. describes a modified protocol for generating pancreatic progenitor cells such that 73-80% of the cell population is composed of PDX1-positive (PDX1+) HNKX6.1+ pancreatic progenitors. Pancreatic progenitor cells are further differentiated into islet cells (ICs), which reproducibly contained 73-89% endocrine cells, of which approximately 40-50% expressed insulin. Most of these insulin-positive cells were single-hormone positive and expressed the transcription factors PDX1 and NKX6.1. An article by Agulnick et al. describes a protocol in which the 2012 protocol by Schulz et al. was modified by additional treatment with activin A, Wnt3A, and heregulin β1 at stage 3 (days 5-7) and with activin A and heregulin β1 at stage 4 (days 7). -13). In one embodiment, said hypoimmunogenic cells are substantially similar to those described in US Pat. No. 8,859,286, which is incorporated herein by reference in its entirety.

[0058] Growth, passage, and proliferation of human embryonic stem cells can be performed essentially as described in US Pat. Nos. 7,964,402; 8,211,699; 8,334,138; 8.008.07; and 8,153,429.

[0059] Standard Acquisition Protocol

[0060] A standard protocol for obtaining a pancreatic endoderm cell (PEC) derived from hESC is described below in Table 2.

[0061] HESC Agg.: hESC aggregates; XF HA: DMEM/F12 containing GlutaMAX supplemented with 10% v/v Xeno-free KnockOut Serum replacement, 1% v/v non-essential amino acids, 1% v/v penicillin/streptomycin (all from Life Technologies), 10 ng/ml heregulin -1β (Peprotech) and 10 ng/ml Activin A (R&D Systems); SP: StemPro® hESC SFM (Life Technologies); r0.2FBS: RPMI 1640 (Mediatech); 0.2% FBS (HyClone), 1x GlutaMAX-1 (Life Technologies), 1% v/v penicillin/streptomycin; ITS: insulin-transferrin-selenium (Life Technoiogies), diluted 1:5000 or 1:1000; A100: 100 ng/mL recombinant human activin A (R&D Systems); W50: 50 ng/ml recombinant mouse Wnt3A (R&D Systems); K25: 25 ng/ml recombinant human KGF (R&D Systems); IV: 2.5 μM TGF-β RI kinase inhibitor IV (EMD Bioscience); db: DMEM HI glucose (HyClone) supplemented with 0.5x B-27 supplement (Life Technologies), 1x GlutaMAX, and 1% v/v penicillin/streptomycin; CTT3: 0.25 μM KAAD-cyclopamine (Toronto Research Chemicals) and 3 nM TTNPB (Sigma-Aldrich); N50: 50 ng/ml recombinant human Noggin (R&D Systems); K50: 50 ng/ml recombinant human KGF (R&D Systems); E50: 50 ng/ml recombinant human EGF (R&D Systems).

[0062] Calcein release assay

[0063] The calcein release assay is an alternative to studying NK cell cytotoxicity that does not use radioactive isotopes. Target cells transport the fluorescent dye (calcein AM) inside and convert it in the cytoplasm into an active fluorochrome, which is released from the cell only in case of its lysis. The lysed cells release said fluorochrome into the supernatant, which is then collected and its fluorescence quantified with a fluorometer. Percent cell lysis is calculated from the amount of fluorescence present in the supernatant after incubation with or without NK cells (effectors), blocking antibody, or both.

[0064] Specific lysis is calculated by the formula, % lysis = 100×[(average fluorescence with antibody - average spontaneous fluorescence)/(average maximum fluorescence - average spontaneous fluorescence)]. Maximal fluorescence was determined by lysis of cells incubated with detergent (1% Triton X-100) and spontaneous lysis was defined as fluorescence obtained with target cells without any antibodies or effector cells.

[0065] Various cell compositions derived from pluripotent stem cells and methods for their preparation are described herein and can also be found in the present Applicant's US Patent Applications Nos. August 6, 2002; 11/021,618 titled DEFINITIVE ENDODERM, filed December 23, 2004; 11/115,868 titled PDX1 EXPRESSING ENDODERM, filed April 26, 2005; 11/165,305 titled METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIATING DEFINITIVE ENDODERM, filed June 23, 2005; 11/573,662 entitled METHODS FOR INCREASING DEFINITIVE ENDODERM DIFFERENTIATION OF PLURIPOTENT HUMAN EMBRYONIC STEM CELLS WITH PI-3 KINASE INHIBITORS, filed August 15, 2005; 12/729, 084 titled PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM, filed October 27, 2005; 12/093,590 titled MARKERS OF DEFINITIVE ENDODERM, filed November 14, 2005; 11/993,399 entitled EMBRYONIC STEM CELL CULTURE COMPOSITIONS AND METHODS OF USE THEREOF, filed June 20, 2006; 11/588,693 titled PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM filed October 27, 2006; 11/681,687 entitled ENDOCRINE PROGENITOR/PRECURSOR CELLS, PANCREATIC HORMONE-EXPRESSING CELLS AND METHODS OF PRODUCTION, filed March 2, 2007; 11/807,223 entitled METHODS FOR CULTURE AND PRODUCTION OF SINGLE CELL POPULATIONS OF HESC, filed March 24, 2007; 11/773,944 entitled METHODS OF PRODUCING PANCREATIC HORMONES, filed July 5, 2007; 11/860,494 entitled METHODS FOR INCREASING DEFINITIVE ENDODERM PRODUCTION, filed September 24, 2007; 12/099,759 titled METHODS OF PRODUCING PANCREATIC HORMONES, filed April 8, 2008; 12/107,020 entitled METHODS FOR PURIFYING ENDODERM AND PANCREATIC ENDODERM CELLS DERIVED FORM HUMAN EMBRYONIC STEM CELLS, filed April 21, 2008; 12/618,659 entitled ENCAPSULATION OF PANCREATIC LINEAGE CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS, filed November 13, 2009; 12/765,714 and 13/761,078, both titled CELL COMPOSITIONS FROM DEDIFFERENTIATED REPROGRAMMED CELLS, filed April 22, 2010 and February 6, 2013; 11/838,054, titled COMPOSITIONS AND METHODS USEFUL FOR CULTURING DIFFERENTIABLE CELLS, filed August 13, 2007; 12/264,760 entitled STEM CELL AGGREGATE SUSPENSION COMPOSITIONS AND METHODS OF DIFFERENTIATION THEREOF, filed November 4, 2008; 13/259.15 titled SMALL MOLECULES SUPPORTING PLURIPOTENT CELL GROWTH filed April 27, 2010; PCT/US11/25628, entitled LOADING SYSTEM FOR AN ENCAPSULATION DEVICE, filed February 21, 2011; 13/992,931, entitled AGENTS AND METHODS FOR INHIBITING PLURIPOTENT STEM CELLS, filed December 28, 2010; and U.S. Design Applications Nos. 29/408,366, filed December 12, 2011; 29/408,368, filed December 12, 2011; 29/423,365, filed May 31, 2012; and 29/447,944, filed March 13, 2013; U.S. Application No. 14/201,630 titled 3-DIMENSIONAL LARGE CAPACITY CELL ENCAPSULATION DEVICE ASSEMBLY, filed March 7, 2014; and US Application No. 14/106,330 titled IN VITRO DIFFERENTIATION OF PLURIPOTENT STEM CELLS TO PANCREATIC ENDODERM CELLS (PEC) AND ENDOCRINE CELLS, filed December 13, 2013; PCT/US2016/061442, titled PDX1 PANCREATIC ENDODERM CELLS IN CELL DELIVERY DEVICES AND METHODS THEREOF, filed November 10, 2016; each of which is incorporated herein by reference in its entirety.

[0066] Various cell compositions derived from pluripotent stem cells and methods for their preparation are described herein and can be found in applications for which this Applicant has acquired exclusive licenses: US patent application No. 2009/0269845 titled Pluripotent cells, filed 24 April 2008; US patent application No. 2011/0014703 entitled Differentiation of Human Embryonic Stem Cells, filed July 20, 2010; US patent application No. 2011/0014702 entitled Differentiation of Human Embryonic Stem Cells, filed July 19, 2010; US patent application No. 2011/0151561 entitled Differentiation of Human Embryonic Stem Cells, filed December 16, 2010; US patent application No. 2010/0112692 titled Differentiation of Human Embryonic Stem Cells, filed October 22, 2009; US patent application No. 2012/0052576 entitled Differentiation of Pluripotent Stem Cells, filed August 17, 2011; US patent application No. 2010/0112693 entitled Differentiation of Human Pluripotent Stem Cells, filed October 23, 2009; US patent application No. 2011/0151560 entitled Differentiation of Human Embryonic Stem Cells, filed December 16, 2010; US patent application No. 2010/0015100 titled Differentiation of Human Embryonic Stem Cells, filed July 31, 2008; US patent application No. 2009/0170198 entitled Differentiation of Human Embryonic Stem Cells, filed November 25, 2008; US Patent Application No. 2015/0329828 titled Use of Small Molecules to Enhance MAFA Expression in Pancreatic Endocrine Cells, filed May 7, 2015; U.S. Patent Application No. 2013/0330823 entitled Differentiation of Human Embryonic Stem Cells into Pancreatic Endocrine Cells, filed June 6, 2013; publication of international application No. WO 2013/192005 entitled Differentiation of Human Embryonic Stem Cells into Pancreatic Endocrine Cells, filed June 13, 2013; U.S. Patent Application No. 2014/0242693 titled Suspension and Clustering of Human Pluripotent Stem Cells for Differentiation into Pancreatic Endocrine Cells, filed December 30, 2013; US Patent Application No. 2014/0295552 titled Suspension and Clustering of Human Pluripotent Stem Cells for Differentiation into Pancreatic Endocrine Cells, filed June 17, 2014; International Application Publication No. WO 2015/065524 titled Suspension and Clustering of Human Pluripotent Stem Cells for Differentiation into Pancreatic Endocrine Cells, filed May 21, 2014; U.S. Patent Application No. 2013/0330823 entitled Differentiation of Human Embryonic Stem Cells into Pancreatic Endocrine Cells, filed June 6, 2013; U.S. Patent Application No. 2014/0186953 entitled Differentiation of Human Embryonic Stem Cells Into Pancreatic Endocrine Cells Using HB9 Regulators, filed December 18, 2013; US patent application No. 14/963730, filed December 9, 2015; U.S. Patent Application No. 14/898,015, filed December 11, 2015, each of which is incorporated herein by reference in its entirety.

[0067] In one embodiment, hypoimmunogenic cells are packaged within a cell delivery device. Said cell delivery devices may comprise a nonwoven fabric. Cell delivery devices contain various layers, each of which performs one or more functions. In some embodiments, said cell delivery devices include both a cell-separating membrane and a nonwoven web. In another embodiment, said delivery device is TheraCyte (formerly Baxter) (Irvine, CA). TheraCyte cell delivery devices are described in US Pat. Nos. 6,773,458; 6,156,305; 6,060,640; 5,964,804; 5,964,261; 5,882,354; 5,807,406; 5,800,529; 5,782,912; 5,741,330; 5,733,336; 5,713,888; 5,653,756; 5,593,440; 5,569,462; 5,549,675; 5,545,223; 5,453,278; 5,421,923; 5,344,454; 5,314,471; 5,324,518; 5,219,361; 5,100,392; and 5,011,494, each of which is incorporated herein by reference in its entirety.

[0068] In another embodiment, said device is a device as described in US Pat. U.S. Design Patents 29/447,944, 29/509,102, 29/484,363, 29/484,360, 29/484,359, 29/484,357, 29/484,356, 29/484,355, 29/484,362, 29/484, 358.29/408.366 29/517.319; 29/408.368; 29/518.513; 29/518.516; In other embodiments, the cell delivery devices or large capacity aggregates include one or two or more seals that further separate the lumen of the cell delivery device, ie, a septal seal. See, for example, Applicant's U.S. Design Applications Nos. 29/408366, 29/408368, 29/408370, 29/423,365 and 29/584,046.

[0069] In one embodiment, hypoimmunogenic cells are implanted into perforated cell delivery devices that provide direct intercellular contact between the host vasculature and encapsulated cells. Perforation means are holes or pores in said device. In some embodiments, not all layers of the device are perforated. For example, see PCT/US2016/0061442, which is incorporated herein by reference in its entirety and which discloses perforated cell delivery devices with perforations in only one layer, eg, the cell retaining membrane; or only in the cell retaining membrane and the nonwoven layer. In one embodiment, the hypoimmunogenic cells are encapsulated in a perforated device and surrounded by a nonwoven material. In such embodiments, said nonwoven material is located on the outside of the cell delivery device. Instead of affecting the implanted cells, said nonwoven material enhances the vascularity of the host surrounding the cell-containing body. See, for example, application PCT/US2016/0061442 and US Pat. No. 8,278,106 (both documents are incorporated herein by reference in their entirety), which describe perforated devices and polymers used in said device.

[0070] In one embodiment, said holes/perforations are smaller than the cell aggregates contained in the device, such as aggregates derived from hPSC, eg, the definitive endoderm lineage cell aggregates contained therein. In one instance, a perforated cell delivery device implanted in a rat or human contains perforations in the cell-retaining membrane only (other layers of the device are not perforated), said holes being spaced about 2 mm or more apart, said holes having a diameter of less than than about 100 µm.

[0071] Depletion of hypoimmunogenic cells ("suicide gene")

[0072] The versatility of embryonic stem cells and induced pluripotent stem (iPS) cells to replace and repair any tissue in the body is accompanied by an increased risk of cancer. An increased risk of cancer is also associated with gene therapy. Therefore, reprogrammed tissues derived from ES cells or iPS cells (Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676, (2006), and Hanna, J.H., Saha, K. & Jaenisch, R. Pluripotency and Cellular Reprogramming: Facts, Hypotheses, Unresolved Issues Cell 143, 508-525, (2010), each article incorporated herein by reference in its entirety) or from others multipotent or progenitor cells, and from cells treated with gene therapy vectors, pose safety concerns (Knoepfler, P.S. Deconstructing Stem Cell Tumorigenicity: A Roadmap to Safe Regenerative Medicine. Stem Cells 27, 1050-1056, (2009) is included in its entirety to the present description by reference). For example, subcutaneously implanted iPS cells induce teratomas, and chimeric animals with iPS develop primitive malignant cancer at a high rate (Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676, (2006); Knoepfler, P.S. Deconstructing Stem Cell Tumorigenicity: A Roadmap to Safe Regenerative Medicine Stem Cells 27, 1050-1056, (2009)). While benign teratomas can be easily removed with surgery, invasive cancer remains a risk with cell therapy.

[0073] Strategies to overcome stem cell tumorigenicity have been considered, including a suicide gene strategy (Knoepfler, P.S. Deconstructing Stem Cell Tumorigenicity: A Roadmap to Safe Regenerative Medicine. Stem Cells 27, 1050-1056, (2009)). In particular, a gene can be selectively introduced into said implantable cells that codes for an enzyme that locally metabolizes a systemically available prodrug into an active anticancer agent. For example, treatment with ganciclovir, which is converted by thymidine kinase to compounds that become toxic after triphosphorylation by cellular kinases, resulted in the destruction of tumor cells in vitro. Thus, implanted cells can be modified to artificially create biochemical differences between host tissues and implanted cells. Targeting of these implantable cells is achieved by the choice of the vector used to deliver the "suicide gene" as well as by the biology of the suicide gene/prodrug system. As a result, high doses of the drug, generated only in the environment where the cells are implanted, limit side effects in other tissues.

[0074] The depletion of hypoimmunogenic cells can be achieved by selectively introducing a gene into a hypoimmunogenic cell, the expression of which either directly leads to the death of a hypoimmunogenic cell, or makes a hypoimmunogenic cell specifically susceptible to other pharmacological agents. In accordance with this method, in vivo or ex vivo depletion of a hypoimmunogenic cell can be achieved by delivering the gene of interest to the hypoimmunogenic cell using viral gene delivery systems such as, but not limited to, a retrovirus, adenovirus, or adeno-associated virus gene delivery system. The desired viral delivery system may comprise a virus whose genome encodes a protein that, for example, directly causes cell death, for example, by inducing apoptosis of a hypoimmunogenic cell. Alternatively, the virus delivery system may comprise a virus whose genome encodes, for example, the herpes simplex virus thymidine kinase gene. Expression of the herpes simplex virus thymidine kinase gene in a hypoimmunogenic cell makes the hypoimmunogenic cell sensitive to pharmacological doses of ganciclovir. Thus, subsequent contact of a virus-transduced hypoimmunogenic cell with ganciclovir results in the death of the hypoimmunogenic cell. Depletion of hypoimmunogenic cells can be achieved by introducing a so-called "suicide gene" through genome editing systems such as the ZFN, CRISPR/cas and TALEN systems.

[0075] Agents such as ganciclovir that cause cell death upon expression of a gene such as thymidine kinase are referred to herein as a "cell death inducing agent".

[0076] Genes that can be used to kill hypoimmunogenic cells include the herpes simplex virus thymidine kinase gene and cytosine deaminase or any gene that causes cell death that can be placed under the control of an inducible promoter/regulatory sequence (referred to herein as "promoter/regulatory sequence" or "promoter"), but are not limited to them. Said gene is transferred into a hypoimmunogenic cell, the cells are selected under appropriate selective pressure, then said cells are transferred to a patient and allowed to take root in him. The patient is then administered an agent that induces promoter activity, thereby inducing the expression of a gene whose product causes hypoimmunogenic cell death. In the case of thymidine kinase, other agents that facilitate cell death by this enzyme can also be used, such as, for example, ganciclovir (Bonini et al., 1997, Science 276:1719-1724; Bordignon et al., 1995, Human Gene Therapy 6:813-819; Minasi et al., 1993, J. Exp. Med. 177:1451-1459; Braun et al., 1990, Biology of Reproduction 43:684-693). Other genes suitable for this purpose include the genes for the constitutively active forms of caspase 3, 8, and 9, the bax gene, the granzyme gene, the genes for diphtheria toxin, Pseudomonas A toxin, ricin, and other toxins, which are disclosed elsewhere herein, but not limited to them. The design of appropriate constructs for delivering such genes to humans will be apparent to those skilled in the art and are described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and Ausubel et al. ( 1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0077] It is important that the gene that is transferred into hypoimmunogenic cells to kill cells is under the control of an appropriate promoter sequence so that expression of said gene can be induced by adding an appropriate inducer to the cells (administering to a mammal). Such inducible promoter sequences include, but are not limited to, promoters that are induced when a metal is added to cells, steroid-inducible promoters, and the like. In one preferred embodiment, an ecdysone promoter system can be used. In this embodiment, the ecdysone promoter is cloned upstream of an ecdysone receptor protein sequence that is upstream of a second promoter sequence that directs the expression of an ecdysone binding site operably linked to the desired gene, eg, the desired toxin. Induction of the promoter induces the expression of the toxin, thereby causing the destruction of the death of the cell in which the toxin gene is located.

[0078] Cells that have been transduced with a gene to kill cells, when such cells are transduced ex vivo, can be selected (i.e. separated from cells that do not contain said gene) by providing the cells with a selection marker in addition to the transduced gene. Selection markers are well known in the art and are described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.).

[0079] In addition, depletion of hypoimmunogenic cells can be achieved by introducing into a population of hypoimmunogenic cells an oligonucleotide (e.g., but not limited to an antisense molecule) or a ribozyme that is capable of causing the death of a hypoimmunogenic cell, or inducing dysfunction of a hypoimmunogenic cell. Such oligonucleotides include oligonucleotides that target an essential function of a hypoimmunogenic cell, referred to herein as oligonucleotides, which either kill the hypoimmunogenic cell or impair the function of the hypoimmunogenic cell to stimulate T cells. Such hypoimmunogenic cell functions include, but are not limited to, the joint stimulatory function of B71 and B72, CD40, among others. Thus, oligonucleotides and ribozymes that are useful in the methods of the present invention include, but are not limited to, oligonucleotides and ribozymes that target these targets.

[0080] As noted herein, depletion of hypoimmunogenic cells includes impaired function of hypoimmunogenic cells. Hypoimmunogenic cell dysfunction includes all forms of hypoimmunogenic cell dysfunction, with or without physical removal or depletion of hypoimmunogenic cells. Thus, disrupting the function of hypoimmunogenic cells involves the use of an antibody that blocks the function of hypoimmunogenic cell surface molecules that are critical to the hypoimmunogenic function of the cell.

[0081] Alternatively, peptides that block the function of hypoimmunogenic cell surface molecules, blocking which results in impaired function of hypoimmunogenic cells, can be used to effectively deplete hypoimmunogenic cells in the host. Such peptides include, but are not limited to, peptides that are designed to specifically bind receptor molecules on the surface of hypoimmunogenic cells, and peptides that are designed, for example, to inhibit important enzymatic functions in these cells.

[0082] Similarly, genes and oligonucleotides that serve the same purpose as the genes and oligonucleotides described herein are also included as tools in the methods of the present invention. Thus, peptides, oligonucleotides, and genes that interfere with the biological function of a hypoimmunogenic cell, as the term is defined herein, are also contemplated for use in the methods of the invention disclosed herein.

[0083] The present invention also encompasses the use of pharmaceutical compositions suitable as hypoimmunogenic cell depleting compositions for the practice of the methods of the present invention, said compositions comprising a suitable hypoimmunogenic cell depleting composition and a pharmaceutically acceptable carrier. In some embodiments, said cell-depleting composition is a chimeric composition comprising an antibody and a toxin.

[0084] As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which a suitable hypoimmunogenic cell depleting composition can be combined and which, after combination, can be used to administer the appropriate hypoimmunogenic cell depleting composition to a mammal.

[0085] Pharmaceutical compositions suitable for use in the methods of the present invention can be administered systemically as oral solid formulations, ophthalmic, suppository, aerosol, topical, or other similar formulations. In addition to the composition to deplete hypoimmunogenic cells, such pharmaceutical compositions may contain pharmaceutically acceptable carriers and other ingredients known to enhance and facilitate drug administration. Other possible formulations such as nanoparticles, liposomes, resealed erythrocytes, and systems based on immunological principles can also be used to administer an appropriate hypoimmunogenic cell depleting composition according to the methods of the present invention.

[0086] Methods for introducing "suicide genes" into cells are disclosed in US 20060222633, which is incorporated herein by reference in its entirety.

[0087] The present invention includes a method for depleting hypoimmunogenic cells in a mammalian host. Once said hypoimmunogenic cells have been transplanted into a host, said method comprises contacting the hypoimmunogenic cells with a cell depleting composition to affect the function of the hypoimmunogenic cells or kill the hypoimmunogenic cells, thereby depleting the hypoimmunogenic cells in the mammalian host.

[0088] In another aspect, said composition that destroys hypoimmunogenic cells is selected from the group consisting of a toxin, an antibody, a radioactive molecule, a nucleic acid, a peptide, a peptidomimetic, and a ribozyme.

[0089] In one aspect, said toxin is an immunotoxin. Said toxin is selected from the group consisting of ricin, diphtheria toxin and pseudomonas exotoxin A.

[0090] In another embodiment, said antibody is selected from the group consisting of an antibody specific for CD1a, an antibody specific for CD11c, an antibody specific for HKGCII, an antibody specific for CD11b, an antibody specific for DEC205, an antibody specific for B71, an antibody specific for B72, an antibody specific for CD40, an antibody specific for type 1 lectins, and an antibody specific for type II lectins.

[0091] In yet another embodiment, the nucleic acid molecule is selected from the group consisting of a gene and an oligonucleotide.

[0092] In the following embodiment, the specified radioactive molecule is a radioactively labeled antibody.

[0093] In another embodiment, said antigenic depleting composition is a chimeric composition comprising an antibody and a toxin. Said toxin may be selected from the group consisting of ricin, diphtheria toxin and Pseudomonas exotoxin A.

[0094] In another embodiment, said antibody is selected from the group consisting of an antibody specific for CD1a, an antibody specific for CD11c, an antibody specific for HKGCII, an antibody specific for CD11b, an antibody specific for DEC205, an antibody specific for B71, an antibody specific for B72, an antibody specific for CD40, an antibody specific for type I lectins, and an antibody specific for type II lectins.

Combined product

[0095] The embodiments described herein also disclose a combination product, which refers to a device loaded with hypoimmunogenic cells or a therapeutic agent, i.e. each can be a suitable medical device or cell product, but when used together, they form a combination product . In one instance, said combination product refers to a perforated device loaded with hypoimmunogenic cells. This is called "combination product" or "perforated combination product". Said device (perforated or not) can be any macro cell delivery device described herein, including cell encapsulation devices as described in US Pat. No. D714956, D718472, D718467, D718466, D718468, D718469, D718470, D718471, D720469, D726306, D726307, D728095, D734166, D734847, D747467, D 747468, D747798, D750769, D750770, D755986, D760399, D761423, D761424 (entirely included in the present description by reference), but are not limited to them. Cells loaded into said device (perforated or not) can be any of the hypoimmunogenic cells discussed above, including definitive endoderm, PDX1 positive endoderm, PDX1 positive foregut endoderm, pancreatic endoderm, pancreatic endoderm cells expressing PDX1 nNKX6.1 , endocrine progenitors, endocrine progenitors expressing NKX6.1 and INS, immature beta cells, immature beta cells expressing NKX6.1, INS and MAFB, mature endocrine cells, mature endocrine cells expressing INS, GCG, SST and PP, and mature beta cells and mature beta cells expressing INS and MAFA, but are not limited to.

[0096] Perforated delivery devices loaded with pancreatic endoderm cells or pancreatic progenitors of hypoimmunogenic cells that mature upon in vivo implantation are intended to reduce insulin dependence and/or reduce hypoglycemia in diabetic patients. This includes, but is not limited to, patients at high risk for type I diabetes who are unaware of hypoglycemia, are labile or have received an organ transplant, and who may be undergoing or are already on immunosuppressive therapy. As essentially described in PCT/US2016/0061442 (incorporated in its entirety by reference), the main method of action is the use of human pancreatic endoderm (PEC) cells or hypoimmunogenic pancreatic progenitor cells contained in a permeable, durable implantable medical device, which facilitates direct vascularization owner. These hypoimmunogenic PEC cells after implantation differentiate and mature into therapeutic, glucose sensitive, insulin releasing hypoimmunogenic cells. Thus, the perforated combination product supports human insulin secretion. The perforated combined product limits the spread (output) of RES of hypoimmunogenic cells in vivo. The perforated combination product is implanted at a site that provides sufficient vascularization to maintain a population of therapeutic hypoimmunogenic cells in the device and facilitate distribution of insulin and other pancreatic products into the circulation. The perforated combination product is intended for implantation and retrieval with conventional surgical instruments and to provide a therapeutic dose for two years or more. This device is designed to maintain an adequate dose of hypoimmunogenic PEC cell product during creation, shelf life, processing and surgical implantation to achieve clinical efficacy and ensure that the cell product is within the tissue capsule in accordance with safety requirements.

Knockin

[0097] In some embodiments, tolerogenic factors can be introduced or re-introduced into genome-edited stem cell lines to create immune-privileged universal donor stem cell lines. In some instances, the universal stem cells as claimed herein have been further modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, but are not limited to, one or more of LAHS-C, LAHS-E, LAHS-F, LAHS-G, PD-L1, CTLA-4-Ig, CD47, CI inhibitor, and IL-35. To facilitate the introduction of tolerogenic factors, such as the above tolerogenic factors, any method of gene editing at the AAVS1 locus can be used to actively inhibit immune rejection.

[0098] In particular, in some embodiments, the inventions disclosed herein relate to stem cells whose genome has been altered to attenuate or remove critical components of at least one MHC class I gene and at least , a single NK activating ligand gene, and which has been further altered to increase the expression of one or more tolerogenic factors. In some embodiments, the inventions disclosed herein relate to a stem cell whose genome has been altered to attenuate or remove critical components of at least one MHC class I gene and at least one NK activating ligand gene. , and which has been further modified to increase the expression of one or more of the LAHS-C, LAHS-E, LAHS-P, LAHS-G, PD-L1, CTLA-4-Ig, CD47, CI inhibitor, and IL-35 proteins.

Embodiments

[0099] Other embodiments of the present invention are described with reference to the numbered paragraphs below.

[00100] Related to blocking antibodies: composition

[00101] Paragraph 1: A composition comprising a pluripotent cell lacking at least one human leukocyte antigen (LAH) class I gene and at least one agent that binds to a natural killer (NK) cell activating ligand.

[00102] Paragraph 2: The composition according to paragraph 1, wherein said agent is an antibody.

[00103] Paragraph 3: The composition according to paragraph 1, wherein the class I LAH gene is B2M.

[00104] Paragraph 4: A composition according to paragraphs 1-3, wherein said NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[00105] Paragraph 5: The composition according to paragraphs 1-2, wherein said NK cell activating ligand is ICAM-1 and CEACAM1.

[00106] Paragraph 6: A composition according to paragraphs 1-2, wherein said NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[00107] Paragraph 7: A composition according to paragraphs 1-2, wherein said cells derived from pluripotent cells further express a protein that is expressed in the presence of a cell death agent, said agent being capable of causing the death of said pluripotent cells.

[00108] Paragraph 8: A composition according to paragraph 7 wherein said cell death agent is ganciclovir.

[00109] Paragraph 9: A composition according to any paragraph 1-8, wherein cells derived from pluripotent cells additionally overexpress one or more tolerogenic factors.

[00110] Paragraph 10: A composition according to paragraph 9 wherein said tolerogenic factors are LAHS-C, LAHS-E, LAHS-G, PD-L1, CTLA-4-Ig, CD47, CI inhibitor, or IL-35.

[00111] Paragraph 11: The composition according to paragraph 9, wherein said tolerogenic factors are LAS-C, LAS-E, and LAS-G.

[00112] Related to blocking antibodies: Method

[00113] Paragraph 1: A method for preventing transplant rejection of cells derived from human pluripotent cells, comprising administering to a subject in need of treatment a composition comprising a population of target cells lacking at least one LAH class I gene and at least one NK cell activating ligand binding agent on the target cell in an amount effective to inhibit attack by the subject's NK cells, thereby preventing transplant rejection of human pluripotent cell-derived cells.

[00114] Paragraph 2: The method of paragraph 1 wherein said agent is an antibody.

[00115] Paragraph 3: The method according to paragraph 1, wherein the subject's immune response to an NK cell activating ligand is suppressed.

[00116] Paragraph 4: The method of paragraph 1 wherein said NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, or MICB.

[00117] Paragraph 5: The method of paragraph 1 wherein said NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[00118] Paragraph 6: The method according to paragraph 1, wherein said subject is a human.

[00119] Paragraph 7: The method of paragraph 2, wherein said antibody is a human antibody.

[00120] Related to double knockout of hES cells: Compositions [00121] Paragraph 1: An in vitro cell population comprising cells derived from pluripotent cells, in which the cells derived from pluripotent cells lack at least one class I LAH gene and at least one natural killer (NK) cell activating ligand gene.

[00122] Paragraph 2: The specified in vitro cell population according to paragraph 1, wherein the specified LAH class gene is B2M.

[00123] Paragraph 3: Said in vitro cell population according to paragraph 1-2, wherein said NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[00124] Paragraph 4: Said in vitro cell population according to paragraph 1-2, wherein said NK cell activating ligand is ICAM-1 and CEACAM1.

[00125] Paragraph 5: Said in vitro cell population according to paragraph 1-2, wherein said NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[00126] Paragraph 6: Said population of cells in vitro according to paragraph 1-5, wherein said cells derived from pluripotent cells further express a protein that is expressed in the presence of a cell death agent, said agent being capable of causing said cell death derived from pluripotent cells.

[00127] Paragraph 7: Said in vitro cell population according to paragraph 6, wherein said cell death agent is ganciclovir.

[00128] Paragraph 8: Said population of cells in vitro according to any one of paragraphs 1-7, wherein said cells derived from pluripotent cells additionally overexpress one or more tolerogenic factors.

[00129] Paragraph 9: Said in vitro cell population according to paragraph 8, wherein said tolerogenic factors are LAS-C, LAS-E, LAS-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, IL -35 or a combination.

[00130] Paragraph 10: Said in vitro cell population according to paragraph 9, wherein said tolerogenic factors are LAS-C, LAS-E, and LAS-G.

[00131] Related to triple knockout of pluripotent stem cells: compositions

[00132] A human pluripotent stem cell comprising a modified genome comprising: (a) a first genome modification whereby the B2M gene has been edited to reduce or eliminate B2M expression on the cell surface and/or B2M activity in the cell; (b) a second genome modification in which the ICAM-1 gene has been edited to reduce or eliminate expression of ICAM-1 on the cell surface and/or ICAM-1 activity in the cell; and (c) a third genome modification in which the CEACAM1 gene has been edited to reduce or eliminate expression of CEACAM1 on the cell surface and/or CEACAM1 activity in the cell.

[00133] Knockout-Related Transcriptional Regulators: Compositions

[00134] Paragraph 12: A cell derived from pluripotent cells containing reduced expression of one or more MHC class I or MHC class II genes or protein complexes and one or more NK cell activating ligands compared to a wild-type pluripotent stem cell, wherein the pluripotent stem cell has one or more genes encoding one or more transcription regulators of MHC class I or MHC class II and NK activating ligand genes deleted from at least one allele of the cell.

[00135] Paragraph 13: A pluripotent stem cell having reduced expression of one or more NK cell activating ligands compared to wild-type human pluripotent stem cells.

[00136] Paragraph 14: Human pluripotent stem cell that does not express B2M or ICAM-1.

[00137] Paragraph 15: Human pluripotent stem cell that does not express CIITA or ICAM-1.

[00138] Paragraph 16: Human pluripotent stem cell that does not express LRC5 or ICAM-1.

[00139] Paragraph 17: A human pluripotent stem cell that does not express one or more of NLRC5, CIITA, and B2M, and additionally does not express one or more of ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[00140] Paragraph 18: A human pluripotent stem cell that does not express one or more of LAHS-A, LAHS-B, and LAHS-C and additionally does not express one or more of ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[00141] Paragraph 19: A human pluripotent stem cell that does not express one or more of the MHC class I antigens and one or more NK cell activating ligands, and additionally one or more tolerogenic factors, is inserted into a locus that is a safe place, according to at least one cell allele.

[00142] Paragraph 20: Human pluripotent stem cell containing a modified genome containing a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M expression on the cell surface and/or B2M activity in the cell, and in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 expression on the cell surface and/or ICAM-1 activity in the cell.

[00143] Paragraph 21: A genome-modified human pluripotent stem cell comprising a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M expression on the cell surface and/or B2M activity in the cell, and in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 expression on the cell surface and/or ICAM-1 activity in the cell.

[00144] Related to double knockout of hES cells: compositions

[00145] Paragraph 1: An in vitro cell population comprising pluripotent cells in which said pluripotent cells lack at least one functional MHC class I surface protein and at least one functional natural killer (NK) cell-activating cell surface protein .

[00146] Paragraph 2: Said in vitro cell population according to paragraph 1, wherein said class I MHC cell surface protein is LAHS-A, LAHS-B, LAHS-C, or a combination thereof.

[00147] Paragraph 3: Said in vitro cell population according to paragraphs 1-2, wherein said Class I MHC cell surface protein is B2M protein.

[00148] Paragraph 4: Said in vitro cell population of any one of paragraphs 1-3, wherein said pluripotent cells lack at least two functional NK cell activating cell surface proteins.

[00149] Paragraph 5: Said in vitro cell population of any one of paragraphs 1-4, wherein said pluripotent cells lack at least three functional NK cell-activating cell surface proteins.

[00150] Paragraph 6: Said in vitro cell population of any one of paragraphs 1-5, wherein said NK cell activating cell surface protein is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[00151] Paragraph 7: Said in vitro cell population of any one of paragraphs 1-5, wherein said NK cell activating cell surface protein is ICAM-1 and CEACAM1.

[00152] Paragraph 8: Said in vitro cell population of any one of paragraphs 1-7, wherein said pluripotent cells further express a protein that is expressed in the presence of a cell death agent, said agent being capable of causing said pluripotent cells to die.

[00153] Paragraph 9: Said population of cells in vitro according to any one of paragraphs 1-8, wherein said protein that is expressed in the presence of a cell death agent, wherein said agent capable of causing said pluripotent cell death is a simple virus protein herpes, thymidine kinase or cytosine deaminase.

[00154] Paragraph 10: Said in vitro cell population of any of paragraphs 8-9, wherein said cell death agent is ganciclovir.

[00155] Related to double knockout of hES cells: compositions

[00156] Paragraph 1: An in vitro cell population comprising pluripotent cells, wherein said pluripotent cells have reduced expression of at least one MHC class I cell surface protein and reduced function and/or expression of at least one cell surface protein ligand that activates NK cells, compared to the original genotype or compared to a wild-type human cell.

[00157] Paragraph 2: An in vitro cell population comprising pluripotent cells, wherein said pluripotent cells have reduced expression of one or more of the cell surface proteins LAS-A, LAS-B, and LAS-C and reduced function and/or expression of at least at least one NK cell activating cell surface protein ligand compared to the original genotype or compared to a wild-type human cell.

[00158] Paragraph 3: An in vitro population of cells comprising pluripotent cells, in which said pluripotent cells lack expression of functional LAS cell surface proteins, expression of NK cell activating cell surface protein ligands, and have a protein that is expressed in said pluripotent cells in the presence of a cell death agent, said agent being capable of causing the death of said pluripotent cells.

[00159] Paragraph 4: A stem cell in which the expression of one or more class I LAS cell surface proteins and one or more NK cell activating cell surface protein ligand is reduced compared to a wild-type stem cell.

[00160] Paragraph 5: A pluripotent cell in which the expression of one or more class I LAS cell surface protein, one or more NK cell activating cell surface protein ligand, and one or more tolerogenic cell surface protein factor is reduced compared to the pluripotent cell wild-type and wherein said pluripotent cells further express a protein that is expressed in the presence of a cell death inducing agent, said agent being capable of causing the death of said pluripotent cells.

[00161] Paragraph 6: An in vitro population of cells comprising pluripotent cells, in which said pluripotent cells lack functional class I MHC genes and natural killer (NK) cell activating ligand genes, and in which said pluripotent cells overexpress a tolerogenic a cell surface protein factor compared to a wild-type pluripotent cell, and wherein said pluripotent cells further express a protein that is expressed in the presence of a cell death agent, said agent being capable of causing the death of said pluripotent cells.

[00162] Related to double knockout of PEC cells: composition

[00163] Paragraph 1: An in vitro population of cells comprising pancreatic endoderm (PEC) cells in which said PEC cells lack at least one functional LAH class I gene and at least one natural killer cell (NK) activating ligand gene. ).

[00164] Paragraph 2: Said in vitro cell population according to paragraph 1, wherein said at least one class I LAS gene is the B2M gene.

[00165] Paragraph 3: Said in vitro cell population according to paragraph 1-2, wherein said at least one NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[00166] Paragraph 4: Said in vitro cell population according to paragraph 1-2, wherein said at least one NK cell activating ligand is ICAM-1 and CEACAM1.

[00167] Paragraph 5: Said in vitro cell population according to paragraph 1-2, wherein said at least one NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[00168] Paragraph 6: Said population of cells in vitro according to paragraphs 1-5, wherein said PEC cells further express a protein that is expressed in the presence of a cell death agent, said agent being capable of causing said PEC cells to die.

[00169] Paragraph 7: Said in vitro cell population according to paragraph 6, wherein said cell death agent is ganciclovir.

[00170] Paragraph 8: Said population of cells in vitro according to paragraph 6, wherein said gene that is expressed in the presence of a cell death agent, wherein said agent is capable of causing said PEC cell death, is a herpes simplex virus, thymidine kinase, or cytosine deaminase gene .

[00171] Paragraph 9: Said population of cells in vitro according to any one of paragraphs 1-8, wherein said PEC cells additionally overexpress one or more tolerogenic cell surface proteins.

[00172] Paragraph 10: Specified in vitro cell population according to paragraphs 1-9 wherein said tolerogenic factors are LAHS-C, LAHS-E, LAHS-G, PD-L1, CTLA-4-Ig, CD47, CI inhibitor , IL-35, or a combination thereof.

[00173] Paragraph 11: Said in vitro cell population according to paragraphs 1-9, wherein said tolerogenic factors are LAS-C, LAS-E, and LAS-G.

[00174] Paragraph 12: An in vitro population of cells containing pancreatic endoderm (PEC) cells in which said PEC cells lack at least one functional MHC class I gene, MHC class II gene, and natural killer cell activating ligand gene ( NK).

[00175] Paragraph 13: An in vitro population of cells containing pancreatic endoderm (PEC) cells in which said PEC cells lack at least one functional MHC class I gene and an MHC class II gene, and also lack at least two ligand genes activating natural killer (NK) cells.

[00176] Paragraph 14: A pancreatic endoderm cell (PEC) in which the expression of one or more LAS class I genes and one or more NK cell activating ligand genes is reduced compared to wild-type RES cells.

[00177] Paragraph 15: A pancreatic endoderm (PEC) cell in which the expression of one or more class I LAS cell surface proteins, one or more NK cell activating ligand, and one or more tolerogenic factors is reduced compared to wild-type RES cells and wherein said pancreatic endoderm cell further expresses a protein that is expressed in the presence of a cell death agent, said agent being capable of causing said PEC cells to die.

[00178] Paragraph 16: Pancreatic endoderm (PEC) cell that does not express B2M or ICAM-1.

[00179] Paragraph 17: Pancreatic endoderm (PEC) cell that does not express CIITA or ICAM-1.

[00180] Paragraph 18: Pancreatic endoderm (PEC) cell that does not express LRC5 or ICAM-1.

[00181] Paragraph 19: A pancreatic endoderm (PEC) cell that does not express one or more of NLRC5, CIITA, and B2M and additionally does not express one or more of ICAM-1, CEACAM1, CADM1, MICa, and MICB.

[00182] Paragraph 20: A pancreatic endoderm (PEC) cell that does not express one or more of LAHS-A, LAHS-B, and LAHS-C and additionally does not express one or more of ICAM-1, CEACAM1, CADM1, MICa, and MICB .

[00183] Paragraph 21: A pancreatic endoderm (PEC) cell that does not express one or more class I MHC cell surface proteins and one or more NK cell activating ligands, and additionally has one or more tolerogenic factors introduced into the safe locus at at least one allele of said cell.

[00184] Paragraph 22: A pancreatic endoderm cell (PEC) containing a modified genome containing a first genome modification in which said B2M gene has been altered to reduce or eliminate B2M expression on the surface of said cell and/or activity in said cell, and in which said ICAM-1 gene has been altered to reduce or abolish ICAM-1 Expression on the surface of said cell and/or activity in said cell.

[00185] Paragraph 23: A pancreatic endoderm cell (PEC) containing a modified genome comprising: a first genome modification in which said B2M gene has been altered to reduce or abolish B2M expression on the surface of said cell and/or activity in said cell; and (b) a second genome modification in which said ICAM-1 gene has been altered to reduce or eliminate ICAM-1 expression on the surface of said cell and/or activity in said cell.

[00186] Paragraph 24: A pancreatic endoderm cell (PEC) containing a modified genome comprising: a first genome modification in which said B2M gene has been altered to reduce or abolish B2M expression on the surface of said cell and/or activity in said cell; (b) a second genome modification in which said ICAM-1 gene has been altered to reduce or abolish ICAM-1 expression on the surface of said cell and/or activity in said cell; and (c) a third genome modification in which said CEACAM1 gene has been altered to reduce or abolish CEACAM1 expression on the surface of said cell and/or activity in said cell.

Related to hypoimmunogenic cells

[00187] Paragraph 1: An in vitro cell population containing hypoimmunogenic cells, wherein said hypoimmunogenic cells lack at least one functional cell surface LAS class I protein and at least one functional cell surface NK cell activating ligand protein.

[00188] Paragraph 2: Said in vitro cell population according to paragraph 1, wherein said Class I LAH cell surface protein is B2M.

[00189] Paragraph 3: Said in vitro cell population according to paragraph 1-2, wherein said NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[00190] Paragraph 4: Said in vitro cell population according to paragraph 1-2, wherein said NK cell activating ligand is ICAM-1 and CEACAM1.

[00191] Paragraph 5: Said in vitro cell population according to paragraph 1-2, wherein said NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[00192] Paragraph 6: Said population of cells in vitro according to paragraphs 1-5, wherein said hypoimmunogenic cells further express a protein that is expressed in the presence of a cell death agent, said agent being capable of causing said hypoimmunogenic cells to die.

[00193] Paragraph 7: Said in vitro cell population according to any one of paragraphs 1-6, wherein said hypoimmunogenic cells further overexpress one or more tolerogenic factors.

[00194] Paragraph 8: Specified in vitro cell population according to paragraphs 1-7, wherein said tolerogenic factors are LAHS-C, LAHS-E, LAHS-G, PD-L1, CTLA-4-Ig, CD47, CI inhibitor , IL-35 and combinations thereof.

[00195] Paragraph 9: Said in vitro cell population according to paragraphs 1-7, wherein said tolerogenic factors are LAS-C, LAS-E, and LAS-G.

[00196] Paragraph 10: An in vitro population of cells containing hypoimmunogenic cells, in which said hypoimmunogenic cells lack at least one functional MHC class I gene and at least one NK cell activating ligand gene.

[00197] Paragraph 11: An in vitro population of cells containing hypoimmunogenic cells, wherein said hypoimmunogenic cells lack at least one functional MHC class I gene, MHC class II gene, and NK cell activating ligand gene.

[00198] Paragraph 12: A specified in vitro cell population according to paragraphs 10 or 11, wherein the specified class I MHC gene is LAHS-A, LAHS-B, LAHS-C, or a combination thereof.

[00199] Paragraph 13: Specified in vitro cell population according to paragraphs 10, 11 or 12, wherein the specified class I MHC gene is B2M.

[00200] Paragraph 14: Said in vitro cell population of any of paragraphs 10-13, wherein said hypoimmunogenic cells lack at least two functional NK cell activating ligand genes.

[00201] Paragraph 15: Said in vitro cell population of any of paragraphs 10-13, wherein said hypoimmunogenic cells lack at least three functional NK cell activating ligand genes.

[00202] Paragraph 16: Said in vitro cell population of any of paragraphs 10-13, wherein the NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[00203] Paragraph 17: Said in vitro cell population of any of paragraphs 10-13, wherein said NK cell activating ligand is ICAM-1 and CEACAM1.

[00204] Paragraph 18: Said in vitro cell population of any of paragraphs 10-13, wherein said NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[00205] Paragraph 19: Said in vitro cell population of any one of paragraphs 10-18, wherein said hypoimmunogenic cells further express a protein that is expressed in the presence of a cell death agent, said agent being capable of causing said hypoimmunogenic cells to die.

[00206] Paragraph 20: Said in vitro cell population of any of paragraphs 7-15, wherein said hypoimmunogenic cells are hES cells or a pancreatic cell line.

[00207] Paragraph 21: A hypoimmunogenic cell in which the expression of one or more LAS class 1 cell surface proteins and one or more NK cell activating ligand proteins on the cell surface is reduced compared to a wild-type hypoimmunogenic cell.

[00208] Paragraph 22: A hypoimmunogenic cell in which the expression of one or more LAH class I cell surface protein, one or more NK cell activating ligand, and one or more tolerogenic cell surface protein factor is reduced compared to a wild-type hypoimmunogenic cell, and wherein said pluripotent cells further express a protein that is expressed in the presence of a cell death inducing agent, said agent being capable of causing the death of said hypoimmunogenic cells.

[00209] Paragraph 23: A hypoimmunogenic stem cell in which the expression of one or more MHC class I or MHC class II cell surface proteins and one or more NK cell activating ligands is reduced compared to a pluripotent stem cell in which said pluripotent in a stem cell, one or more genes encoding one or more MHC class I or MHC class II transcriptional regulators and one or more NK cell activating ligand genes are deleted from at least one allele of said cell.

[00210] Paragraph 24: A hypoimmunogenic cell in which the expression of one or more NK cell activating ligands is reduced compared to wild-type human hypoimmunogenic cells.

[00211] Paragraph 25: Human hypoimmunogenic cell that does not express B2M or ICAM-1.

[00212] Paragraph 26: Human hypoimmunogenic cell that does not express SITA or ICAM-1.

[00213] Paragraph 27: Human hypoimmunogenic cell that does not express LRC5 or ICAM-1.

[00214] Paragraph 28: Human hypoimmunogenic cell that does not express one or more of NLRC5, SITA, and B2M and additionally does not express one or more of ICAM-1, CEACAM1, CADM1, MICa, and MICB.

[00215] Paragraph 29: A human hypoimmunogenic cell that does not express one or more of LAHS-A, LAHS-B, and LAHS-C and additionally does not express one or more of ICAM-1, CEACAM1, CADM1, MICa, and MICB.

[00216] Paragraph 30: A hypoimmunogenic human cell that does not express one or more of one or more class I MHC cell surface proteins and one or more NK cell activating ligands, and additionally contains one or more tolerogenic factors introduced at a safe locus at least one allele of said cell.

[00217] Paragraph 31: A hypoimmunogenic cell containing a modified genome containing a first genome modification in which said B2M gene has been altered to reduce or abolish B2M expression on the surface of said cell and/or activity in said cell and in which said ICAM-1 gene was modified to reduce or eliminate the expression of ICAM-1 on the surface of the specified cell and/or activity in the specified cell.

[00218] Paragraph 32: A hypoimmunogenic stem cell containing a modified genome comprising: a first genome modification in which said B2M gene has been altered to reduce or eliminate B2M expression on the surface of said cell and/or activity in said cell; and (b) a second genome modification in which said ICAM-1 gene has been altered to reduce or eliminate ICAM-1 expression on the surface of said cell and/or activity in said cell.

[00219] Paragraph 33: A hypoimmunogenic stem cell comprising a modified genome comprising: a first genome modification in which said B2M gene has been altered to reduce or abolish B2M expression on the surface of said cell and/or activity in said cell; (b) a second genome modification in which said ICAM-1 gene has been altered to reduce or abolish ICAM-1 expression on the surface of said cell and/or activity in said cell; and (c) a third genome modification in which said CEACAM1 gene has been altered to reduce or abolish CEACAM1 expression on the surface of said cell and/or activity in said cell.

Ways to get a double knockout

[00220] Paragraph 1: A method for reducing the likelihood of graft rejection, comprising:

a) administering to a subject in need of a graft an effective amount of a graft comprising a population of pancreatic endoderm cells in which at least one class I LAS cell surface protein and at least one NK cell activating ligand protein function is impaired.

[00221] Paragraph 2: The method of paragraph 1, wherein said Class I LAH cell surface protein is B2M.

[00222] Paragraph 3: The method of paragraph 1-2, wherein said cell surface NK cell activating ligand protein is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[00223] Paragraph 4: The method of paragraph 1-2, wherein said NK cell activating ligand is ICAM-1 and CEACAM1.

[00224] Paragraph 5: The method of paragraph 1-2, wherein said NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[00225] Paragraph 6: A method of reducing the number of hypoimmunogenic cells in a population of cells, comprising contacting said hypoimmunogenic cells with a composition that reduces the number of hypoimmunogenic cells to reduce the function of hypoimmunogenic cells or lead to the death of said hypoimmunogenic cells, thereby reducing the number of said hypoimmunogenic cells in the specified cell population.

[00226] Paragraph 7: The method of paragraph 6, wherein said hypoimmunogenic cells lack the expression of at least one functional LAH class I cell surface protein and at least one NK cell activating ligand.

[00227] Paragraph 8: A method of removing hypoimmunogenic cells from a mammalian host, comprising: (a) transferring the hypoimmunogenic cells to said mammalian host; and (b) contacting the host with the hypoimmunogenic cell reducing composition to impair the function of said hypoimmunogenic cells or cause the death of said hypoimmunogenic cells, thereby removing said hypoimmunogenic cells in said mammalian host.

[00228] Paragraph 9: The method of paragraph 8, wherein said function of at least one class I LAS cell surface protein and at least one NK cell activating ligand is reduced.

[00229] Paragraph 10: A method of increasing the amount of NK activating ligands in a population of target cells, comprising exposing the population of target cells to IFN-γ, thereby increasing the amount of NK activating ligands in the target cells compared to a wild-type cell.

Related to pluripotent cells in which the function of both the MHC class I gene and the NK cell activating ligand gene is impaired or inhibited: compositions

[00230] Paragraph 1: An in vitro population of cells containing pluripotent cells in which the function of at least one major histocompatibility complex (MCHC) class I gene and at least one natural killer (NK) ligand gene is impaired or inhibited.

[00231] Paragraph 2: Said in vitro cell population according to paragraph 1, wherein said MHC gene encodes beta-2 microglobulin (B2M).

[00232] Paragraph 3: Said in vitro cell population according to paragraph 1, wherein said NK cell activating ligand is ICAM1, CD58, PVR, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[00233] Paragraph 4: Said in vitro cell population according to paragraph I, wherein said NK cell activating ligand is ICAM1 and CD58.

[00234] Paragraph 5: Said in vitro cell population according to paragraph 1, wherein said NK cell activating ligand is ICAM1, CD58, CD155, CEACAM1, CADM1, MICA, and MICB.

[00235] Paragraph 6: Said in vitro cell population according to paragraph 1, wherein said pluripotent cells are human embryonic stem cells.

[00236] Paragraph 7: Said population of cells in vitro according to paragraph 1, wherein said pluripotent cells are differentiated into pancreatic endoderm cells.

[00237] Paragraph 8: Said population of cells in vitro according to paragraph 1, wherein said pluripotent cells further comprise a protein that is expressed in the presence of a cell death agent, said agent being capable of causing said cell death.

[00238] Paragraph 9: Said in vitro cell population according to paragraph 1, wherein said class I MHC gene has been disrupted using genome editing tools.

[00239] Paragraph 10: Said population of cells in vitro according to paragraph 9, wherein said genome editing agent is Zinc Finger Protein Domain Nuclease (ZFN), Clustered, Regularly Interspersed, Short, Palindromic Repeat (CRISPR)/cas, or transcription activator-like effector nuclease (TALEN) system.

[00240] Paragraph 11: Said population of cells in vitro according to paragraph 1, wherein the gene for said NK cell activating ligand is disrupted using genome editing tools.

[00241] Paragraph 12: Said in vitro cell population according to paragraph 1, wherein the function of said NK cell activating ligand is disrupted using an anti-NK cell activating ligand agent.

[00242] Paragraph 13: Specified population of cells in vitro according to paragraph 8, in which the specified agent is an antibody.

Related to a pancreatic cell line in which the functions of both the MHC class I gene and the NK cell activating ligand gene are impaired or inhibited: compositions

[00243] Paragraph 14: An in vitro cell population comprising a pancreatic cell line in which the function of at least one major histocompatibility complex (MCHC) class I gene and at least one natural killer (NK) ligand gene is impaired or inhibited.

[00244] Paragraph 15: Said in vitro cell population according to paragraph 14, wherein said class I MHC gene encodes B2M.

[00245] Paragraph 16: Said in vitro cell population according to paragraph 14, wherein said NK cell activating ligand is ICAM1, CD58, CD155, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[00246] Paragraph 17: Said in vitro cell population according to paragraph 14, wherein said NK cell activating ligand is ICAM1 and CD58.

[00247] Paragraph 18: Said in vitro cell population according to paragraph 14, wherein said NK cell activating ligand is ICAM1, CD58, CD 155, CEACAM1, CADM1, MICA, and MICB.

[00248] Paragraph 19: Said in virto cell population according to paragraph 14, wherein said pancreatic cell line is definitive endoderm, anterior digestive tract endoderm, pancreatic endoderm, endocrine progenitors, or insulin producing cells.

[00249] Paragraph 20: Said in vitro cell population according to paragraph 14, wherein said pancreatic cell line further expresses a protein that is expressed in the presence of a cell death agent, said agent being capable of causing said cell death.

Ways to prevent pancreatic cell transplant rejection

[00250] Paragraph 21: A method for preventing rejection of a human pancreatic cell transplant, comprising:

[00251] a. providing a population of human pancreatic cells that do not express at least one major histocompatibility complex (MCHC) class I cell surface protein and do not express at least one natural killer (NK) cell activating ligand; And

[00252] b. transplantation of said population of human pancreatic cells into a mammalian subject in which the lack of expression of MHC class I cell surface protein and NK cell activating ligand prevents cell transplant rejection.

[00253] Paragraph 22: The method of paragraph 21, wherein said class I major histocompatibility complex (MCHC) cell surface protein is beta-2 microglobulin (B2M) or LAHS-ABC cell surface protein.

[00254] Paragraph 23: The method of paragraph 21, wherein said NK cell activating ligand is ICAM1, CD58, CD155, PVR, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[00255] Paragraph 24: The method of paragraph 21 wherein said human pancreatic cell population is pancreatic endoderm (PEC) cells.

[00256] Paragraph 25. The method of paragraph 21, wherein said population of human pancreatic cells further comprises a gene for a protein that is expressed in said population of human cells in the presence of a cell death agent, wherein said agent is capable of causing the death of said population of pancreatic cells. human glands.

Ways to get insulin

[00257] Paragraph 26. A method for producing insulin, comprising:

[00258] a) providing a population of human pancreatic endoderm cells that do not express at least one major histocompatibility complex (MCHC) class I cell surface protein and do not express at least one natural killer (NK) cell activating ligand; And

[00259] b) transplanting said population of human pancreatic endoderm cells into a mammalian subject, wherein said pancreatic endoderm cells mature in the mammalian subject and produce insulin in response to glucose stimulation.

[00260] Paragraph 27: The method of paragraph 26, wherein said class I major histocompatibility complex (MCHC) cell surface protein is beta-2 microglobulin (B2M) or LAHS-ABC cell surface protein.

[00261] Paragraph 28. The method of paragraph 26, wherein said NK cell activating ligand is ICAM1, CD58, CD155, PVR, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[00262] Paragraph 29: The method of paragraph 26, wherein said population of human pancreatic cells further comprises a gene for a protein that is expressed in said population of human pancreatic cells in the presence of a cell death agent, wherein said agent is capable of causing the death of said population human pancreatic cells.

[00263] Paragraph 30. The method of paragraph 29, wherein said protein is herpes simplex thymidine kinase and said agent is ganciclovir.

[00264] A method for producing a hypoimmunogenic stem cell, comprising reducing the expression of one or more class I MHC cell surface proteins and one or more NK activating ligands in said hypoimmunogenic stem cell, and thereby obtaining said hypoimmunogenic stem cell.

[00265] A method for producing a hypoimmunogenic stem cell, comprising reducing the expression of one or more class I MHC cell surface proteins, one or more NK cell activating ligands, and reducing the expression of one or more tolerogenic factors in said stem cell and thereby obtaining said hypoimmunogenic stem cell.

[00266] A method for reducing the expression of one or more MHC class I cell surface proteins and an NK cell activating ligand, a cell surface protein in a stem cell, comprising deleting one or more genes encoding one or more MHC class I gene transcription regulators and ligands, NK cell activating ligand from at least one allele of said cell and thereby reducing the expression of said one or more MHC class I cell surface protein and the NK cell activating ligand cell surface protein.

[00267] A hypoimmunogenic stem cell containing a modified genome comprising (a) a first genome modification in which said B2M gene has been altered to reduce or eliminate B2M expression on the surface of said cell and/or activity in said cell by bringing said cell into contact, or with a Cas protein or a nucleic acid encoding a Cas protein, in combination with either a plasmid encoding a ribonucleic acid that is homologous to any of the sequences of SEQ IDNO: 1-3, or with a ribonucleic acid that is homologous to any of the sequences of SEQ ID NO: 1-3 ; and (b) a second genome modification wherein said ICAM-1 gene has been altered to reduce or eliminate ICAM-1 expression on the surface of said cell and/or activity in said cell by contacting said cell with either a Cas protein or a nucleic acid an acid encoding a Cas protein in combination with either a plasmid encoding a ribonucleic acid that is homologous to any of SEQ ID NOs: 4-6 or a ribonucleic acid that is homologous to any of SEQ ID NOs: 4-6.

[00268] A hypoimmunogenic stem cell containing a modified genome, comprising (a) a first genome modification in which said B2M gene has been altered to reduce or eliminate B2M expression on the surface of said cell and/or activity in said cell by bringing said cell into contact, or with a Cas protein, or with a nucleic acid encoding a Cas protein, in combination with either a plasmid encoding a ribonucleic acid that is homologous to any of the sequences of SEQ ID NO: 1-3, or with a ribonucleic acid that is homologous to any of the sequences of SEQ ID NO: 1-3; (b) a second genome modification wherein said ICAM-1 gene has been altered to reduce or eliminate ICAM-1 expression on the surface of said cell and/or activity in said cell by contacting said cell with either a Cas protein or a nucleic acid , encoding the Cas protein, in combination with either a plasmid encoding a ribonucleic acid that is homologous to any of the sequences of SEQ ID NO: 4-6, or with a ribonucleic acid that is homologous to any of the sequences of SEQ ID NO: 4-6; and (c) a third genome modification wherein said CEACAM1 gene has been altered to reduce or eliminate CEACAM1 expression on the surface of said cell and/or activity in said cell by contacting said cell with either a Cas protein or a nucleic acid encoding the protein Cas, in combination with either a plasmid encoding a ribonucleic acid that is homologous to any of the sequences of SEQ ID NOs: 7-9, or with a ribonucleic acid that is homologous to any of the sequences of SEQ ID NOs: 7-9.

[00269] A genome-modified pluripotent stem cell comprising (a) a first genome modification in which said B2M gene has been altered to reduce or eliminate B2M expression on the surface of said cell and/or activity in said cell by bringing said cell into contact, or with a Cas protein, or with a nucleic acid encoding a Cas protein, in combination with either a plasmid encoding a ribonucleic acid that is homologous to any of the sequences of SEQ ID NO: 1-3, or with a ribonucleic acid that is homologous to any of the sequences of SEQ ID NO: 1-3; and (b) a second genome modification wherein said ICAM-1 gene has been altered to reduce or eliminate ICAM-1 expression on the surface of said cell and/or activity in said cell by contacting said cell with either a Cas protein or a nucleic acid an acid encoding a Cas protein in combination with either a plasmid encoding a ribonucleic acid that is homologous to any of SEQ ID NOs: 4-6 or a ribonucleic acid that is homologous to any of SEQ ID NOs: 4-6.

[00270] The indicated sequences of SEQ ID Nos: 1-9 are shown below in Table 4, and these and additional sequences are described below.

[00271] SEQ ID NO. 1: Exon 1, negative strand.

[00272] SEQ ID NO. 2: Exon 2, negative strand.

[00273] SEQ ID NO. 3: Exon 1, negative strand.

[00274] SEQ ID NO. 4: Exon 2, positive strand.

[00275] SEQ ID NO. 5: Exon 2, negative strand.

[00276] SEQ ID NO. 6: Exon 1, positive strand.

[00277] SEQ ID NO. 7: Exon 1, negative strand.

[00278] SEQ ID NO. 8: Exon 1, positive strand.

[00279] SEQ ID NO. 9: Exon 1, positive strand.

[00280] SEQ ID NO. 10: encodes the human ICAM1 sequence.

[00281] SEQ ID NO. 11: encodes the human CEACAM1 sequence.

[00282] SEQ ID NO. 12: encodes the human B2M sequence.

[00283] SEQ ID NO. 13: encodes the human CADM1 sequence.

[00284] SEQ ID NO. 14: encodes the human CD58 sequence.

[00285] SEQ ID NO. 15: encodes the human CD155 sequence.

[00286] Target sequences for CRISPR7Cas9 cleavage, including PAM (NGG), are shown in the table below.

[00287] A pancreatic endoderm (PEC) cell containing a modified genome comprising (a) a first genome modification in which said B2M gene has been altered to reduce or abolish B2M expression on the surface of said cell and/or activity in said cell by bringing into contact said cell with a Cas protein or a nucleic acid encoding a Cas protein and a ribonucleic acid containing any sequence from SEQ ID NOs: 1-3; (b) a second genome modification in which said ICAM-1 gene has been altered to reduce or eliminate ICAM-1 expression on the surface of said cell and/or activity in said cell by contacting said cell with a Cas protein or nucleic acid encoding the protein Cas, and ribonucleic acid containing any sequence from SEQ ID NO: 4-6.; and (c) a third genome modification wherein said CEACAM1 gene has been altered to reduce or eliminate CEACAM1 expression on the surface of said cell and/or activity in said cell by contacting said cell with a Cas protein or a nucleic acid encoding a Cas protein, and ribonucleic acid containing any sequence from SEQ ID NO: 7-9.

[00288] A method for reducing hypoglycemia, comprising:

a) administering to a subject in need of a graft an effective amount of a graft comprising a population of pancreatic endoderm cells in which at least one class I LAS cell surface protein and at least one NK cell activating cell surface ligand protein is impaired in which said population of pancreatic endoderm cells matures in vivo and produces insulin in response to glucose stimulation in vivo, thus reducing hypoglycemia in the patient.

[00289] A method for reducing insulin dependence, comprising: a) administering to a subject in need of a graft an effective amount of a graft comprising a population of pancreatic endoderm cells in which at least one class I LAH cell surface protein and at least one protein is impaired a cell surface ligand that activates NK cells, wherein said population of pancreatic endoderm cells matures in vivo and produces insulin in response to glucose stimulation in vivo, thereby reducing insulin dependence in a patient.

DEFINITIONS

[00290] "Hypoimmunogenic" or "universal donor cells" or "mutant cells", or equivalents, means cells with reduced or abolished expression of at least one class I LAS cell surface protein and at least one NK cell activating ligand. It is expected that such a cell will be less susceptible to immune or graft rejection by the subject to whom such cells or graft is transplanted. For example, compared to unaltered wild-type cells, such a hypoimmunogenic cell may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 %, 95%, 97.5%, 99% or more less susceptible to immune rejection by the subject into which such cells are transplanted.

[00291] The term "treatment" or "cure" or equivalents refers to a therapeutic intervention that reduces (improves) a sign or symptom.

[00292] The term "patient", "host", "mammalian host", "subject", or equivalents thereof, refers to living multicellular vertebrate organisms, a category that includes both mammals and humans. In some embodiments, said subject is a human. The preferred patient for treatment is a human. Target patient populations may change over time of clinical use/experience in ways that are independent of the combination product itself, but rather related to the nature of the immunosuppression regimen, or lack of it. For example, a combination product may be used in the T1D population using hypoimmunogenic cell therapy in combination with an immunosuppressant (ISD) regimen that provides operational tolerability or a low toxicity and side effect profile.

[00293] The term "blocking agent" as used herein refers to any agent capable of binding to an NK cell activating ligand on the surface of a target cell, including, but not limited to, an antibody; or refers to an agent that prevents or inhibits the expression of an NK cell activating ligand protein, including, but not limited to, a protein, enzyme, or chemical currently known or later developed.

[00294] The term "antibody" is used herein in its broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments if they exhibit the desired biological blocking activity.

[00295] As used herein, the term "antibody fragments" refers to the portion of an intact antibody, preferably containing its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[00296] The term "blocking antibody" as used herein refers to an antibody which, when bound to an NK cell activating ligand on a target cell in vivo or in vitro, results in the prevention or reduction of the ability of NK cells to cause lysis of the target cell.

[00297] As used herein, the term "syngeneic" refers to cells, tissues, or organs that are genetically identical or derived from a genetically identical source to a transplant recipient (e.g., an identical twin), especially with respect to antigens or immunological responses. Such cells, tissues or organs are called isografts. As used herein, the term "allogeneic" refers to cells, tissues, or organs that are not genetically identical or derived from a non-genetically identical source for a transplant recipient (e.g., an unrelated donor), especially with respect to antigens or immunological responses. Such cells, tissues, or organs are called allografts, allogeneic transplants, homografts, or allografts.

[00298] As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence that is necessary for the expression of a gene product operably linked to a promoter/regulatory sequence. In some cases, this sequence may be the main promoter sequence, and in other cases, this sequence may also include the enhancer sequence and other regulatory elements that are necessary for the expression of the gene product. Said promoter/regulatory sequence may, for example, be a sequence that expresses the gene product in a tissue-specific manner.

[00299] The term "effective amount" or "therapeutically effective amount" or equivalents refers to an amount of an agent sufficient to achieve the desired effect in the subject or cell being treated. For example, this may be the number of cells required to inhibit or significantly lower blood glucose and ultimately achieve homeostatic glycemic control. It can also mean an effective amount of an agent to change the function or structure of a cell or subject. A therapeutically effective amount of the agent may be administered in a single dose or in multiple doses. However, the effective amount will depend on the particular agent used, the subject being treated, the severity and type of disease, and the route of administration.

[00300] The terms "reduction", "destruction", "reduced", "reduction" and "inhibition" are used in the present description, as a rule, to denote a decrease, in particular a decrease by a statistically significant value. However, for the avoidance of doubt, "reduced", "reduced", "reduced", "inhibited" includes a reduction of at least 10% from a control level, such as a reduction of at least about 20% or at least about 30 %, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least at least about 80%, or at least about 90%, or up to and including including a 100% reduction (i.e., none compared to the control) or any 10-100% reduction from control level, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least at least about 10 times, or any decrease of 2-10 times or more compared to the control level.

[00301] The terms "increased", "increase" or "enhance" or "activate" are used herein to mean an increase by a statically significant amount; for the avoidance of doubt, the terms "increased", "increase" or "enhance" or "activate" means an increase of at least 10% over a control level, such as an increase of at least about 20%, or at least at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70% , or at least about 80%, or at least about 90%, or up to a 100% increase, or any 10-100% increase over a control level, or at least , about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase of 2-10 times or more compared to the control level.

[00302] The term "statistically significant" or "significantly" refers to statistical significance and usually means two standard deviations (2SD) below normal or lower concentration of the reference. The term can also mean two standard deviations (2SD) above the normal or higher concentration of the reference. The term refers to statistical evidence that a difference is being detected. It is defined as the probability of deciding to reject the null hypothesis when the null hypothesis is indeed true. The decision is often made using the p value.

[00303] As used herein, the term "reduced hypoglycemia" or equivalents means a decrease in the number of episodes of hypoglycemia, together with no deterioration in glycemic control, as defined by a 0.2% increase in HbA1c.

[00304] As used herein, the term "reduced insulin dependence" or equivalents means a reduction in the number and/or dose of exogenous insulin injections, together with no deterioration in glycemic control, as defined by a 0.2% increase in HbA1c.

[00305] As used herein, the term "tissue capsule" or equivalents means a foreign body capsule that is formed around an implant or graft. The combined product and/or device or perforated device containing certain cells is intended to be retained in the capsule during the implantation period.

[00306] "Transplantation" or its equivalents refers to the differentiation of a progenitor population or the maturation of a cell population into mature cell types. For example, transplantation of a PDX1-positive population of pancreatic endoderm cells maturing into a population of pancreatic endocrine cells.

[00307] "Transplant" refers to a differentiated cell population encapsulated or delivered in the devices described herein. For example, cell populations including pancreatic endoderm, pancreatic progenitor, PDX-1-positive pancreatic endoderm, pancreatic endocrine progenitor, pancreatic endocrine graft, single or polyhormonal endocrine graft, pre-beta, beta, and/or insulin secreting grafts, but are not limited to them.

[00308] The term "essentially" or equivalents means essentially or de minimus or a reduced amount of a component or cell present in any cell population or culture, e.g., mature beta cell culture, which are "essentially mature beta cells expressing INS, NKX6.1 and PDX1 and essentially not expressing NGN3". Other examples include "essentially hES cells", "essentially definitive endoderm cells", "essentially anterior digestive tract endoderm cells", "essentially PDX1 negative anterior digestive tract endoderm cells", "essentially PDX1 positive cells pancreatic endoderm", "essentially pancreatic endocrine cell precursors", "essentially pancreatic endocrine cells" and the like, but are not limited to.

[00309] With regard to cells in cell cultures or cell populations, the term "substantially free" or equivalents means that the specified cell type, from which the culture cells or cell population is free, is present in an amount of less than about 10%, less than less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total number of cells present in said cell cultures or cell population.

[00310] The term "nonwoven fabric" or its equivalent includes, but is not limited to, reinforced fabrics, molded fabrics, or artificial fabrics that are produced by methods other than weaving or knitting.

[00311] It should be understood that the inventions disclosed herein are not limited in their application to the details set forth in the present description or as examples. The present invention covers other embodiments and can be practiced or carried out in various ways. In addition, it should be understood that the phraseology and terminology used herein is for the purposes of description and should not be construed as limiting the scope of the present invention in any way.

[00312] Although some of the compositions, methods and assays of the present invention have been specifically described in accordance with certain embodiments, the following examples serve only to illustrate the methods and compositions of the present invention and do not limit the scope of the present invention in any way.

[00313] Singular articles (singular) used in the present description and in the claims, unless otherwise indicated, should be understood as including the plural. Claims or descriptions that include the conjunction "or" between one or more members of a group are considered to be satisfied if one, more than one, or all of the members of the group are present, used, or otherwise related to a given product or process, unless otherwise indicated or obviously out of context. The present invention includes embodiments in which one member of the group is specifically present, used, or otherwise related to a given product or process. The present invention also includes embodiments in which more than one member of the group or all members of the group are present, used or otherwise related to a given product or process.

[00314] In addition, it should be understood that the present invention covers all variants, combinations and permutations in which one or more of the restrictions, elements, clauses, descriptive terms, etc. of one or more of the listed claims are included in another claim dependent on the same independent claim (or, as the case may be, on any other claim), unless otherwise indicated, or unless it is clear to a person skilled in the art that there may be a conflict or inconsistency. If elements are presented as lists (eg, in the form of a Markush structure or a similar format), it should be understood that each subgroup of elements is also disclosed, and any element(s) may be removed from the group. It should be understood that, in general, when the present invention or aspects of the present invention are referred to as containing specific elements, features, etc., some embodiments of the present invention or aspects of the present invention consist or essentially consist of such elements, features, etc. .d. For the sake of simplicity, these embodiments are not in all cases specifically set forth in this description in detail using so many words. It should also be understood that any embodiment or aspect of the present invention may be explicitly excluded from the claims, regardless of whether a specific exception is indicated in the description. Publications and other references referenced herein to describe the background of the present invention and to provide additional details regarding its practice are incorporated herein by reference in their entirety. The present description is illustrated by the following examples, which do not limit the scope of the present invention.

EXAMPLES

EXAMPLE 1: PRODUCTION OF HES CELLS DEFICIENT FOR B2M

[00315] B2M-deficient hES cells were generated using the CyT49 cell line, however, any human pluripotent stem cell line can be used. A targeted disruption of the B2M gene generates cells that do not express any of the class I LAH proteins on the surface of these cells. Both alleles of the B2M locus in the CyT49 hESC line were disrupted using CRISPR/Cas9 technology using the known methods described in PCT Application Publication WO2016183041A (which is incorporated herein in its entirety). However, other nucleases can be used for gene editing, including zinc finger nuclease (ZFN) and effector nuclease like transcription activators (TALEN), as well as traditional homologous recombination and the like. Examples of published B2M gene sequences are shown in SEQ ID NOs: 1, 2, and 3. NEXTGEN™ CRISPR (Transposagen Inc., Lexington, KY) was used for gene editing, which includes a double guide RNA and a catalytically inactive Cas9 protein coupled to FokI nuclease . Plasmids containing guide RNAs and Cas9 were introduced into CyT49 hESCs by electroporation and the resulting cells were seeded onto tissue culture dishes. 12 days after electroporation, cells were sorted for negative anti-B2M antibody activity (BioLegend, Cat. No. 316306) by fluorescence-activated cell sorting (FACS). Sorted cells were seeded at the density required for cloning. Individual clones were collected and seeded for 25 days. Clones were grown and cryopreserved. Grown clones that showed a normal G-stained karyotype and lacked expression of B2M protein and expression of LH class I proteins on the surface of these cells as determined by flow cytometry and/or immunofluorescence were selected for further experiments.

[00316] B2M expression on the surface of said wild-type (WT) and knockout cells was assessed by flow cytometry under normal and inflammatory conditions (after treatment with interferon (IFN)-γ). See Fig. 2A: Normal conditions: untreated growth medium (Line B); inflammation conditions: treated with 100 ng/ml IFN-γ for 18-24 hours (Line A). The inflammatory response was consistent with tissue injury. It resulted in the release of pro-inflammatory cytokines, some of which are IL-1-α, IL-1-β, TNF-α, IL-6, IL-8 and IFN-γ. Although WT and B2M -/- ESC and PEC cells were treated with IFN-γ, these observations can also be extended to other cytokines.

[00317] In Fig. 2A shows B2M expression in wild-type hES cells (Line B) without IFN-γ and B2M expression after treatment of wild-type hES cells with IFN-γ (Line A). A change (increase) in fluorescence intensity in untreated wild-type hES cells (Line B) from baseline (shaded area) indicates that wild-type hES cells express B2M. A further change (increase) in fluorescence intensity above WT B2M expression (Line B) after treatment of wild-type hES cells with IFN-γ indicates that B2M expression is increased in wild-type hES cells after treatment with IFN-γ (Line A). As such, the expression of cell surface class I LAH proteins can be upregulated by cellular stress and inflammation, such as occurs with at least IFN-γ treatment.

[00318] In Fig. 2B shows B2M expression in B2M knockout hES cells generated using the CRISPR/Cas system. There is essentially no change (increase) in fluorescence intensity from baseline (shaded area) with or without IFN-γ treatment, suggesting that B2M knockout hES cells had reduced or absent expression of B2M on the surface of said cell and that expression B2M was not induced by IFN-γ treatment. In such B2M knockouts, class I LAH cell surface protein expression is not upregulated by cellular stress and inflammation mediated by IFN-γ processing.

[00319] This Example demonstrates that B2M knockout hES cells had reduced or absent expression of B2M on the cell surface as shown using anti-B2M antibodies.

EXAMPLE 2: EXPRESSION ANALYSIS OF LAH CLASS I CELL SURFACE PROTEINS IN WT AND B2M DEFICIENT CELLS

[00320] Next, wild-type hES cells and B2M knockout cells were analyzed using Pan-LAH-ABC monoclonal antibody (BD Pharmingen, Cat # 560169) to confirm that these knockout cells do not express class I LH proteins on the cell surface. . Said Pan-LAH-ABC antibody reacts with class I major histocompatibility complex (MCHC) class LAH-A, -B, and -C proteins. Expression using the Pan-LAH-ABC antibody was assessed in wild-type and knockout cells using flow cytometry under normal and inflammatory conditions after treatment with IFN-γ. Normal conditions: no IFN-γ (Line B); inflammatory conditions: treatment with 100 ng/ml IFN-γ for 18-24 hours (Line A).

[00321] In Fig. 3A shows pan-expression of LAHS-ABC cell surface proteins in wild-type hES cells (Line B) and pan-expression of LAHS-ABC cell surface proteins after treatment of wild-type hES cells with IFN-γ (Line A). A change (increase) in fluorescence intensity in these untreated wild-type hES cells (Line B) from baseline (shaded area) indicates that wild-type hES cells express pan-LAH-ABC. The change (increase) in fluorescence intensity relative to hES expression of WT cells after IFN-γ treatment indicates that pan-LAH-ABC expression is increased in wild-type hES cells after IFN-γ treatment.

[00322] In Fig. 3B shows LAH-ABC cell surface protein pan expression in B2M knockout hES cells using the CRISPR/Cas system. No shift (increase) in fluorescence intensity from baseline (shaded area) was observed with or without IFN-γ treatment, which may indicate that the knockout cells had reduced or absent expression of LAH class I cell surface proteins and that protein expression Class I LAS was not induced by IFN-γ treatment.

[00323] This Example demonstrates that B2M knockout hES cells had reduced or absent expression of LH class I cell surface proteins as shown using anti-pan-LAH-ABC antibodies.

EXAMPLE 3: DIFFERENTIATION OF B2M DEFICIENT CELLS INTO PANCREATIC CELL LINE

[00324] B2M knockout hES cells were grown, passaged and propagated under the same conditions as wild-type hES cells, described in Schulz m dp. A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells PLoS One 7:5 1-17 (2012) and as described in US Pat. No. 8,895,300, both documents are incorporated herein by reference in their entirety. In particular, Schulz et al. describe the adhesive expansion of hESCs and their differentiation in suspension.

[00325] Briefly, wild-type hES cells and B2M ^-/- hES cells were differentiated in suspension aggregates using a four (4) step procedure over about 2 weeks (or 14 days) to obtain a population of pancreatic-type cells, including pancreatic cell progenitors, progenitors endocrine cells and cells expressing hormones, collectively referred to as cells of the pancreatic endoderm (PEC). Human ES cells were dissociated using accutase and individual cells were collected in roller bottles. To initiate differentiation, aggregates were pooled into conical tube(s) and allowed to settle by gravity followed by washing with Stage-1 media without growth factors (RPMI + 0.2% v/v PBS containing 1 :5000 dilution of insulin-transferrin-selenium (ITS)). These aggregates were re-assembled, then resuspended in one-day medium, which contained RPMI+0.2% v/v. PBS containing a 1:5000 dilution of insulin-transferrin-selenium (ITS), activin A (100 ng/ml) and wnt3a (50 ng/ml) and dispensed into roller bottles at a density of 2 μl/ml. These roller vials were placed in a FlexiRoll digital cell roller (Argos Technologies) operating at 31 rpm. The cultures were rotated at approximately 31 rpm for the remainder of the differentiation process with daily media changes as described in Table 2 below, adapted from Schulz et al., (2012), supra. Growth, passaging and propagation of hES cells was carried out essentially as described in US patent No. 7,964,402; 8,211,699; 8,334,138; 8,008.07 and 8,153,429. A standard method for obtaining pancreatic endoderm (PEC) cells derived from human embryonic stem cells is described in Table 2 below.

[00327] hESC Agg.: hESC aggregates; XF HA: DMEM/F12 containing GlutaMAX supplemented with 10% v/v Xeno-free knockout serum replacement, 1% v/v nonessential amino acids, 1% vol./about. penicillin/streptomycin (all from Life Technologies), 10 ng/ml heregulin-1β (Peprotech) and 10 ng/ml activin A (R&D Systems); SP: StemPro® hESC SFM (Life Technologies); r0.2FBS: RPMI 1640 (Mediatech); 0.2% FBS (HyCione), 1x GlutaMAX-1 (Life Technologies), 1% v/v penicillin/streptomycin; ITS: insulin-transferrin-selenium (Life Technologies) 1:5000 or 1:1000 dilution; A100: 100 ng/ml recombinant human Activin A (R&D Systems); W50: 50 ng/ml recombinant human Wnt3A (R&D Systems); K25: 25 ng/ml recombinant human KGF (R&D Systems); IV: 2.5 μM TGF-β RI kinase inhibitor IV (EMD Bioscience); db: DMEM HI Glucose (HyClone) supplemented with 0.5x B-27 Supplement (Life Technologies), 1x GlutaMAX, and 1% v/v. penicillin/streptomycin; CTT3: 0.25 μM KAAD-Cyclopamine (Toronto Research Chemicals) and 3 nM TTNPB (Sigma-Aldrich); N50: 50 ng/ml recombinant human Nogin (R&D Systems); K50: 50 ng/ml recombinant human KGF (R&D Systems); E50: 50 ng/ml recombinant human EGF (R&D Systems).

[00328] Differentiated B2M -/- and wild-type RES were analyzed using flow cytometry to determine the relative abundance of endocrine and pancreatic cell progenitors in the stage 4 population as shown in Table 3.

[00330] The relative levels of pancreatic endocrine cells, progenitors, PDX-1 cells only, and triple negative cells in differentiated B2M -/- cells in all 3 clones are essentially similar to the levels observed in WT cells (top row).

[00331] This Example demonstrates that these B2M -/- hES cells can differentiate into pancreatic cell lines as well as wild-type hES cells.

EXAMPLE 4: B2M EXPRESSION ANALYSIS IN WT AND B2M DEFICIENT PANCREATIC ENDODERM CELLS

[00332] Wild type and B2M knockout pancreatic endoderm (PEC) cells from Example 3 were then analyzed using flow cytometry with and without IFN-γ treatment: no IFN-γ (Line B); treated with 100 ng/ml IFN-γ for 18-24 hours (Line A).

[00333] In Fig. 4A shows B2M expression in wild-type PEC cells without treatment (Line B) and after treatment with IFN-γ (Line A). A change (increase) in fluorescence intensity in these untreated wild-type RES cells (Line B) from baseline (shaded area) indicates that wild-type RES cells express B2M. A change (increase) in fluorescence intensity above WT expression after wild-type RES treatment with IFN-γ (Line A) indicates that B2M expression is increased in wild-type RES cells after IFN-γ treatment. That is, IFN-γ treatment increases B2M expression in wild-type PEC cells.

[00334] In Fig. 4B shows B2M expression in PEC cells derived from B2M knockout hES cells. No shift (increase) in fluorescence intensity from baseline (shaded area) was observed with or without IFN-γ treatment, indicating that B2M knockout PEC cells had reduced or absent B2M expression on the cell surface and that B2M expression cannot be induced in PEC cells derived from B2M knockout cells by treatment with IFN-γ.

[00335] This Example shows that PEC cells derived from hES cells in which B2M expression was reduced/eliminated had reduced or absent B2M expression on the surface of said cell.

EXAMPLE 5: EXPRESSION OF LAH CLASS I CELL SURFACE PROTEINS IN WT CELLS AND B2M KNOCKOUT PANCREATIC ENDODERM CELLS

[00336] As in Example 2, wild-type cells and B2M knockout PEC cells were analyzed using a pan-LAH-ABC monoclonal antibody (BD Pharmingen cat #560169) to determine cell surface class I LH expression. Expression was measured by flow cytometry in wild-type and knockout cells under two conditions: (1) untreated (Line B) and (2) treated with 100 ng/ml IFN-γ for 18-24 hours (Line A).

[00337] In Fig. 5A shows expression of LH class I cell surface proteins in wild-type RES cells under untreated conditions (Line B) and expression after treatment with IFN-γ (Line A). The change (increase) in fluorescence intensity in these untreated wild-type RES (Line B) from baseline (shaded area) indicates that wild-type RES express class I LAS on the cell surface. A change (increase) in fluorescence intensity above the level of wild-type RES expression after treatment with IFN-γ indicates that class I LAS cell surface expression is increased in wild-type RES cells after treatment with IFN-γ.

[00338] In FIG. 5B shows expression of class I LAH cell surface proteins in PEC cells derived from B2M knockout hES cells. There was no change (increase) in fluorescence intensity from baseline (shaded area) in PEC cells with or without IFN-γ treatment, suggesting that these knockout cells had reduced or absent expression of LH on the cell surface and that expression of the LH class I in B2M-/- PEC cells cannot be induced by IFN-γ treatment.

[00339] As such, reduced or absent expression of class I LAH cell surface proteins was observed in those PEC cells in which B2M expression was reduced/eliminated.

EXAMPLE 6: CELL SURFACE ICAM-1 PROTEIN EXPRESSION ANALYSIS IN WT CELLS AND B2M KNOCKOUT HES CELLS

[00340] To further determine the effect of IFN-γ treatment on target cells, ICAM-1 expression was assessed by flow cytometry in WT cells and B2M knockout hES cells under two conditions: (1) no treatment (Line B) and (2 ) treatment with 100 ng/ml IFN-γ for 18-24 hours (Line A). The ICAM-1 protein is credited with several immunological functions, including antigen presentation in target cells, and is known as a NK cell activating ligand. In vivo, it is possible to obtain immunological benefits by disrupting the cell-to-cell binding interaction of ICAM/LFA by the use of specific monoclonal antibodies ("mAbs"), ie anti-ICAM-1 or anti-LFA-1 antibodies. See Isobe et al., Specific Acceptance of Cardiac Allograft After Treatment With Antibodies to ICAM-1 and LFA-1, 255 SCIENCE 1125-1127 (Feb. 1992). Applicant used anti-ICAM-1 antibodies obtained from Milteney Biotec Inc., Cat. No. 130-103-909.

[00341] In Fig. 6A shows ICAM-1 cell surface protein expression in untreated wild-type hES cells (Line B) and after treatment with IFN-γ (Line A). A change (increase) in fluorescence intensity from baseline (shaded area) in untreated WT hES cells (Line B) indicates that wild-type hES cells express ICAM-1 protein on their surface. A change (increase) in fluorescence intensity above WT expression after treatment of wild-type hES cells with IFN-γ indicates that ICAM-1 expression is increased after treatment with IFN-γ.

[00342] In Fig. 6B shows ICAM-1 cell surface protein expression in untreated B2M knockout hES cells (Line B) and after treatment with IFN-γ (Line A). On FIG. 6B shows that ICAM-1 cell surface protein expression in these B2M knockout hES cells was similar to expression in wild type hES cells.

[00343] This Example demonstrates that treatment of WT cells and B2M knockout hES cells with IFN-γ increases the expression of ICAM-1 cell surface proteins.

EXAMPLE 7: ICAM-1 CELL SURFACE PROTEIN EXPRESSION ANALYSIS IN WT CELLS AND B2M KNOCKOUT PANCREATIC ENDODERM CELLS

[00344] In analogy to Examples 4 and 5, wild-type cells and B2M knockout PEC cells were analyzed using an antibody to a known NK cell activating ligand, eg, ICAM-1. Cell surface protein expression of ICAM-1 was assessed by flow cytometry in WT cells and knockout PEC cells under two conditions: (1) no treatment and (2) treatment with 100 ng/ml IFN-γ for 18-24 hours.

[00345] In FIG. 7A shows expression of ICAM-1 cell surface protein in wild-type PEC cells (Line B) and expression of ICAM-1 cell surface protein after treatment of wild-type PEC cells with IFN-γ (Line A). The change (increase) in fluorescence intensity in these untreated wild-type RES (Line B) from baseline (shaded area) indicates that wild-type RES express ICAM-1 protein on their surface. A further change (increase) in fluorescence intensity above expression in wild-type RES after IFN-γ treatment indicates that ICAM-1 expression is increased in wild-type RES cells after IFN-γ treatment. This demonstrates that NK-activating ligands, and in particular ICAM-1, are highly inducible by stimulation with IFN-γ.

[00346] In FIG. 7B shows ICAM-1 cell surface protein expression in B2M knockout PEC cells. Expression of ICAM-1 cell surface protein in these B2M knockout PECs was similar to expression in wild-type RES, with and without IFN-γ treatment, with a slight decrease in fluorescence. Thus, treatment of B2M knockout PEC cells with IFN-γ increases the expression of cell surface protein ICAM-1.

[00347] In Fig. 8 shows RNA expression data on a biochip (Affymetrix) demonstrating that, at the mRNA level, IFN-γ treatment upregulates ICAM-1 expression in WT hESC, B2M-/- hESC, wild-type PEC and B2M-/- PEC cells. The present inventors found that cell surface protein expression of the NK cell activating ligand ICAM-1 was increased in differentiated cell types (WILD-TYPE and B2M-/-PEC) after treatment with IFN-γ. Thus, ICAM-1 is strongly induced by stimulating IFN-γ in RES.

EXAMPLE 8: EFFECT OF IFN-GAMMA TREATMENT ON ADDITIONAL NK ACTIVATED LIGANDS

[00348] In order to further characterize the effect of IFN-γ treatment on target cells, wild-type PEC cells were studied by flow cytometry under two conditions: (1) no treatment (Line B) and (2) treatment with 100 ng/ ml of IFN-γ for 18-24 hours (Line A), and analyzed the expression of cell surface proteins of other known NK activating ligands.

[00349] In Fig. 9 shows expression of the cell surface protein CD58 (also known as LFA-3) using an antibody from BioLegend, cat. No. 330909, in untreated wild-type RES cells (Line B) and after treatment with IFN-γ (Line A). A large shift (increase) in fluorescence intensity from baseline (Line C) in these untreated wild-type hES cells (Line B) indicates that most of these cells express the CD58 protein on their surface. There was a slight additional change (increase) in fluorescence intensity after treatment with IFN-γ.

[00350] In FIG. 10 shows expression of CD155 cell surface protein (also known as PVR, NECL-5, HVED) using an antibody from Milteneyi Biotech Inc., cat. No. 130-105-905 in untreated wild-type RES cells (Line B) and after treatment with IFN-γ (Line A). The change (increase) in fluorescence intensity from baseline (Line C) in these untreated wild-type RES (Line B) indicates that wild-type RES express CD155. No additional change (increase) in fluorescence intensity compared to untreated cells after IFN-γ treatment was observed, indicating that CD 155 expression was not increased in wild-type PEC cells after IFN-γ treatment.

[00351] In FIG. 11 shows cell surface protein expression CEACAM1 (also known as CD66a, BGP, and BGP1) using an antibody from Milteneyi Biotech Inc., cat. No. 130-098-858 in untreated wild-type RES cells (Line B) and after treatment with IFN-γ (Line A). A change (increase) in fluorescence intensity from baseline (Line C) in these untreated wild-type RESs (Line B) indicates that wild-type RESs express the CEACAM1 protein on the cell surface. The change (increase) in fluorescence intensity from untreated conditions after wild-type RES treatment with IFN-γ indicates that cell surface CEACAM1 protein expression is increased in wild-type RES cells after IFN-γ treatment.

[00352] In Fig. 12 shows expression of cell surface protein BAT3 (also known as BAG6) using an antibody from Abeam Inc., cat. No. ab210838 in untreated wild-type RES cells (Line B) and after treatment with IFN-γ (Line A). The change (increase) in fluorescence intensity from baseline (Line C) in these untreated PECs (Line B) indicates that these cells express CEACAM1. No additional change (increase) in fluorescence intensity compared to untreated cells was observed after wild-type RES treatment with IFN-γ, indicating that BAT3 expression was not increased in wild-type RES cells after IFN-γ treatment.

[00353] In FIG. 13 shows expression of CADM1 cell surface protein (also known as NECL2, TSLC1, IGSF4, RA175) using an antibody from MBL international Corporation, cat. No. CM004-4 in untreated wild-type RES cells (Line B) and after treatment with IFN-γ (Line A). The change (increase) in fluorescence intensity from baseline (Line C) under the indicated untreated conditions (Line B) indicates that wild-type PECs express the CADM1 protein on their surface. No additional change (increase) in fluorescence intensity from untreated conditions after PEC treatment with IFN-γ was observed, indicating that CADM1 expression is not increased after IFN-γ treatment.

[00354] In FIG. 14 shows the expression of CD112 (also known as Nectin-2, PVRR2, HVEB) using an antibody from Milteneyi Biotech Inc., cat. No. 130-109-056 in untreated wild-type RES cells (Line B) and after treatment with IFN-γ (Line A). The change (increase) in fluorescence intensity from baseline (Line C) under the indicated untreated conditions (Line B) indicates that wild-type RES express the CD112 protein on their surface. No additional change (increase) in fluorescence intensity from untreated conditions after PEC treatment with IFN-γ was observed, indicating that CD112 expression is not increased after IFN-γ treatment.

EXAMPLE 9: MHC CLASS I DEFICIENT CELLS DEFICIENT FOR NK CELL ACTIVATED LIGANDS PREVENT NK CELL-MEDIATED LYSIS

[00355] To test whether the combination of reduced or eliminated LAH class I protein expression and reduced or eliminated expression of NK cell activating ligand is sufficient to prevent NK cell mediated lysis, ICAM-1 expression on target cells was blocked using antibodies blocking ICAM-1 at concentrations of 5 and 10 µg/ml. Addition of an antibody blocking ICAM-1 to WT or B2M -/- ES or PEC cells caused a decrease in target NK cell lysis after treatment with IFN-γ (see Fig. 15).

Target cell staining with Calcein-AM

[00356] The Calcein Release Assay is a non-radioactive alternative for studying NK cell cytotoxicity. The target cells take up the fluorescent dye (calcein AM) and cytoplasmically convert it into the active fluorochrome, which is released from the cells after lysis. The lysed cells release the fluorochrome into the supernatant, which is then collected and the amount of fluorescence is quantified in a fluorimeter. Percent cell lysis is calculated from the amount of fluorescence present in the supernatant after incubation in the presence or absence of NK cells (effectors), blocking antibody, or both.

[00357] Target cells contained either WT ESC, B2M-/-ESC, wild-type PEC or B2M-/-PEC treated with or not treated with 100 ng/ml IFN-γ prior to labeling. To obtain said target cells, populations of said target cells were stained with 2 μg/ml calcein AM staining medium (Enzo biosciences, 1 mg/ml concentrated solution (Cat. No. C3100MP)). These target cells were incubated for 1 hour at 37° C. in an 8% CO ₂ incubator with intermittent shaking. These target cells were washed twice to remove all free calcein AM and resuspended to a concentration of 1×10 ⁵ cells/ml in complete RPMI medium (RPMI, 10% heat-inactivated PBS and 1% antibiotics).

Co-cultured cells, NK cells and blocking antibody

[00358] Calcein-labeled AM target cells were incubated with NK cells (effector cells) in an effector:target cell ratio (E:T ratio) of 10:1. Specifically, 100 µl of NK cells at a density of 1×10 ⁶ cells/ml was added to each well of a 96-well V-bottom plate, followed by 100 µl of calcein-stained ESC or PEC cells (1×10 ⁵ cells per well) . Where indicated, a blocking antibody to human ICAM-1 surface antigen (R&D Systems, Inc., cat. no. BBA3) at 5 and 10 μg/mL was added to the wells to determine whether the rate of cell lysis mediated by NK cells would be reduced. .

[00359] the Tablets were incubated for 4 hours at 37°C in an incubator with 8% CO ₂ . After incubation, the plates were centrifuged at 200×g for 2 minutes. 100 μl of the supernatant was carefully removed and transferred to a black pigmented 96-well plate and fluorescence was measured using a Molecular Device plate reader (excitation filter: 485 nm/emission filter: 530 nm). Specific lysis was calculated using the following formula, % lysis = 100×[(Average fluorescence with antibody - Average spontaneous fluorescence)/(Average maximum fluorescence - Average spontaneous fluorescence)]. Maximal fluorescence was determined by lysis of cells incubated with detergent (1% Triton X-100) and spontaneous lysis was defined as fluorescence obtained with target cells without any antibody or effector cells.

results

[00360] As shown in FIG. 1, the goal is to move from Scenario C (NK cells attack target cells) to Scenario A (no response or reduced response). On FIG. 15, this scenario is shown under conditions 4 and 8. Under conditions 4 and 8, the target cells (B2M -/- hES or PEC cells) lack the expression of functional class I LAH surface protein and are treated with IFN-γ. As discussed above and seen under conditions 4 and 8, IFN-γ treatment induces an increase in NK-activating ligands (ICAM-1), which results in increased cell lysis compared to class I LAS knockout cells without IFN-γ treatment (compare the first columns in condition 3 compared to condition 4 and condition 7 compared to condition 8). However, when treated with an ICAM-1 inhibitory antibody that serves to block the expression of NK-activating signals on the target cell, NK-mediated cell lysis drops from 83% to 73% in B2M-/- hES cells and from 72% to 61 % in B2M-/- RES cells treated with IFN-γ. The percentage of NK-mediated cell lysis does not drop to zero because expression of the cell surface protein ICAM-1 cannot be completely blocked using said blocking antibody and, as described above, said target cells express more than one NK cell activating ligand. It is expected that the percentage of NK-mediated cell lysis will fall to a greater extent when the expression of ICAM-1 is further inhibited and other NK-activating ligands are also blocked in these target cells. Therefore, said ICAM-1 inhibitory antibody can be combined with additional antibodies that inhibit NK cell activating ligands, including inhibitory antibodies to any of the ligands listed in Table 1, and preferably Category 1 ligands (known activating ligands) and 2 (potential activating ligand candidates identified based on DNA chip data, ligands upregulated in PECs and/or ESCs after IFNγ treatment). In one embodiment, said ICAM1 gene and other NK cell activating ligand genes are disrupted to completely block their activity.

[00361] Scenario D in FIG. 1 is represented in the first columns of conditions 2 and 6 in FIG. 15. Under conditions 2 and 6, these wild-type and RES hES cells overexpress cell surface proteins of both LHS class I antigens and NK-activating ligands as a result of their treatment with IFN-γ. When these cells are incubated with an ICAM-1 inhibitory antibody, this demonstrates the situation of moving from Scenario D to Scenario B in FIG. 1. After incubation with an antibody that inhibits ICAM1, cell death is reduced from 79% to 60% for wild-type hES cells and from 53% to 27% for wild-type RES cells treated with IFN-γ. Indeed, when wild-type RES is treated with IFN-γ and incubated with an antibody that inhibits ICAM-1, NK-mediated cell lysis is reduced compared to untreated wild-type RES: 27% for wild-type RES, IFN-γ, and an antibody that inhibits ICAM-1. 1 compared to 37% for wild-type PEC, ICAM-1 inhibitory antibody, without IFN-γ treatment.

[00362] Scenario B in FIG. 1 is represented by bars with ICAM-1 inhibitory antibody treatment under conditions 1, 2, 5 and 6 in FIG. 15. Here, said hES and PEC target cells have class I LAS and NK activating ligands, but when said target cells are not treated with IFN-γ, there is no increase in expression of ICAM-1 cell surface proteins. As a result, said ICAM-1 inhibitory antibody has less effect. Therefore, cell lysis remains approximately the same: 58% and 57% in hES cells and 33% and 37% in RES cells.

[00363] Scenario C in FIG. 1 is similar to the first columns in conditions 3, 4, 7 and 8 in FIG. 15. Under these conditions, these cells do not express class I LAS on the cell surface as a result of the B2M-/- genotype. Therefore, when NK-activating ligands are not activated by IFN-γ, treatment of said cells with said ICAM-1 inhibitory antibody has little effect, 71% and 70% in hES cells and 54% and 57% in PEC cells.

[00364] As a general observation, NK-mediated cell lysis is less pronounced in differentiated cell populations (wild type PEC and B2M -/- PEC) compared to undifferentiated cell populations (WT hES and B2M -/- hES). NK-mediated cell lysis is increased to a greater extent when cells (WT or B2M -/- knockouts, hES or PEC) are activated by IFN-γ.

EXAMPLE 10: PRODUCTION OF B2M-/- HES KNOCKOUT CELLS DEFICIENT FOR NK ACTIVATED LIGAND

[00365] Example 9 describes that inhibition or attenuation of ICAM-1 expression protects IFN-γ-treated B2M -/- PEC cells from NK-mediated cell death. Therefore, in order to protect B2M -/- PEC cells from NK cell-mediated death after transplantation, it is desirable to disrupt the NK cell activating ligand gene. For example, based on the information in the Examples above, ICAM-1, a known NK cell activating ligand gene, may be disrupted or "knocked out". Preferably, both alleles of the ICAM-1 locus in the B2M-/-CyT49 hESC cell line are disrupted using CRTSPR/Cas9 or any other gene editing technology currently known or to be known in the future, see PCT Publication WO2016183041A which incorporated herein by reference in its entirety. Examples of published ICAM-1 sequences are shown in SEQ ID NOs: 4, 5, and 6. For example, in one embodiment of the present invention, NEXTGEN™ CRISPR (Transposagen Inc., Lexington, Kentucky) is used to edit the genes of these cells, which includes a double guide RNA and catalytically inactive Cas9 protein coupled to FokI nuclease.

[00366] Plasmids containing guide RNAs and Cas9 can be introduced into B2M -/- CyT49 hESC cells by electroporation, after which the resulting cells are seeded on tissue culture dishes. After electroporation, these cells can be sorted for negative reactivity to ICAM-1 antibody by flow cytometry. Sorted cells can be scattered at the required density for cloning. Individual clones can be collected and reseeded. Clones can be propagated and cryopreserved. Propagated clones that show a normal karyotype by G-staining and are negative for ICAM-1 protein expression and surface expression of ICAM-1 proteins can be harvested for further experiments by flow cytometry and/or immunofluorescence.

[00367] As described above, cells deficient in ICAM1 and B2M cannot express at least one NK cell activating ligand and at least one or all of the class I MHC proteins on the surface of said cells and therefore cannot bind to receptors that activate NK cells and are protected from NK-mediated cell death.

EXAMPLE 11: PRODUCTION OF B2M-/- HES KNOCKOUT CELLS DEFICIENT FOR MULTIPLE NK-ACTIVATED LIGANDS

[00368] Examples 9 and 10 demonstrate that inhibition of functional cell surface expression (NK cell activating anti-ligand) and disruption of the NK cell activating ligand gene (e.g., ICAM1-/-) in combination with disruption of expression MHC class I on the cell surface (eg, B2M-/-) can provide target cells with protection from NK-mediated cell death. Transplantation of cells deficient in more than one NK cell activating ligand can be performed and this will provide further protection against NK-mediated cell death.

[00369] The CD58 gene was selected for knockout as the NK cell activating ligand gene. Both alleles of the CD58 locus can be disrupted using CRISPR/Cas9 technology using known techniques described in WO2016183041A in said B2M-/-:ICAM-/- double knockout CyT49 hESC cell line. Examples of published CEACAM1 sequences are shown in SEQ ID NOs: 7, 8, and 9. Once again, the specified edit version may be NEXTGEN™ CRISPR (Transposagen Inc., Lexington, KY).

[00370] Plasmids containing guide RNAs and Cas9 can be introduced into B2M -/-, ICAM -/- CyT49 knockout hESC by electroporation, and the resulting cells are plated on tissue culture dishes. After electroporation, cells can be sorted for negative reactivity to the CD58 antibody by flow cytometry. Sorted cells can be seeded at the density required for cloning. Individual clones can be collected and reseeded. Clones can be propagated and cryopreserved. Propagated clones that showed a normal karyotype by G-staining and are negative for CD58 protein expression and for surface expression of CD58 proteins can be selected for further experiments by flow cytometry and/or immunofluorescence.

[00371] As described above, cells with disrupted, deleted, or modified ICAM1, CD58, and B2M cannot express either ICAM1 and CD58 or MHC class I proteins on the cell surface and, therefore, must not bind to NK cell-activating receptors, and protected from NK-mediated cell death.

[00372] The CD155 gene (also referred to as PVR) can also be selected for knockout as an NK cell activating ligand gene. The CD155 gene may be selected in addition to or instead of CD58. Also as described above, the CD28 gene can be disrupted using CRISPR/Cas9 technology. The CD155 gene can also be selected for knockout as a NK cell activating ligand gene. The CD155 gene may be selected in addition to or instead of CD58. Said CAECAM1 gene can also be selected for knockout as a NK cell activating ligand gene. The CAECAM1 gene may be selected in addition to or instead of CD58 and/or CD155.

[00373] EXAMPLE 12: INACTIVATION OF NK ACTIVATE LIGANDS OF ICAM1 AND CD58

[00374] In order to completely eliminate NK activation, it may be necessary to eliminate/reduce the activity of multiple NK activating ligands, either by knockout in target cells (for example, in cell therapy based on the use of hES cells), or using a blocking antibody or other strategies currently known or developed in the future. To determine whether NK cell toxicity can be reduced by inhibiting the effect of NK activating ligands on target cells, expression of NK activating ligands in WT and hES B2M -/- and PEC cells can be blocked using ICAM1 and a CD58 blocking antibody. to block ICAM1 and CD58 proteins on the surface of target cells. It can be expected that cell lysis of target cells will be reduced (similar to Fig. 15). Therefore, cell lysis by NK cells can be reduced by blocking NK-activating ligands such as ICAM1 and CD58. Blocking the expression of ICAM1 and CD58 using antibodies against NK-activating ligands in B2M -/- cells is evidence for the idea of creating a cell that has 3 knockouts (LAH class 1 gene knockout and NK cell activating ligand gene knockout). In this case, the target cells (eg, the hES line and/or the pancreatic cell line) move from Scenario C to Scenario A in FIG. 1. In particular, these cells, tissues and organs according to the present invention inhibit or do not express class I LAS cell surface proteins (B2M -/-) and inhibit or do not express cell surface protein ligands that activate the expression of NK cells (for example, ICAM1 -/- and CD58 -/-). Inhibition of cell surface protein expression can be achieved by knocking out genes or blocking protein expression using antibodies. Other strategies for interfering with cell surface protein expression include the use of antisense RNA, decoy RNA, ribozymes, RNA aptamers, miRNAs, shRNA/miRNAs, transdominant negative proteins (TNPs), chimeric/fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFv), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides and chemical agents) and protease inhibitors. Multiple knockouts would effectively prevent both cytotoxic T cell (CTL) and NK cell mediated toxicity because there would be little or no class I LAS proteins and NK cell activating ligands expressed on the surface. cells for binding to CTL cells or NK cells.

[00375] EXAMPLE 13: INACTIVATION OF NK-ACTIVATION LIGANDS CADM1 and CD58

[00376] In order to completely inactivate NK activation, it is necessary to eliminate/reduce the expression of multiple NK-activating ligands, either through gene knockout in the target cell (e.g., hES cell-based cell therapy), or using blocking antibodies or other strategies, currently known, or strategies that will be developed in the future. To determine whether NK cell toxicity can be reduced by inhibiting the effect of NK activating ligands on target cells, expression of NK activating ligands in hES WT cells and hES B2M -/- and PEC cells can be blocked using an antibody that blocks CADM1 and CD58, to block CADM1 and CD58 proteins on the cell surface. It can be expected that cell lysis of target cells will be reduced (similar to that shown in Fig. 15). Therefore, cell lysis by NK cells can be reduced by blocking NK-activating ligands such as CADM1 and CD58. Blocking the expression of CADM1 and CD58 by antibodies against NK-activating ligands in B2M -/- cells is evidence for the idea of creating a cell with 3 knockouts (LAH class I gene knockout and NK cell activating ligand gene knockout). In this case, target cells (e.g., hES and/or pancreatic cell line) move from Scenario C to Scenario A in FIG. -/-) and inhibited or not expressed ligands that activate the expression of NK cells in surface cells (for example, CADM1 -/-) and CD58 -/-). Inhibition of cell surface protein expression can be achieved by knocking out genes or blocking protein expression using antibodies. Other strategies to suppress expression of cell surface proteins include the use of antisense RNA, decoy RNA, ribozymes, RNA aptamers, miRNAs, shRNA/miRNAs, transdominant negative proteins (TNPs), chimeric/fusion proteins, nucleases, chemokine ligands, anti-infective cellular proteins, intracellular antibodies (sFvs), nucleoside analogs (NRT1), non-nucleoside analogs (NNRT1), integrase inhibitors (oligonucleotides, dinucleotides and chemical agents) and protease inhibitors. Multiple gene knockouts would effectively prevent both cytotoxic T cell (CTL) and NK cell mediated toxicity because there would be virtually no class I LAS proteins and NK cell activating ligand proteins expressed on the cell surface for binding to CTL or NK cells.

[00377] EXAMPLE 14: INACTIVATION OF NK ACTIVATED LIGANDS CD155 and CD58

[00378] In order to completely inactivate NK activation, it is necessary to eliminate/reduce the expression of multiple NK-activating ligands, either through gene knockout in the target cell (e.g., hES cell-based cell therapy), or using blocking antibodies or other strategies, currently known, or strategies that will be developed in the future. To determine whether NK cell toxicity can be reduced by inhibiting the effect of NK activating ligands on target cells, expression of NK activating ligands in hES WT cells and hES B2M -/- and PEC cells can be blocked using an antibody that blocks CD155 and CD58, to block CD155 and CD58 proteins on the surface of target cells. It can be expected that cell lysis of target cells will be reduced (similar to that shown in Fig. 15). Therefore, cell lysis by NK cells can be reduced by blocking NK activating ligands such as CD155 and CD58. Blocking the expression of CD155 and CD58 by antibodies against NK-activating ligands in B2M -/- cells is evidence for the idea of creating a cell with 3 knockouts (class I LAS gene knockout and NK cell activating ligand gene knockout). In this case, target cells (e.g., hES and/or pancreatic cell line) move from Scenario C to Scenario A in FIG. -/-) and inhibited or not expressed ligands that activate the expression of NK cells in surface cells (for example, CD155 -/- and CD58 -/-). Inhibition of cell surface protein expression can be achieved by knocking out genes or blocking protein expression using antibodies. Other strategies to suppress expression of cell surface proteins include the use of antisense RNA, decoy RNA, ribozymes, RNA aptamers, miRNAs, shRNA/miRNAs, transdominant negative proteins (TNPs), chimeric/fusion proteins, nucleases, chemokine ligands, anti-infective cellular proteins, intracellular antibodies (sFvs), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides and chemical agents) and protease inhibitors. Multiple gene knockouts would effectively prevent both cytotoxic T cell (CTL) and NK cell mediated toxicity because there would be virtually no class I LAS proteins and NK cell activating ligand proteins expressed on the cell surface for binding to CTL or NK cells.

[00379] EXAMPLE 15: INACTIVATION OF NK ACTIVATED LIGANDS ICAM1, CD155, and CD58

[00380] In order to completely inactivate NK activation, it is necessary to eliminate/reduce the expression of multiple NK-activating ligands, either through gene knockout in the target cell (e.g., hES cell-based cell therapy), or using blocking antibodies or other strategies, currently known, or strategies that will be developed in the future. To determine whether NK cell toxicity can be reduced by inhibiting the effects of NK activating ligands on target cells, expression of NK activating ligands in hES WT cells and hES B2M -/- and PEC cells can be blocked using an antibody that blocks ICAM1 , CD155 and CD58 to block the ICAM1, CD155 and CD58 proteins on the surface of the target cell. It can be expected that cell lysis of target cells will be reduced (similar to that shown in Fig. 15). Therefore, cell lysis by NK cells can be reduced by blocking NK-activating ligands such as ICAM1, CD58 and CD155. Blocking the expression of ICAM1, CD58 and CD 155 with antibodies against NK-activating ligands in B2M -/- cells is evidence for the idea of creating a cell with 3 knockouts (class I LAH gene knockout and NK cell activating ligand gene knockout). In this case, target cells (e.g., hES and/or pancreatic cell line) move from Scenario C to Scenario A in FIG. -/-) and inhibited or not expressed ligands that activate the expression of NK cells in surface cells (for example, ICAM1 -/-, CD58-/- and CD 155-/-). Inhibition of cell surface protein expression can be achieved by knocking out genes or blocking protein expression using antibodies. Other strategies to suppress expression of cell surface proteins include the use of antisense RNA, decoy RNA, ribozymes, RNA aptamers, miRNAs, shRNA/miRNAs, transdominant negative proteins (TNPs), chimeric/fusion proteins, nucleases, chemokine ligands, anti-infective cellular proteins, intracellular antibodies (sFvs), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides and chemical agents) and protease inhibitors. Multiple gene knockouts would effectively prevent both cytotoxic T cell (CTL) and NK cell mediated toxicity because there would be virtually no class 1 LAS proteins and NK cell activating ligand proteins expressed on the cell surface for binding to CTL or NK cells.

[00381] EXAMPLE 16: INACTIVATION OF NK ACTIVATED LIGANDS ICAM1, CD155, CD58, and CADM1

[00382] In order to completely inactivate NK activation, it is necessary to eliminate/reduce the expression of multiple NK-activating ligands, either through gene knockout in the target cell (e.g., hES cell-based cell therapy), or using blocking antibodies or other strategies, currently known, or strategies that will be developed in the future. To determine whether NK cell toxicity can be reduced by inhibiting the effects of NK activating ligands on target cells, expression of NK activating ligands in hES WT cells and hES B2M -/- and PEC cells can be blocked using an antibody that blocks ICAM1 , CD155, CD58, and CADM1 to block ICAM1, CD155, CD58, and CADM1 proteins on the target cell surface. It can be expected that cell lysis of target cells will be reduced (similar to that shown in Fig. 15). Therefore, cell lysis by NK cells can be reduced by blocking NK-activating ligands such as ICAM1, CD58, CD155 and CADM1. Blocking the expression of ICAM1, CD58, CD155 and CADM1 by antibodies against NK-activating ligands in B2M -/- cells is evidence for the idea of creating a cell with 3 knockouts (knockout of the LAH class I gene and a knockout of the NK cell activating ligand gene). In this case, target cells (e.g., hES and/or a pancreatic cell line) move from Scenario C to Scenario A in Figure 1. Specifically, the cells, tissues, and organs of the present invention inhibit or do not express class 1 LH cell surface proteins (B2M -/-) and inhibited or not expressed ligands that activate the expression of NK cells in surface cells (for example, ICAM1 -/-, CD58-/-, CD155-/- and CADM1-/-). Inhibition of cell surface protein expression can be achieved by knocking out genes or blocking protein expression using antibodies. Other strategies to suppress expression of cell surface proteins include the use of antisense RNA, decoy RNA, ribozymes, RNA aptamers, miRNAs, shRNA/miRNAs, transdominant negative proteins (TNPs), chimeric/fusion proteins, nucleases, chemokine ligands, anti-infective cellular proteins, intracellular antibodies (sFvs), nucleoside analogs (NRTI), non-nucleoside analogs (NNRT1), integrase inhibitors (oligonucleotides, dinucleotides and chemical agents) and protease inhibitors. Multiple gene knockouts would effectively prevent both cytotoxic T cell (CTL) and NK cell mediated toxicity because there would be virtually no class I LAS proteins and NK cell activating ligand proteins expressed on the cell surface for binding to CTL or NK cells.

[00383] EXAMPLE 17: INACTIVATION OF NK ACTIVATED LIGANDS ICAM1, CD155, and CADM1

[00384] In order to completely inactivate NK activation, it is necessary to eliminate/reduce the expression of multiple NK-activating ligands, either through gene knockout in the target cell (e.g., hES cell-based cell therapy), or using blocking antibodies or other strategies, currently known, or strategies that will be developed in the future. To determine whether NK cell toxicity can be reduced by inhibiting the effects of NK activating ligands on target cells, expression of NK activating ligands in hES WT cells and hES B2M -/- and PEC cells can be blocked using an antibody that blocks ICAM1 , CD 155, and CADM1, to block ICAM1, CD155, and CADM1 proteins on the target cell surface. It can be expected that cell lysis of target cells will be reduced (similar to that shown in Fig. 15). Therefore, cell lysis by NK cells can be reduced by blocking NK-activating ligands such as ICAM1, CD155 and CADM1. Blocking the expression of ICAM1, CD155, and CADM1 with antibodies against NK-activating ligands in B2M -/- cells is evidence for the idea of creating a cell with 3 knockouts (LAH class I gene knockout and NK cell activating ligand gene knockout). In this case, target cells (e.g., hES and/or a pancreatic cell line) move from Scenario C to Scenario A in Figure 1. Specifically, the cells, tissues, and organs of the present invention inhibit or do not express class 1 LH cell surface proteins (B2M -/-) and inhibited or not expressed ligands that activate the expression of NK cells in surface cells (for example, ICAM1 -/-, CD155-/- and CADM1-/-). Inhibition of cell surface protein expression can be achieved by knocking out genes or blocking protein expression using antibodies. Other strategies to suppress expression of cell surface proteins include the use of antisense RNA, decoy RNA, ribozymes, RNA aptamers, miRNAs, shRNA/miRNAs, transdominant negative proteins (TNPs), chimeric/fusion proteins, nucleases, chemokine ligands, anti-infective cellular proteins, intracellular antibodies (sFvs), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides and chemical agents) and protease inhibitors. Multiple gene knockouts would effectively prevent both cytotoxic T cell (CTL) and NK cell mediated toxicity because there would be virtually no class I LAS proteins and NK cell activating ligand proteins expressed on the cell surface for binding to CTL or NK cells.

[00385] Blocking antibodies suitable for the present invention are: CADM1 antibody, clone 9D2, Creative Biolabs, Cat. No. BRD-0007MZ; CD155 antibody, clone SKII.4, Biolegend, Cat. No. 337602; and CD58 antibody, clone TS2/9, Biolegend, Cat. No. 330911.

--->

SEQUENCE LISTING

<110> ViaCyte, Inc.

<120> UNIVERSAL DONOR CELLS AND RELATED METHODS

<130> P-191628F

<150> US 15/648,337<151> 2017-07-21

<160> 15

<170>PatentIn version 3.5

<210> 1<211> 23<212> DNA<213> Homo sapiens

<400> 1cgcgagcaca gctaaggcca cgg 23

<210> 2<211> 23<212> DNA<213> Homo sapiens

<400> 2cagtaagtca acttcaatgt cgg 23

<210> 3<211> 23<212> DNA<213> Homo sapiens

<400> 3gagtagcgcg agcacagcta agg 23

<210> 4<211> 23<212> DNA<213> Homo sapiens

<400> 4cctcaaaagt catcctgccc cgg 23

<210> 5<211> 23<212> DNA<213> Homo sapiens

<400> 5agcaactcct ttttaggcaa cgg 23

<210> 6<211> 23<212> DNA<213> Homo sapiens

<400> 6ccgcactcct ggtcctgctc ggg 23

<210> 7<211> 23<212> DNA<213> Homo sapiens

<400> 7gagtgcgtgt accctggcag ggg 23

<210> 8<211> 23<212> DNA<213> Homo sapiens

<400> 8ggtacacgca ctctgtgaag tgg 23

<210> 9<211> 23<212> DNA<213> Homo sapiens

<400> 9tacacgcact ctgtgaagtg ggg 23

<210> 10<211> 1599<212> DNA<213> Homo sapiens

<400> 10atggctccca gcagcccccg gcccgcgctg cccgcactcc tggtcctgct cggggctctg 60

Ttcccaggac ctggcaatgc ccagacatct gtgtccccct caaaagtcat cctgccccgg 120

Ggaggctccg tgctggtgac atgcagcacc tcctgtgacc agcccaagtt gtggggcata 180

Gagaccccgt tgcctaaaaa ggagttgctc ctgcctggga acaaccggaa ggtgtatgaa 240

Ctgagcaatg tgcaagaaga tagccaacca atgtgctatt caaactgccc tgatgggcag 300

Tcaacagcta aaaccttcct caccgtgtac tggactccag aacgggtgga actggcaccc 360

Ctcccctctt ggcagccagt gggcaagaac cttaccctac gctgccaggt ggaggtggg 420

Gcaccccgggg ccaacctcac cgtggtgctg ctccgtgggg agaaggagct gaaacggggag 480

Ccagctgtgg gggagcccgc tgaggtcacg accacacggtgc tggtgaggag agatcaccat 540

Ggagccaatt tctcgtgccg cactgaactg gacctgcggc cccaagggct ggagctgttt 600

660

Cttgtcagcc cccgggtcct agaggtggac acgcagggga ccgtggtctg ttccctggac 720

Gggctgttcc cagtctcgga ggcccaggtc cacctggcac tgggggacca gaggttgaac 780

840

Gaggacgagg gcacccagcg gctgacgtgt gcagtaatac tggggaacca gagccaggag 900

Acactgcaga cagtgaccat ctacagcttt ccggcgccca acgtgattct gacgaagcca 960

Gaggtctcag aagggaccga ggtgacagtg aagtgtgagg cccaccctag agccaaggtg 1020

1080

Accccagagg acaacggggcg cagcttctcc tgctctgcaa ccctggaggt ggccggccag 1140

Cttatacaca agaaccagac ccgggagctt cgtgtcctgt atggcccccg actggacgag 1200

Agggattgtc cgggaaactg gacgtggcca gaaaattccc agcagactcc aatgtgccag 1260

Gcttggggga acccattgcc cgagctcaag tgtctaaagg atggcacttt cccactgccc 1320

1380

Agcactcaag gggaggtcac ccgcaaggtg accgtgaatg tgctctcccc ccggtatgag 1440

Attgtcatca tcactgtggt agcagccgca gtcataatgg gcactgcagg cctcagcacg 1500

Tacctctata accgccagcg gaagatcaag aaatacagac tacaacaggc ccaaaaaggg 1560

Acccccatga aaccgaacac acaagccg cctccctga 1599

<210> 11<211> 1407<212> DNA<213> Homo sapiens

<400> 11atggggcacc tctcagcccc acttcacaga gtgcgtgtac cctggcaggg gcttctgctc 60

Acagcctcac ttctaacctt ctggaacccg cccaccactg cccagctcac tactgaatcc 120

Atgccattca atgttgcaga ggggaaggag gttcttctcc ttgtccacaa tctgccccag 180

Caactttttg gctacagctg gtacaaaggg gaaagagtgg atggcaaccg tcaaattgta 240

300

Atatacccca atgcatccct gctgatccag aacgtcaccc agaatgacac aggattctac 360

Acctacaag tcataaagtc agatcttgtg aatgaagaag caactggaca gttccatgta 420

Tacccggagc tgcccaagcc ctccatctcc agcaacaact ccaaccctgt ggaggacaag 480

Gatgctgtgg ccttcacctg tgaacctgag actcaggaca caacctacct gtggtggata 540

Aacaatcaga gcctcccggt cagtcccagg ctgcagctgt ccaatggcaa caggaccctc 600

Actctactca gtgtcacaag gaatgacaca ggaccctatg agtgtgaaat acagaaccca 660

Gtgagtgcga accgcagtga cccagtcacc ttgaatgtca cctatggccc ggacaccccc 720

Accatttccc cttcagacac ctattaccgt ccaggggcaa acctcagcct ctcctgctat 780

840

Acacaagagc tctttatccc taacatcact gtgaataata gtggatccta tacctgccac 900

Gccaataact cagtcactgg ctgcaacagg accacagtca agacgatcat agtcactgag 960

Ctaagtccag tagtagcaaa gccccaaatc aaagccagca agaccacagt cacaggagat 1020

Aaggactctg tgaacctgac ctgctccaca aatgacactg gaatctccat ccgttggttc 1080

1140

Ctcagcataa accctgtcaa gagggaggat gctgggacgt attggtgtga ggtcttcaac 1200

1260

Caagaaaatg gcctctcacc tggggccatt gctggcattg tgattggagt agtggccctg 1320

Gttgctctga tagcagtagc cctggcatgt tttctgcatt tcgggaagac cggcaggacc 1380

Actccaatga cccacctaac aagatga 1407

<210> 12<211> 360<212> DNA<213> Homo sapiens

<400> 12atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct 60

120

Aatttcctga attgctatgt gtctgggttt catccatccg acattgaagt tgacttactg 180

Aagaatggag agagaattga aaaagtggag cattcagact tgtctttcag caaggactgg 240

Tctttctatc tcttgtacta cactgaattc acccccactg aaaaagatga gtatgcctgc 300

360

<210> 13<211> 1329<212> DNA<213> Homo sapiens

<400> 13atggcgagtg tagtgctgcc gagcggatcc cagtgtgcgg cggcagcggc ggcggcggcg 60

Cctcccgggc tccggctccg gcttctgctg ttgctcttct ccgccgcggc actgatcccc 120

Acaggtgatg ggcagaatct gtttacgaaa gacgtgacag tgatcgaggg agaggttgcg 180

Accatcagtt gccaagtcaa taagagtgac gactctgtga ttcagctact gaatcccaac 240

Aggcagacca tttatttcag ggacttcagg cctttgaagg acagcaggtt tcagttgctg 300

360

Agatactttt gccagctcta taccgatccc ccacaggaaa gttacaccac catcacagtc 420

Ctggtcccac cacgtaatct gatgatcgat atccagaaag acactgcggt ggaaggtgag 480

Gagattgaag tcaactgcac tgctatggcc agcaagccag ccacgactat caggtggttc 540

Aaagggaaca cagagctaaa aggcaaatcg gaggtggaag agtggtcaga catgtacact 600

Gtgaccagtc agctgatgct gaaggtgcac aaggaggacg atggggtccc agtgatctgc 660

Caggtggagc accctgcggt cactggaaac ctgcagaccc agcggtatct agaagtacag 720

Tataagcctc aagtgcacat tcagatgact tatcctctac aaggcttaac ccgggaaggg 780

840 Gacgcgcttg agttaacatg tgaagccatc gggaagcccc

Gtgagagtcg atgatgaaat gcctcaacac gccgtactgt ctgggcccaa cctgttcatc 900

Aataacctaa acaaaacaga taatggtaca taccgctgtg aagcttcaaa catagtgggg 960

Aaagctcact cggattatat gctgtatgta tacgatcccc ccacaactat ccctcctccc 1020

1080

1140

Gtggcggtgg tggtgttcgc catgctgtgc ttgctcatca ttctggggcg ctattttgcc 1200

Agacataaag gtacatactt cactcatgaa gccaaaggag ccgatgacgc agcagacgca 1260

1320

Ttcatctag 1329

<210> 14<211> 753<212> DNA<213> Homo sapiens

<400> 14atggttgctg ggagcgacgc ggggcgggcc ctgggggtcc tcagcgtggt ctgcctgctg 60

Cactgctttg gtttcatcag ctgtttttcc caacaaatat atggtgttgt gtatgggaat 120

Gtaactttcc atgtaccaag caatgtgcct ttaaaagagg tcctatggaa aaaacaaaag 180

Gataaagttg cagaactgga aaattctgaa ttcagagctt tctcatcttt taaaaatagg 240

Gtttatttag acactgtgtc aggtagcctc actatctaca acttaacatc atcagatgaa 300

Gatgagtatg aaatggaatc gccaaatatt actgatacca tgaagttctt tctttatgtg 360

Cttgagtctc ttccatctcc cacactaact tgtgcattga ctaatggaag cattgaagtc 420

Caatgcatga taccagagca ttacaacagc catcgaggac ttataatgta ctcatgggat 480

Tgtcctatgg agcaatgtaa acgtaactca accagtatat attttaagat ggaaaatgat 540

Cttccacaaa aaatacagtg tactcttagc aatccattat ttaatacaac atcatcaatc 600

Attttgacaa cctgtatccc aagcagcggt cattcaagac acagatatgc acttataccc 660

Ataccattag cagtaattac aacatgtatt gtgctgtata tgaatggtat tctgaaatgt 720

Gacagaaaac cagacagaac caactccaat tga 753

<210> 15<211> 1254<212> DNA<213> Homo sapiens

<400> 15atggcccgag ccatggccgc cgcgtggccg ctgctgctgg tggcgctact ggtgctgtcc 60

Tggccacccc caggaaccgg ggacgtcgtc gtgcaggcgc ccacccaggt gcccggcttc 120

Ttgggcgact ccgtgacgct gccctgctac ctacaggtgc ccaacatgga ggtgacgcat 180

Gtgtcacagc tgacttgggc gcggcatggt gaatctggca gcatggccgt cttccaccaa 240

300

Gcggagctgc ggaatgcctc gctgaggatg ttcgggttgc gcgtagagga tgaaggcaac 360

Tacacctgcc tgttcgtcac gttcccgcag ggcagcagga gcgtggatat ctggctccga 420

Gtgcttgcca agccccagaa cacagctgag gttcagaagg tccagctcac tggagagcca 480

Gtgcccatgg cccgctgcgt ctccacaggg ggtcgcccgc cagcccaaat cacctggcac 540

Tcagacctgg gcgggatgcc caatacgagc caggtgccag ggttcctgtc tggcacagtc 600

Actgtcacca gcctctggat attggtgccc tcaagccagg tggacggcaa gaatgtgacc 660

720

Tactaccccc cagaggtatc catctctggc tatgataaca actggtacct tggccagaat 780

840

Acgaccatgg gtcccctgcc accctttgct gtggcccagg gcgcccagct cctgatccgt 900

Cctgtggaca aaccaatcaa cacaacttta atctgcaacg tcaccaatgc cctaggagct 960

Cgccaggcag aactgaccgt ccaggtcaaa gagggacctc ccagtgagca ctcaggcatg 1020

Tcccgtaacg ccatcatctt cctggttctg ggaatcctgg ttttctctgat cctgctgggg 1080

1140

tgtccctcga gtacagagca tgccagcgcc tcagctaatg ggcatgtctc ctattcagct 1200

Gtgagcagag agaacagctc ttcccaggat ccacagacag agggcacaag gtga 1254

<---

Claims (56)

1. A cell composition for transplantation containing a population of human pancreatic cells in vitro, containing CHGA ^- , NKX6.1 ⁺ , PDX1 ⁺ pancreatic cell precursor cells, and CHGA ⁺ pancreatic endocrine cells, in which the function of at least one major histocompatibility complex gene (MHC) class I and at least one natural killer (NK) cell activating ligand is disrupted or inhibited, resulting in reduced binding of the NK activating ligand to the NK activating receptor in said population of human pancreatic cells in vitro. 2. A cell composition for transplantation according to claim 1, characterized in that said MHC class I gene encodes beta-2 microglobulin (B2M). 3. A cell composition for transplantation according to claim 1 or 2, characterized in that said NK cell activating ligand is: a) intercellular adhesion molecule 1 (ICAM1), CD58, CD155, cell adhesion molecule associated with carcinoembryonic antigen 1 (CEACAM1), cell adhesion molecule 1 (CADM1), protein associated with the major histocompatibility complex class IA (MICA) polypeptide, protein associated with a class I B major histocompatibility complex (MICB) polypeptide, or a combination thereof; b) ICAM1 and CD58; c) ICAM1, CD58, CD155, CEACAM1, CADM1, MICA and MICB; d) ICAM1, CD58, CD155; e) ICAM1, CD58, CD155 and CADM1; f) CD58 and CADM1; or g) ICAM1, CADM1 and CD155. 4. Composition of cells for transplantation according to any one of paragraphs. 1-3, characterized in that said population of pancreatic cells expresses a protein which, when expressed in the presence of a cell death agent, is capable of causing cell death of said cell population. 5. A cell composition for transplantation according to claim 4, characterized in that said protein is herpes simplex virus thymidine kinase and said cell death agent is ganciclovir. 6. The use of a cell composition for transplantation according to any one of paragraphs. 1-5 for reducing transplant rejection of a population of human pancreatic cells, in which disruption or inhibition of the function of the MHC class I gene and ligand that activates NK cells, resulting in a decrease in the binding of the NK activating ligand to the NK activating receptor in the specified population of human pancreatic cells in vitro, reduces cell transplant rejection compared to human pancreatic cells without disrupting or inhibiting the function of said MHC class I gene and NK cell activating ligand. 7. The use of a cell composition for transplantation according to any one of paragraphs. 1-5 for producing insulin in a mammalian subject, wherein said population of human pancreatic cells in vitro mature in the mammalian subject into cells that produce insulin in response to glucose stimulation. 8. A method for reducing transplant rejection of pancreatic endoderm cells (PEC) in an immunocompetent or immunocompromised human, comprising: transplantation of an encapsulated cell device containing a cell population containing PEC cells, which are CHGA ^- , NKX6.1 ⁺ , PDX1 ⁺ pancreatic cell progenitor cells, and RES, which are CHGA ⁺ pancreatic endocrine cells, to a location in a human having a healthy immune system or a weakened immune system that allows vascularization in the recipient, wherein the function of at least one major histocompatibility complex (MCHC) class I gene and at least one natural killer (NK) cell activating ligand is impaired or inhibited in said cell population, resulting in decreased binding of the NK activating ligand to NK activating receptor, and disruption or inhibition of at least one MHC class I gene and at least one natural NK cell activating ligand results in inhibition of cell transplant rejection of said cell population in an immunocompetent or immunocompromised human. 9. The method according to claim 8, characterized in that the specified location in a person with a healthy immune system or a weakened immune system is a subcutaneous location. 10. The method according to claim 8 or 9, wherein said class I MHC gene encodes beta-2 microglobulin (B2M) or encodes a human leukocyte antigen (LAH)-ABC cell surface protein. 11. The method according to any one of paragraphs. 8-10, characterized in that the specified NK cell activating ligand is: a) intercellular adhesion molecule 1 (ICAM1), CD58, CD155, cell adhesion molecule associated with carcinoembryonic antigen 1 (CEACAM1), cell adhesion molecule 1 (CADM1), major histocompatibility complex class IA (MICA) polypeptide-associated protein, or a protein associated with a class I B major histocompatibility complex (M1CB) polypeptide; b) ICAM1 and CD58; c) ICAM1, CD58, CD155; d) ICAM1, CD58, CD155 and CADM1; e) CD58 and CADM1; or f) ICAM1, CADM1 and CD155. 12. The method according to any one of paragraphs. 8-11, characterized in that said cell population expresses a protein which, when expressed in the presence of a cell death agent, is capable of causing cell death of said cell population. 13. The method of claim 12, wherein said protein is herpes simplex virus thymidine kinase and said cell death agent is ganciclovir. 14. The method according to any one of paragraphs. 8-13, characterized in that said person is a person with healthy immunity. 15. The method according to any one of paragraphs. 8-13, characterized in that the specified person is a person with a weakened immune system. 16. A method for producing insulin in a person with healthy immunity or weakened immunity, including: transplantation of an encapsulated cell device containing a cell population containing PEC cells, which are CHGA ^- , NKX6.1 ⁺ , PDX1 ⁺ pancreatic cell progenitor cells, and RES, which are CHGA ⁺ pancreatic endocrine cells, to a location in a human having a healthy immune system or a weakened immune system that allows vascularization in the recipient, wherein the function of at least one major histocompatibility complex (MCHC) class I gene and at least one natural killer (NK) cell activating ligand is impaired or inhibited in said cell population, resulting in decreased binding of the NK activating ligand to An NK-activating receptor, wherein said population of cells matures in said immunocompetent or immunocompromised individual into a population of mature cells, resulting in insulin production in response to glucose stimulation. 17. The method according to p. 16, characterized in that the specified location in a person with a healthy immune system or a weakened immune system is a subcutaneous location. 18. The method of claims 16 and 17, wherein said class I MHC gene encodes beta-2 microglobulin (B2M) or encodes a human leukocyte antigen (LAH)-ABC cell surface protein. 19. The method according to any one of paragraphs. 16-18, characterized in that the specified NK cell activating ligand is: a) intercellular adhesion molecule 1 (ICAM1), CD58, CD155, cell adhesion molecule associated with carcinoembryonic antigen 1 (CEACAM1), cell adhesion molecule 1 (CADM1), major histocompatibility complex class IA (MICA) polypeptide-associated protein, or a protein associated with a class I B major histocompatibility complex (M1CB) polypeptide; b) ICAM1 and CD58; c) ICAM1, CD58 and CD155; d) ICAM1, CD58, CD155 and CADM1; e) CD58 and CADM1; or f) ICAM1, CADM1 and CD155. 20. The method according to any one of paragraphs. 16-19, characterized in that said cell population expresses a protein which, when expressed in the presence of a cell death agent, is capable of causing cell death of said cell population. 21. The method of claim 20, wherein said protein is herpes simplex virus thymidine kinase and said cell death agent is ganciclovir. 22. The method according to any one of paragraphs. 16-21, characterized in that said person is a person with healthy immunity. 23. The method according to any one of paragraphs. 16-21, characterized in that the specified person is a person with a weakened immune system. 24. A composition of cells for transplantation containing a population of pancreatic cells in vitro, containing CHGA ^- , NKX6.1 ⁺ , PDX1 ⁺ pancreatic cell precursor cells, and CHGA ⁺ pancreatic endocrine cells, in which the function of at least one gene of the major histocompatibility complex ( MHC) class I and at least one natural killer (NK) cell activating ligand is disrupted or inhibited, resulting in reduced binding of the NK activating ligand to the NK activating receptor in said population of pancreatic cells in vitro. 25. A cell composition for transplantation according to claim 24, characterized in that said MHC class I gene encodes beta-2 microglobulin (B2M). 26. A cell composition for transplantation according to claim 24 or 25, characterized in that said NK cell activating ligand is: a) intercellular adhesion molecule 1 (ICAM1), CD58, CD155, cell adhesion molecule associated with carcinoembryonic antigen 1 (CEACAM1), cell adhesion molecule 1 (CADM1), protein associated with the major histocompatibility complex class IA (MICA) polypeptide, protein associated with a class I B major histocompatibility complex (MICB) polypeptide, or a combination thereof; b) ICAM1 and CD58; c) ICAM1, CD58, CD155, CEACAM1, CADM1, MICA and MICB; d) ICAM1, CD58 and CD155; c) ICAM1, CD58, CD155 and CADM1; f) CD58 and CADM1; or g) ICAM1, CADM1 and CD155.

Images