Universal donor cells and related methods

Abstract

To provide universal donor stem cells and cells derived therefrom and related methods of their use and production, the universal donor stem cells disclosed herein being useful for overcoming allogeneic immune rejection in cell-based transplantation therapies.SOLUTION: In certain embodiments, the universal donor cells disclosed herein are pancreatic endoderm cells that do not express one or more MHC-Class I cell-surface proteins and whose expression of at least one NK activating ligand is disrupted or inhibited.SELECTED DRAWING: Figure 15

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is the subject of U.S. Patent Application No. 2007 / 0109999 entitled "UNIVERSAL DONOR CELLS AND RELATED METHODS," filed July 12, 2010, which is incorporated herein by reference in its entirety. Priority is claimed to application Ser. No. 15 / 648,337.

[0002] This application relates to the fields of gene expression, genome engineering, and gene / cell therapy. [Background technology]

[0003] Human pluripotent stem cells (hPSCs) are a useful tool for generating any adult cell type for transplantation into patients. In principle, hPSC-based cell therapy has the potential to treat most, if not all, degenerative diseases, but the success of such therapy can be limited by the subject's immune response. The immune system protects organisms from infection with a highly specific, layered defense. Simply put, physical barriers prevent pathogens, such as bacteria and viruses, from invading the organism. If a pathogen breaches these barriers, the innate immune system provides an immediate but nonspecific response. If a pathogen successfully evades the innate response, vertebrates have a second layer of protection, the adaptive immune system, activated by the innate response. The adaptive immune system generates an even more specific response. Here, the immune system adapts its response during infection to improve pathogen recognition. This improved response is then retained in the form of immunological memory after the pathogen is eliminated, allowing the adaptive immune system to mount a faster and more powerful attack each time the pathogen is encountered. The adaptive immune response is antigen-specific and requires the recognition of specific "non-self" antigens in a process called antigen presentation. Antigen specificity allows responses to be tailored to specific pathogens or pathogen-infected cells. Interferon gamma (IFN-γ) plays an important role in combating infectious and non-infectious diseases. The primary source of IFN-γ in the human immune response is T cells. NK cells, macrophages, and IFN-γ play important roles in both innate and adaptive immunity.

[0004] The major histocompatibility complex (MHC) is a set of cell surface proteins essential for regulating the immune system. The primary function of MHC molecules is to bind pathogen-derived antigens and present them on the cell surface for recognition by appropriate T cells. The MHC gene family is divided into three subgroups: class I, class II, and class III. Human MHC is also referred to as the HLA (human leukocyte antigen) complex (often simply HLA). Natural killer (NK) cells are lymphocytes that function at the interface between innate and adaptive immunity. NK cells directly contribute to immune defense through effector functions such as cytotoxicity and cytokine secretion, and by regulating innate and adaptive immune responses. When target or host cells encounter NK cells, several outcomes are possible. The magnitude of the NK response depends on the amount and type of activating and inhibitory receptors on the NK cell and the amount and type of activating and inhibitory ligands on the target cell. See Figure 1. In scenario A, if the target cell does not have human leukocyte antigen (HLA) class I and an NK activating ligand, NK cells expressing MHC class I inhibitory and activating receptors will not attack the target cell (no response or no permission). In scenario B, if the target cell expresses HLA class I but does not have an activating ligand, NK cells expressing inhibitory and activating receptors will not be able to attack the target. In scenario C, if the target cell downregulates or does not have HLA class I and expresses an NK activating ligand, NK cells expressing inhibitory and activating receptors will attack the target cell. In scenario D, if the target cell expresses both self-HLA class I and an NK activating ligand, the level of response by NK cells expressing inhibitory and activating receptors will be determined by the level of inhibitory and activating receptors against the NK cell. The immune system responds to various stresses and conditions, depending on the balance of immune responses and activation signals. Haynes et al., THE IMMUNE SYSTEM IN HEALTH AND DISEASE, PART 15: Immune-Mediated, Inflammatory, and Rheumatologic Disorders, 372e Introduction to the Immune System.

[0005] Historically, efforts to overcome the host's immune response to allogeneic cells have focused on preventing the adaptive immune response, i.e., the adhesion between T cells and MHC class I antigens presented on foreign cells. Therefore, CRISPR and TALEN systems have been used to generate loss-of-function genetic modifications, thus creating stem cells that do not express one or more classical MHC / HLA genes. However, these cells and cells derived from them remain vulnerable to the host's innate immune response (NK cells). See, e.g., Parham et al. (2005) Nat Rev Immunol. 5(3):201-214. To overcome the host's innate immune response, others have attempted to reintroduce tolerogenic factors into target cells. The focus has been on the "missing self." See WO2016183041A2 (the disclosure of which is incorporated by reference in its entirety). Applicants have surprisingly discovered that the key to evading the host's NK-mediated immune response is not "loss of selfness," but rather the expression and magnitude of NK cell-activating ligands on target cells.

[0006] Thus, there is a need for compositions and methods to develop cells that lack some or all classical HLA expression but that are not attacked by NK cells for lysis. Summary of the Invention

[0007] Disclosed herein is a strategy for overcoming graft rejection, particularly alloimmune graft rejection in cell-based transplantation therapy, by providing a universal donor cell line. In one embodiment, human pluripotent stem cells are provided that lack some or all of classical HLA class I cell surface protein expression and NK activating ligand expression. In one embodiment, cells derived from human pluripotent stem cells, such as pancreatic cells, are provided that lack some or all of classical HLA class I cell surface protein expression and NK activating ligand expression. In one embodiment, a method for preventing cellular graft rejection is provided 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), have been disrupted, deleted, modified, or inhibited. In another embodiment, a method for preventing cellular graft rejection is provided by providing transplanted pancreatic cells in which the expression of at least one MHC protein, such as B2M, and at least one NK activating ligand protein, such as ICAM-1, has been disrupted, deleted, modified, or inhibited. Disruption, deletion, alteration, or inhibition of B2M results in the loss of all HLA class I surface expression and function. [Brief explanation of the drawings]

[0008] [Figure 1]Figure 1 is a reproduction of Figure 372e-4 of Haynes et al., supra, which is incorporated herein in its entirety, showing different scenarios (of NK-mediated responses to target cells). In the absence of MHC class I on the target cell and in the absence of an NK-activating ligand, inhibitory and activating receptors on the NK cell are not engaged, and the NK cell remains unresponsive (Scenario A). In the presence of MHC class I on the target cell but in the absence of an NK-activating ligand, inhibitory receptors on the NK cell are engaged, but activating receptors on the NK cell are not, and the NK cell remains unresponsive (Scenario B). In the absence of self-MHC class I on the target cell but in the presence of an NK-activating ligand, inhibitory receptors on the NK cell are not engaged, but activating receptors on the NK cell are engaged, and the NK cell attacks (Scenario C). In the presence of MHC class I and an NK-activating ligand on the target cell, inhibitory and activating receptors on the NK cell are engaged, and the balance of signals determines the outcome (Scenario D). [Figure 2A] Figure 2A shows a representative flow cytometry analysis of B2M cell surface protein expression on wild-type (WT) hES cells without IFN-γ (line B) and after exposure to IFN-γ (line A). The shaded area represents background expression without antibody staining. Exposure to IFN-γ increases B2M cell surface protein expression in WT hES cells. [Figure 2B] Figure 2B shows a representative flow cytometry analysis of B2M cell surface protein expression on B2M knockout (B2M- / -) hES cells without IFN-γ (line B) and after exposure to IFN-γ (line A). B2M- / - hES cells express little B2M cell surface protein, and this expression does not change significantly after exposure to IFN-γ. [Figure 3A] Figure 3A shows a representative flow cytometry analysis of HLA-ABC cell surface protein expression on WT hES cells without IFN-γ (line B) and after exposure to IFN-γ (line A) using a pan-HLA class I antibody. The shaded area represents background expression without antibody staining. [Figure 3B]Figure 3B shows a representative flow cytometry analysis of HLA-ABC cell surface protein expression on B2M knockout hES cells without IFN-γ (line B) and after exposure to IFN-γ (line A). B2M- / - hES cells have no detectable HLA-ABC cell surface protein expression. [Figure 4A] Figure 4A shows a representative flow cytometry analysis of B2M cell surface protein expression on WT pancreatic endoderm cells (PECs) without IFN-γ (line B) and after exposure to IFN-γ (line A). The shaded area represents background expression without antibody staining. Exposure to IFN-γ increases B2M cell surface protein expression in WT PECs. [Figure 4B] Figure 4B shows representative flow cytometry analysis of B2M cell surface protein expression on B2M knockout PECs without IFN-γ (line B) and after exposure to IFN-γ (line A). B2M- / - PECs have no detectable B2M cell surface protein expression. [Figure 5A] Figure 5A shows a representative flow cytometry analysis of HLA-ABC cell surface protein expression on WT PECs without IFN-γ (line B) and after exposure to IFN-γ (line A). The shaded area represents background expression without antibody staining. Exposure to IFN-γ increases HLA-ABC cell surface protein expression in WT PECs. [Figure 5B] Figure 5B shows representative flow cytometry analysis of HLA-ABC cell surface protein expression on B2M knockout PECs without IFN-γ (line B) and after exposure to IFN-γ (line A). B2M- / - PECs have no detectable HLA-ABC cell surface protein expression. [Figure 6A] Figure 6A shows a representative flow cytometry analysis of ICAM-1 cell surface protein expression on WT hES cells without IFN-γ (line B) and after exposure to IFN-γ (line A). The shaded area represents background expression without antibody staining. Exposure to IFN-γ increases ICAM-1 cell surface protein expression in WT hES cells. [Figure 6B]Figure 6B shows a representative flow cytometry analysis of ICAM-1 cell surface protein expression on B2M knockout hES cells without IFN-γ (line B) and after exposure to IFN-γ (line A). B2M- / - hES cells have similar ICAM-1 cell surface protein expression to WT hES cells before and after IFN-γ exposure. [Figure 7A] Figure 7A shows a representative flow cytometry analysis of ICAM-1 cell surface protein expression on WT PECs without IFN-γ (line B) and after exposure to IFN-γ (line A). The shaded area represents background expression without antibody staining. Exposure to IFN-γ increases ICAM-1 cell surface protein expression in WT PECs. [Figure 7B] Figure 7B shows representative flow cytometry analysis of ICAM-1 cell surface protein expression on B2M knockout PECs without IFN-γ (line B) and after exposure to IFN-γ (line A). After exposure to IFN-γ, B2M- / - PECs have ICAM-1 cell surface protein expression similar to WT PECs, with greater than background (shaded area) expression. [Figure 8] Figure 8 is a bar graph showing ICAM-1 mRNA expression data (Affymetrix expression array) in WT hES cells, B2M(- / -) hES cells, WT PECs, and B2M(- / -) PECs, each of which was not exposed to IFN-γ (control) or was exposed to IFN-γ. ICAM-1 expression was also assessed in cells known to have low ICAM cell surface protein expression: cancer cells (K562 and SKBR3), transplanted PECs that were able to mature into insulin-producing cells in vivo, human pancreatic islet cells, and two different samples of peripheral blood mononuclear cells (PBMCs) (without exposure to IFN-γ). ICAM-1 mRNA expression increases after exposure of hES cells (WT or B2M- / -) or PECs (WT or B2M- / -) to IFN-γ. [Figure 9]Figure 9 shows a representative flow cytometry analysis of CD58 (also known as LFA-3) cell surface protein expression on WT PECs without IFN-γ (line B) and after exposure to IFN-γ (line A). Line C is background expression without antibody staining. Exposure to IFN-γ only slightly increases CD58 cell surface protein expression on WT PECs compared to untreated controls. Antibody obtained from BioLegend, catalog number #330909. [Figure 10] Figure 10 shows a representative flow cytometry analysis of CD155 cell surface protein expression on WT PECs without IFN-γ (line B) and after exposure to IFN-γ (line A). Line C is background expression without antibody staining. After exposure to IFN-γ, WT PECs have CD155 cell surface protein expression similar to WT untreated PEC controls. Gene symbol PVR (also known as CD155, NECL-5, HVED). Antibody obtained from Milteneyi Biotech Inc., catalog number #130-105-905. [Figure 11] Figure 11 shows a representative flow cytometry analysis of CEACAM1 (also known as CD66a, BGP, BGP1) cell surface protein expression on WT PECs without (line B) and after (line A) exposure to IFN-γ. Line C is background expression without antibody staining. Exposure to IFN-γ slightly increases CEACAM1 cell surface protein expression in WT PECs compared to untreated controls. Antibody obtained from Milteneyi Biotech Inc., catalog number #130-098-858. [Figure 12] Figure 12 shows a representative flow cytometry analysis of BAT3 cell surface protein expression on WT PECs without (line B) and after (line A) exposure to IFN-γ. Line C is background expression without antibody staining. Untreated control WT PECs have similar BAT3 cell surface protein expression as WT PECs exposed to IFN-γ. Gene symbol BAG6 (also known as BAT3, HLA-B associated transcript 3). Antibody obtained from Abcam, Inc., catalog number #ab210838. [Figure 13] Figure 13 shows a representative flow cytometry analysis of CADM1 (also known as NECL2, TSLC1, IGSF4, RA175) cell surface protein expression on WT PECs without (line B) and after (line A) exposure to IFN-γ. Line C is background expression without antibody staining. After exposure to IFN-γ, WT PECs have similar CADM1 cell surface protein expression to untreated controls. Antibody obtained from MBL International Corp., catalog number #CM004-4. [Figure 14] Figure 14 shows a representative flow cytometry analysis of CD112 cell surface protein expression on WT PECs without (line B) and after (line A) exposure to IFN-γ. Line C is background expression without antibody staining. After exposure to IFN-γ, WT PECs have CD112 cell surface protein expression similar to untreated controls. Gene symbol PVRL2 (also known as CD112, nectin-2, PVRR2, HVEB). Antibody obtained from Milteneyi Biotech Inc., catalog number #130-109-056. [Figure 15] Figure 15 is a bar graph showing the reduction in NK cell lysis of target cells after blocking ICAM-1 expression on the target cells with an anti-ICAM1 antibody (from left to right: WT hESCs for NK cytotoxicity assay, WT hESCs exposed to IFN-γ, B2M- / - hESCs, B2M- / - hESCs exposed to IFN-γ, WT PECs, WT PECs exposed to IFN-γ, B2M- / - PECs, B2M- / - PECs exposed to IFN-γ, K562 control cell line). From left to right, each set of three bars represents NK-062216; NK-062216 + ICAM1AB (5 μg / ml); NK-062216 + ICAM1AB (10 μg / ml). DETAILED DESCRIPTION OF THE INVENTION

[0009] Sequence Listing The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleobases and three-letter codes for amino acids as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but any reference to the shown strand is understood to include the complementary strand. The sequence listing has been submitted as an ASCII text file, Sequence_Listing, July 3, 2018, 11 KB, which is incorporated herein by reference.

[0010] MHC class I molecules are one of the two major classes of major histocompatibility complex (MHC) molecules (the other being MHC class II). Their function is to present peptide fragments of non-self proteins from within cells to cytotoxic T cells. This triggers an immediate immune response against specific non-self antigens presented with the help of MHC class I proteins. In humans, the HLAs corresponding to MHC class I are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. Human HLA-E, HLA-F, and HLA-G are non-classical MHC class I molecules characterized by limited polymorphism and lower cell surface expression than their classical paralogs (HLA-A, HLA-B, and HLA-C). All MHC class I proteins require binding with β2-microglobulin (B2M) to generate functional heterodimeric MHC class I protein complexes prior to functional expression on the cell surface. MHC class I molecules also function as inhibitory ligands for NK cells: reduction of normal levels of cell surface MHC class I activates NK cell-mediated killing.

[0011] Historically, target cells bearing MHC class I inhibitory ligands were thought to avoid attack when exposed to NK cells due to the presumed dominant nature of MHC class I inhibitory signals (copied from Figure 372e-4 in Part 15 of Harrison's Principles of Internal Medicine 19 E (Vol. 1 and Vol. 2) A Major Histocompatibility Complex). (See Scenario B in Figure 1.) However, applicant has surprisingly discovered that the opposite is true.

[0012] Exposure of cells to IFN-γ has been shown to increase mRNA expression of MHC class I molecules and also increase the expression of MHC class I protein complexes on the cell surface. This increase in MHC class I expression is expected to inhibit NK cells. Applicant discovered that wild-type (WT) hES cells increased NK cell-mediated cytotoxicity when exposed to IFN-γ (which has been shown to increase MHC class I molecules on the cell surface of hES cells, Figures 2A and 3A). See Figure 15, which shows that NK cell-mediated killing (lysis) increased from 58% to 79% (compare the first bars for conditions 1 and 2 in Figure 15). The same was true when WT PEC cells were exposed to IFN-γ, where the cells also increased B2M and HLA-ABC expression. See Figures 4A and 5A. NK-mediated killing (lysis) increased from 33% to 53% (compare the first bars for conditions 5 and 6 in Figure 15). This data suggests that the key to overcoming the host's NK cell immune response is to inhibit the inhibitory MHC class I. These results suggest that the cause is not overexpression of I signaling but blockage of NK activating ligand signaling.

[0013] This hypothesis was further tested in the context of accentuated NK cell-mediated cytotoxicity. To investigate this, we generated B2M- / - (knockout) hES cells (similar to scenario C in Figure 1). As expected, B2M- / - eliminated cell surface expression of MHC class I molecules on hES cells (Figures 2B and 3B) and PECs (Figures 4B and 5B). Also as expected, B2M- / - cells exhibited increased NK cell-mediated lysis compared with WT cells, from 58% to 71% for hES cells and from 33% to 54% for PECs (compare the first bars in Figure 15 for condition 1 vs. 3 or condition 5 vs. 7). We found that exposing B2M- / - hES cells or PECs to IFN-γ further increased the rate of NK cell-mediated lysis (compare the first bars in Figure 15 for condition 2 vs. 4 and condition 6 vs. 8). Correspondingly, we found that cell surface expression (Figures 6B and 7B) and mRNA expression (Figure 8) of NK cell-activating ligands increased upon exposure to IFN-γ. This data suggested that NK cell-activating ligands on target cells play an important role in NK cell cytotoxicity and led us to hypothesize that inhibiting NK cell-activating ligand expression, in the context of reduced MCH class I expression, for example, in the context of B2M- / -, may protect against NK cell-mediated cytotoxicity.

[0014] To determine whether NK cytotoxicity could be reduced by inhibiting the effect of NK-activating ligands on target cells, Applicant blocked the expression of NK-activating ligands in WT and B2M- / - hES cells and PECs using, for example, an ICAM1-blocking antibody to block ICAM1 protein on the target cell surface. Applicant surprisingly found that target cell cytolysis was reduced (compare the first bar for conditions 2, 4, 6, and 8 with the second and third bars in Figure 15). Thus, Applicant discovered that blocking NK-activating ligands can reduce NK cell-mediated cytolysis. Blocking ICAM1 expression in B2M- / - cells using an antibody against an NK-activating ligand is proof-of-concept for generating cells with a double knockout (HLA class I gene knockout and NK-activating ligand gene knockout). In doing so, Applicant can transition target cells (e.g., hES and / or pancreatic lineage cells) from scenario C to scenario A in Figure 1. Specifically, the cells, tissues, and organs of the present invention have inhibited or absent HLA class I cell surface protein expression (B2M- / -) and inhibited or absent NK-activating ligand cell surface protein expression (e.g., ICAM1- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or using antibodies to block protein expression. Other strategies for interfering with cell surface protein expression include the use of antisense RNA, RNA decoys, ribozymes, RNA aptamers, siRNA, shRNA / miRNA, transdominant negative proteins (TNPs), chimeric / fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFv), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors.Double or multiple gene knockouts effectively prevent both cytotoxic T cell (CTL)-mediated and NK cell-mediated killing because there is little or no HLA class I expressed on the cell surface and little or no NK-activating ligand protein for CTL or NK cells to bind. Furthermore, to completely eliminate NK activation, Applicants envision that it will be necessary to eliminate / reduce the expression of multiple NK-activating ligands, either by gene knockout in the target cells (e.g., hES cell-derived cell therapy) or by using blocking antibodies or other strategies now known or developed in the future.

[0015] NK cell activating ligand blockers According to one aspect of the present invention, treatment methods for suppressing NK cell function are provided. According to another aspect of the present invention, treatment methods for suppressing at least one immune response are provided. Each method comprises administering to a subject in need of treatment an agent that inhibits NK cell function. In some embodiments, the agent is an antibody. In some embodiments, the antibody selectively binds to an NK cell-activating ligand on target cells.

[0016] It is contemplated that a variety of reagents, including antibodies and blocking proteins, can be used to prevent adhesion between NK cells and the NK-activating ligand of a target cell.

[0017] In certain embodiments, such NK-activating ligands are selected from Table 1.

[0018] [Table 1-1]

[0019] [Table 1-2]

[0020] [Table 1-3]

[0021] NK activating ligands are listed in Pegram et al., Activating and inhibitory receptors of NK Cells Immunology and Cell Biology (2011) 89, 216-224, which is incorporated herein by reference in its entirety.

[0022] Hypoimmunogenic hES cells and cells derived therefrom

[0023] HLA are cell surface molecules encoded by a large gene family and can be classified into class I and class II molecules. HLA class I molecules are found on the surface of all nucleated cells and are the focus of the invention described herein. HLA mismatches between donor (target) cells and recipient immune cells (e.g., T cells) during transplantation often result in immune rejection or graft rejection. Structurally, the HLA class I complex is composed of polymorphic heavy chains (e.g., HLA-A, HLA-B, and HLA-C) composed of HLA class I peptides. HLA class I is composed of the HLA-C and HLA-C light chains and the light chain β2-microglobulin (β2m or B2M). Without B2M, class I HLAs are not properly assembled and are not expressed on the cell surface or plasma membrane. In the invention described herein, the applicants have generated hES cell lines and cells derived therefrom by disrupting the B2M gene (by adding or deleting several base pairs, resulting in a frameshift and loss-of-function mutation in the mRNA / protein), thereby depleting HLA class I expression from the cell surface of hESCs.

[0024] The above methodology further disrupts genes encoding NK-activating ligands such as ICAM1. hES cell lines and cells derived therefrom can also be used to generate or produce hES cell lines and cells derived therefrom by disrupting the target cells. Thus, in one embodiment of the present invention, compositions and methods are provided for generating target cells that lack at least one HLA class I antigen and at least one NK-activating ligand, thereby generating hypoimmunogenic cells. Such hypoimmunogenic cells are expected to be less susceptible to immune rejection by the subject into whom they are transplanted. When transplanted, the hypoimmunogenic cells should engraft (not be rejected). In one embodiment, such target cells are capable of engrafting and surviving with little or no need for immunosuppression in the recipient.

[0025] In one embodiment, inhibition, reduction, and / or deletion of both HLA class I and NK-activating ligand expression (or HLA class I-deficient and NK-activating ligand-deficient) in hESC cells and cells derived therefrom can serve as a universal donor cell source for transplantation therapy. These double knockouts (HLA class I-deficient and NK-activating ligand-deficient) can be universally transplanted without minor histocompatibility complex (MiHC) matching, human leukocyte antigen (HLA) matching, or immunosuppression.

[0026] Novel in vitro-derived, hypoimmunogenic compositions and cells are disclosed herein. Specifically, in certain embodiments, the inventions disclosed herein relate to stem cells whose genomes have been modified to reduce or delete key components of both MHC class I genes and NK-activating ligand genes. In certain embodiments, the inventions disclosed herein relate to pancreatic lineage cells, such as pancreatic endoderm cells, pancreatic epithelial cells, pancreatic progenitor cells, pancreatic precursor endocrine cells, pancreatic endocrine cells, pancreatic pre-beta cells, or pancreatic beta cells, whose genomes have been modified to reduce or delete key components of both MHC class I genes and NK-activating ligand genes, thereby generating hypoimmunogenic pancreatic lineage cells. Natural killer activating ligands include, but are not limited to, ligands listed in categories 1, 2, and 3 in Table 1, or combinations thereof. Natural killer activating ligands include, for example, ICAM-1, CEACAM1, CADM1, MICA, and MICB. MHC class I genes include HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and B2M. In certain embodiments, such reduction or knockout of expression of MHC class I and / or MHC class II genes is achieved by directly and / or indirectly targeting the NLRC5, B2M, and CIITA genes, as well as other components of the MHC enhanceosome (enhancers are higher-order protein complexes assembled at enhancers that regulate the expression of target genes, e.g., MHC class I or MHC class II transcriptional regulators).

[0027] Also disclosed herein are methods for preparing hypoimmunogenic cells, the methods comprising modulating the expression of one or more NK-activating ligands expressed by the cells and modulating the expression of one or more MHC class I and / or MHC class II by the cells, thereby preparing hypoimmunogenic cells. In certain aspects, modulating cell surface protein expression of one or more MHC class I and / or MHC class II complexes comprises reducing, inhibiting, and / or disrupting the expression of one or more MHC class I and / or MHC class II genes or proteins. In certain embodiments, modulating the expression of one or more MHC class I and / or MHC class II complexes comprises deleting one or more genes encoding one or more transcriptional regulators of MHC class I or MHC class II from at least one allele of the cell. For example, in certain embodiments, such methods comprise deleting one or more genes encoding one or more transcriptional regulators of MHC class I or MHC class II genes selected from the group consisting of LRC5, CIITA, B2M, and combinations thereof. In certain embodiments, modulating the expression of one or more NK-activating ligands includes deleting, inhibiting, or reducing the expression of one or more genes encoding the NK-activating ligands. In certain embodiments, such an NK-activating ligand is selected from Table 1. In certain embodiments, such an NK-activating ligand is selected from categories 1, 2, 3 of Table 1, or a combination thereof. In certain embodiments, such an NK-activating ligand is selected from categories 1, 2, and 3 of Table 1. In certain embodiments, such an NK-activating ligand is selected from categories 1 and 3 of Table 1. In certain embodiments, such an NK-activating ligand is selected from categories 1 and 2 of Table 1. In certain embodiments, such an NK-activating ligand is selected from categories 2 and 3 of Table 1. In certain embodiments, such an NK-activating ligand is selected from the group consisting of ICAM-1, CEACAM1, CADM1, MICA, MICB, and combinations thereof.

[0028] In certain embodiments, the transplanted hypoimmunogenic cells are in a medium that does not contain animal-source products, eg, xenofree products.

[0029] The present invention contemplates modifying a target polynucleotide sequence in any manner available to one of skill in the art, for example, utilizing meganucleases or endodeoxyribonucleases, zinc finger nucleases (ZFNs or ZNFs), transcription activator-like effector-based nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR / Cas or CRISPR / Cas9) systems, or traditional homologous recombination techniques. Such CRISPR / Cas systems can use a variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005; 1(6)e60 ). In some embodiments, the CRISPR / Cas system is a CRISPR Type I system. In some embodiments, the CRISPR / Cas system is a CRISPR Type II system. In some embodiments, the CRISPR / Cas system is a CRISPR Type V system. NEXTGEN™ CRISPR (Transposagen Inc., Lexington, KY), which introduces a catalytically inactive Cas9 protein fused to dual guide RNA and a FokI nuclease, can also be used to modify target polynucleotide sequences. Other methods of targeting polynucleotide sequences to reduce or eliminate expression in target cells, now known to those of skill in the art or later discovered, can be used to generate the hypoimmunogenic cells described herein.

[0030] In some embodiments, the modification reduces expression of the target polynucleotide sequence. In some embodiments, the modification is a homozygous modification. In some embodiments, the modification is a heterozygous modification.

[0031] In some embodiments, the target polynucleotide sequence is a genomic sequence. In some embodiments, the target polynucleotide sequence is a human genomic sequence. In some embodiments, the target polynucleotide sequence is a mammalian genomic sequence. In some embodiments, the target polynucleotide sequence is a vertebrate genomic sequence.

[0032] In some embodiments, the hypoimmunogenic cells are embryonic stem cells. In certain embodiments, the hypoimmunogenic cells are pluripotent stem cells. In certain embodiments, the hypoimmunogenic cells are induced pluripotent stem cells, reprogrammed cells, dedifferentiated or transdifferentiated cells. In certain embodiments, the hypoimmunogenic cells are multipotent pancreatic progenitor cells. In certain embodiments, the hypoimmunogenic cells are singly hormonal or polyhormonal cells. In the present invention, the hypoimmunogenic cell is a mesendoderm cell, a definitive endoderm cell, a PDX-1-negative foregut endoderm cell, a PDX-1-positive foregut endoderm cell, a pancreatic endoderm cell, an endocrine precursor / progenitor cell, an endocrine cell, a properly specified endocrine cell, an immature endocrine cell, or a functional beta cell. In some embodiments, the hypoimmunogenic cells can be a homogeneous or heterogeneous cell population. In some embodiments, the hypoimmunogenic cells are cells that produce one or more bioactive substances of interest. Hypoimmunogenic cells, such as pancreatic progenitor cells or PDX1-positive pancreatic endoderm, may not initially be therapeutically active when first transplanted, but once transplanted, they further develop and mature to have a therapeutic effect.

[0033] In some embodiments, the hypoimmunogenic cells can be any cell that can be derived from a human pluripotent stem cell, including, but not limited to, any cell, tissue, or organ, including skin cells, beta cells (i.e., cells of the pancreas in the islets of Langerhans), parathyroid cells, intestinal cells, endocrine cells, heart cells, brain cells, kidney cells, liver cells, gastrointestinal and accessory gastrointestinal cells, 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 intestine.

[0034] In some embodiments, the hypoimmunogenic cells may be individual (single) cells in suspension or cell aggregates. In some embodiments, the hypoimmunogenic cells include totipotent cells. In one embodiment, the hypoimmunogenic cells include pluripotent cells. In one embodiment, the hypoimmunogenic cells include unipotent cells.

[0035] In some embodiments, the hypoimmunogenic cells are derived from a pluripotent cell population lacking functional HLA class I expression and NK activating ligand expression. The derived cells can be selected from the group consisting of any cell, tissue, or organ, including skin cells, beta cells (i.e., pancreatic cells in the islets of Langerhans), parathyroid cells, intestinal cells, endocrine cells, heart cells, brain cells, kidney cells, liver cells, gastrointestinal and accessory gastrointestinal cells, 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 intestine.

[0036] The transplanted hypoimmunogenic cells, tissues and / or organs can be syngeneic or allogeneic to the subject receiving the transplant.

[0037] In one embodiment, the hypoimmunogenic cell is a human pluripotent cell. In one embodiment, the hypoimmunogenic cell is a human pancreatic lineage 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 human pancreatic monohormonal or multihormonal cell. In one embodiment, the hypoimmunogenic cell is a human insulin-expressing cell.

[0038] In one embodiment, the hypoimmunogenic cells are well-known and publicly available pluripotent cell lines. The invention described herein is useful with all hES cell and iPSC lines and at least hESCs, e.g., CyT49, CyT25, CyT203, and CyT212. Pluripotent cell lines include those commercially available from WiCell on the World Wide Web at wicell.org / home / stem-cell-lines / order-stem-cell-lines / obtain-stem-cell-lines.cmsx, specifically BG01, BG02, and BG03. Includes:

[0039] 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) Nature Biotechnology 24, 1392-1401, the entire contents of which are incorporated herein by reference. D'Amour et al. describe a five-step differentiation protocol: Stage 1 (generating mostly definitive endoderm), Stage 2 (generating mostly PDX1-negative foregut endoderm), Stage 3 (generating mostly PDX1-positive foregut endoderm), Stage 4 (generating mostly pancreatic endoderm, also called pluripotent pancreatic progenitor cells or pancreatic endocrine progenitor cells), and Stage 5 (generating mostly hormone-expressing endocrine cells). In one embodiment, the hypoimmunogenic cells are substantially similar to those described in U.S. Pat. Nos. 7,510,876, 7,695,965, 7,985,585, 8,586,357, 8,633,024, and 8,129,182, which are incorporated by reference in their entireties.

[0040] In one embodiment, the hypoimmunogenic cells are 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 by reference in its entirety. Schulz et al., Propagation and Banking of hESCs We describe a method for the development of a suspension-based differentiation system. Specifically, undifferentiated pluripotent cells were aggregated into clusters in dynamic rotational suspension culture and subsequently differentiated together for 2 weeks using the Stage 4 protocol. Briefly, from the hES cell aggregate suspension, hESC monolayers were dissociated using Accutase (Innovative Cell Technologies), collected, and plated at 1 × 10 cells in StemPro hESC SFM (Life Technologies; combined DMEM / F12 containing Glutamax, StemPro hESC supplement, BSA, and 1% (v / v) penicillin / streptomycin; omitting FGF-2 and 2-mercaptoethanol). 6 The single cell suspension was either dispensed into non-TC-treated 6-well plates (5.5 mL / well) and spun at 95 rpm on an Innova 2000 rotator (New Brunswick Scientific) or dispensed into 500 mL Nalgene filter receiver storage bottles (150 mL / bottle) and resuspended at 1000 saturates in a Sartorius C The cells were rotated at 65 rpm on a ertomat RM-50 rotator (configured with a 5 cm rotating shaft). The cells were rotated overnight in a 37°C / 8% CO2 incubator to form aggregates of approximately 100-200 μm in diameter. For 100-200 μm diameter aggregates, a rotation speed of 60-140 rpm can be used in 6-well dishes, and a rotation speed of 5-20 rpm can be used in 500 mL bottles. Differentiation of suspension aggregates included only a few modifications from D'Amour. During stage 2, TGF-βRI kinase inhibitor IV was included, and during stage 3, retinoic acid was replaced with the more stable retinoid analog TTNPB (3 nM). Growth factors KGF (50 ng / mL) and EGF (50 ng / mL) were added in stage 4 to maintain cell mass. Noggin (50 ng / mL) was also included in stage 4. In one embodiment, the hypoimmunogenic cells are substantially similar to those described in U.S. Pat. Nos. 8,008,075 and 8,895,300, which are incorporated by reference herein in their entireties.

[0041] In one embodiment, the hypoimmunogenic cells are substantially similar to the cells 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 Translationalmedicine 4:1-9 (2015), which is incorporated herein by reference in its entirety. Agulnick et al. reported that 73% to 80% of the cell population were PDX1 positive (PDX1+) and described a modified protocol for generating pancreatic progenitor cells, consisting of NKX6.1+ and NKX6.1+ pancreatic progenitor cells. The pancreatic progenitor cells were further differentiated into islet-like cells (ICs), which reproducibly contained 73%-89% endocrine cells, of which approximately 40%-50% expressed insulin. The majority of these insulin-positive cells were single-hormone positive and expressed the transcription factors PDX1 and NKX6.1. Agulnick described a protocol modified from Schulz et al.'s 2012 protocol by further treating the cells with activin A, Wnt3A, and heregulin β at stage 3 (days 5-7) and with activin A and heregulin β at stage 4 (days 7-13). In one embodiment, the hypoimmunogenic cells are substantially similar to those described in U.S. Patent No. 8,859,286, which is incorporated herein by reference in its entirety.

[0042] Human embryonic stem cells can be grown, passaged, and expanded substantially as described in U.S. Patent Nos. 7,964,402, 8,211,699, 8,334,138, 8,008,07, and 8,153,429.

[0043] Standard Manufacturing Protocol A standard production method for generating pancreatic endoderm cells (PECs) derived from hESCs is disclosed in Table 2 below.

[0044] [Table 2]

[0045] hESC Agg.: hESC aggregates; XF HA: DMEM / F12 with 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); r0.2 FBS: RPMI1640 (Mediatech), 0.2% FBS (HyClone), 1× GlutaMAX-1 (Life Technologies), 1% v / v penicillin / streptomycin; ITS: Insulin-Transferrin-Selenium (Life Technologies) 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-βRI kinase inhibitor IV (EMD Bioscience); db: DMEM HI Glucose (HyClone) supplemented with 0.5× B-27 Supplement (Life Technologies), 1× 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).

[0046] Calcein release assay The calcein release assay is a non-radioactive alternative for studying NK cell cytotoxicity. Target cells take up a fluorescent dye (calcein AM), which is converted by the cytoplasm to an active fluorescent dye, which is released from the cells only upon lysis. Lysed cells release the fluorescent dye into the supernatant, which is then collected and the amount of fluorescence quantified in a fluorometer. The percentage of cytolysis is calculated from the amount of fluorescence present in the supernatant after incubation with or without NK cells (effectors), blocking antibodies, or both.

[0047] Specific lysis can be calculated by using the formula: % Lysis = 100 × [(mean fluorescence with antibody - mean spontaneous fluorescence) / (mean maximum fluorescence - mean spontaneous fluorescence)]. Maximum fluorescence was determined by lysis of cells incubated with detergent (1% Triton X-100), and spontaneous lysis was the fluorescence obtained with target cells without antibody or effector cells.

[0048] Various cell compositions and methods derived from pluripotent stem cells are described herein and are incorporated by reference in their entirety in the following patent applications: METHODS FOR CULTURE OF HESC ON FEEDER CELLS, filed August 6, 2002; Applicant's U.S. patent application Ser. No. 10 / 486,408, filed Dec. 23, 2004, entitled "DEFINITIVE ENDODERM"; Ser. No. 11 / 021,618, filed Apr. 2005, entitled "DEFINITIVE ENDODERM"; No. 11 / 115,868, filed June 26, 2005, entitled PDX1 EXPRESSING ENDODERM; No. 11 / 165,305, filed June 23, 2005, entitled METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIATING DEFINITIVE ENDODERM; Serial No. 11 / 573,662, entitled "METHODS FOR INCREASING DEFINITIVE ENDODERM DIFFERENTIATION OF PLURIPOTENT HUMAN EMBRYONIC STEM CELLS WITH PI-3 KINASE INHIBITORS," filed on October 27, 2005; Serial No. 12 / 729,084, entitled "PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM," filed on November 27, 2005; No. 12 / 093,590, filed June 14, 2006, entitled MARKERS OF DEFINITIVE ENDODERM; No. 12 / 093,590, filed June 20, 2006, entitled EMBRYONIC STEM CELL CULTURE COMPOSITIONS; and Methods of Use Thereof, No. 11 / 993,399; October 2006 The patent, titled PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM, was filed on the 27th. No. 11 / 588,693, filed March 2, 2007, for Endocrine Progenitor / Precursor Cells, Pancreatic Hormone-Expressing Cells and Methods of Production No. 11 / 681,687, entitled "METHODS FOR CULTURE AND PRODUCTION OF SINGLE CELL POPULATIONS OF HESC," filed May 24, 2007; No. 11 / 773,944, entitled METHODS OF PRODUCING PANCREATIC HORMONES, filed July 5, 2007; No. 11 / 860,444, entitled METHODS FOR INCREASING DEFINITIVE ENDODERM PRODUCTION, filed September 24, 2007; No. 94; No. 12 / 099,759, filed April 8, 2008, entitled "METHODS OF PRODUCING PANCREATIC HORMONES"; No. 12 / 107,020, filed April 21, 2008, entitled "PURIFYING ENDODERM AND PANCREATIC ENDODERM CELLS DERIVED FORM HUMAN EMBRYONIC STEM CELLS"; and No. 12 / 107,020, filed November 13, 2009, entitled "ENCAPSULATION OF PANCREATIC LINEAGE CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS." US Patent No. 12 / 618,659, entitled "CELL COMPOSITIONS FROM DEDIFFERENTIATED REPROGRAMMED CELLS," filed April 22, 2010, and US Patent No. 13 / 761,078, entitled "CELL COMPOSITIONS FROM DEDIFFERENTIATED REPROGRAMMED CELLS," filed February 6, 2013; US Patent No. 11 / 838,054, entitled "COMPOSITIONS AND METHODS USEFUL FOR CULTURING DIFFERENTIABLE CELLS," filed August 13, 2007; US Patent No. 12 / 264,760, entitled "STEM CELL AGGREGATE SUSPENSION COMPOSITIONS AND METHODS OF DIFFERENTIATION THEREOF," filed November 4, 2008; and US Patent No. 12 / 264,760, entitled "SMALL MOLECULES SUPPORTING PLURIPOTENT SUBJECT TO CRITICAL PHASE CELLS," filed April 27, 2010. No. 13 / 259,15, entitled CELL GROWTH; International Application PCT / US11 / 25628, entitled LOADING SYSTEM FOR AN ENCAPSULATION DEVICE, filed February 21, 2011; and International Application PCT / US11 / 25628, entitled AGENTS AND METHODS FOR INHIBITING PLURIPOTENT STEM, filed December 28, 2010. No. 13 / 992,931, entitled "DIMENSIONAL LARGE CAPACITY CELLS"; and U.S. Design Application 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; and U.S. Application No. 14 / 201,630, filed March 7, 2014, entitled "DIMENSIONAL LARGE CAPACITY CELL ENCAPSULATION DEVICE ASSEMBLY." U.S. application entitled IN VITRO DIFFERENTIATION OF PLURIPOTENT STEM CELLS TO PANCREATIC ENDODERM CELLS (PEC) AND ENDOCRINE CELLS, filed December 13, 2013; No. 14 / 106,330, filed November 10, 2016, entitled PDX1 PANCREATIC ENDODERM CELLS IN CELL DELIVERY DEVICES AND METHODS THEREOF; / US2016 / 061442, all of which are incorporated herein by reference in their entireties.

[0049] Various cell compositions and methods derived from pluripotent stem cells are described herein and are incorporated by reference in the following commonly-licensed applications: U.S. Patent Publication No. 2009 / 0269845, filed April 24, 2008, entitled "Pluripotent cells"; U.S. Patent Publication No. 2010 / 0109994, filed April 24, 2010, entitled "Pluripotent cells"; A patent application filed on July 20th entitled "Differentiation of Human Embryonic Stem Cells" US Patent Publication No. 2011 / 0014703, filed July 19, 2010, entitled "Differentiation of Human Embryonic Stem Cells"; No. 702; filed December 16, 2010, Differentiation of Human Embryonic U.S. Patent Publication No. 2011 / 0151561 entitled Stem Cells; October 2, 2009 A U.S. patent entitled "Differentiation of Human Embryonic Stem Cells," filed on the 2nd Publication No. 2010 / 0112692; U.S. Patent Publication No. 2012 / 0052576, filed August 17, 2011, entitled Differentiation of Pluripotent Stem Cells; U.S. Patent Publication No. 2010 / 0112693, filed October 23, 2009, entitled "Differentiation of human pluripotent stem cells"; The patent application filed in U.S. Patent Publication No. 2002 / 0022334 entitled "Differentiation of human embryonic stem cells" is hereby incorporated by reference. 011 / 0151560; U.S. Patent Publication No. 2010 / 0015100, entitled "Differentiation of human embryonic stem cells," filed July 31, 2008; Differentiation of human embryonic stem cells, filed on November 25, 2008 U.S. Patent Publication No. 2009 / 0170198, filed May 7, 2015, entitled "Use of Small Molecules to Enhance MAFA Expression in Pancreatic Endocrine Cells" U.S. Patent Publication No. 2015 / 0329828, filed June 6, 2013, entitled "Differentiation of Human Embryonic Stem Cells into Pancreatic Endocrine Cells" U.S. Patent Publication No. 2013 / 0330823, filed June 13, 2013, entitled "Differentiation of human embryonic stem cells into pancreatic endocrine cells," International Publication No. 2013 / 192005, filed December 30, 2013, entitled "Differentiation of human embryonic stem cells into pancreatic endocrine cells," , U.S. Patent Publication No. 2014 / 0242693, entitled "Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells" U.S. Patent Application Publication No. 2014 / 01100604, filed on June 17, 2014, entitled "Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells"; Publication No. 2014 / 0295552; ​​filed on May 21, 2014, Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells; International Publication No. 2015 / 065524; June 6, 2013 U.S. Patent Publication No. 2013 / 0330823, entitled "Differentiation of Human Embryonic Stem Cells into Pancreatic Endocrine Cells," filed December 2013; No. 2014 / 0186953, filed December 18, entitled "Differentiation of Human Embryonic Stem Cells Into Pancreatic Endocrine Cells Using HB9 Regulators"; U.S. Application No. 14 / 963730, filed December 9, 2015; and U.S. Application No. 14 / 898,015, filed December 11, 2015, all of which are incorporated herein by reference in their entireties.

[0050] In one embodiment, the hypoimmunogenic cells are encapsulated within a cell delivery device. The cell delivery device may comprise a nonwoven fabric. The cell delivery device comprises various layers, each performing one or more functions. In some embodiments, the cell delivery device comprises both a cell-excluding membrane and a nonwoven fabric. In another embodiment, the delivery device is a TheraCyte (formerly Baxter) device (Irvine, Calif.). TheraCyte cell delivery devices are disclosed in U.S. Patent 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, 5,653,756, 5,763,763, 5,773,458, 5,773,458, 5,156,305 ... Nos. 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, all of which are incorporated herein by reference in their entireties.

[0051] In another embodiment, the delivery device is a device substantially as described in U.S. Pat. No. 8,278,106, as well as U.S. application Ser. No. 14 / 201,630, filed Mar. 7, 2014, and International Application No. PCT / US2016 / 061442, filed Nov. 10, 2016, and U.S. Design Nos. 29 / 447,944, 29 / 509,102, and No. 29 / 484,363, No. 29 / 484,360, No. 29 / 484,359, No. 29 / 484,357, No. 29 / 484,356, No. 29 / 484,35 No. 5, No. 29 / 484,362, No. 29 / 484,358, No. 29 / 408,366, No. 29 / 517,319, No. 29 / 408,368, No. 29 / 51 and 29 / 584,046, all of which are incorporated herein by reference in their entireties. In other embodiments, the cell delivery device or large volume assembly comprises one or more seals, i.e., partition seals, that further partition the lumen of the cell delivery device. See, e.g., commonly-owned U.S. Design Application Nos. 29 / 408,366, 29 / 408,368, 29 / 408,370, 29 / 423,365, and 29 / 584,046.

[0052] In one embodiment, the hypoimmunogenic cells are implanted in a perforated cell delivery device that provides direct cell-to-cell contact between the host's vascular cells and the encapsulated cells. By perforated, we mean holes or apertures in the device. In some embodiments, not all layers of the device are perforated. See, for example, International Application No. PCT / US2016 / 0061442, incorporated herein by reference in its entirety, which discusses perforated cell delivery devices with perforations in only one layer, e.g., the cell-exclusion membrane or the cell-exclusion membrane and the nonwoven fabric layer. In one embodiment, the hypoimmunogenic cells are encapsulated in a perforated device surrounded by a nonwoven fabric. In these embodiments, the nonwoven fabric is on the exterior of the cell delivery device. The nonwoven fabric does not affect the implanted cells but promotes host vascularization surrounding the cell housing. See, for example, International Application PCT / US2016 / 0061442 and U.S. Patent No. 8,278,106, both of which are incorporated by reference in their entirety, describing perforated devices and device polymers.

[0053] In one embodiment, the holes / porosity are smaller than the cell aggregates contained in the device, e.g., hPSC-derived aggregates, such as definitive endoderm lineage cell aggregates, contained in the device. In one embodiment, a perforated cell delivery device implanted in a rat or human contains perforations only in the cell exclusion membrane (no other layers of the device are perforated), the holes are about 2 mm or more apart, and the hole diameter is less than about 100 microns.

[0054] Depletion of hypoimmunogenic cells ("suicide genes") The versatility of embryonic stem cells and induced pluripotent stem (iPS) cells to replace and restore tissues in the body is paralleled by an increased risk of cancer, which is also associated with gene therapy. Therefore, reprogrammed tissues, whether 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 and Hanna, JH, Saha, K. & Jaenisch, R. Pluripotency and Cellular Reprogramming: Facts, Hypotheses, Unresolved Issues. Cell 143, 508-525, (2010) (both of which are incorporated herein by reference in their entirety)) or other pluripotent or progenitor cells, and whether derived 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) (which is incorporated by reference in its entirety)). For example, subcutaneously implanted stem cells can be used to treat pluripotent stem cells. iPS cells induce teratomas, and iPS chimeric animals develop primary malignant cancers at a high incidence (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)). Benign teratomas can be easily removed by surgery, but invasive cancers remain at risk from cell therapy.

[0055] Strategies to overcome stem cell tumorigenicity, including suicide gene strategies This is being investigated (Knoepfler, PS Deconstructing Stem Cell Tumorigenicity: A Roadmap to Safe Regenerative Medicine. Stem Cells 27, 1050-1056, (2009)). To achieve this, genes encoding enzymes that metabolize systemically available prodrugs into locally active antitumor agents can be selectively introduced into transplanted cells. For example, treatment with ganciclovir, which is converted by thymidine kinase into a compound that becomes toxic after triphosphorylation by cellular kinases, resulted in the destruction of tumor cells in vitro. Thus, transplanted cells can be modified to artificially create exploitable biochemical differences between the host tissue and the transplanted cells. Targeting of the transplanted cells is achieved by the choice of vector used to deliver the suicide gene and the biology of the suicide gene / prodrug system used. As a result, high doses of the drug produced only in the environment where the cells are transplanted limit side effects in other tissues.

[0056] Depletion of hypoimmunogenic cells can be achieved by selectively introducing a gene into the hypoimmunogenic cells, the expression of which directly results in hypoimmunogenic cell death, or by specifically sensitizing the hypoimmunogenic cells to other agents. In vivo or ex vivo depletion of hypoimmunogenic cells by this method can be achieved by delivering a desired gene to the hypoimmunogenic cells using a viral gene delivery system, such as, but not limited to, retroviral, adenoviral, or adeno-associated viral gene delivery. A desired viral delivery system can include a virus whose genome encodes a protein that directly causes cell death, e.g., by inducing apoptosis in the hypoimmunogenic cells. Alternatively, the viral delivery system can include a virus whose genome encodes, e.g., the herpes simplex virus thymidine kinase gene. Expression of the herpes simplex virus thymidine kinase gene in the hypoimmunogenic cells renders the hypoimmunogenic cells sensitive to pharmacological doses of ganciclovir. Therefore, subsequent contact of the virally transduced hypoimmunogenic cells with ganciclovir results in the death of the hypoimmunogenic cells. Depletion of hypoimmunogenic cells can be achieved by introducing so-called "suicide genes" via genome editing applications such as ZFN, CRISPR / cas, and TALEN systems.

[0057] Agents such as ganciclovir that mediate cell death upon expression of genes such as thymidine kinase are referred to herein as "cell death inducers."

[0058] Genes that can be used to kill hypoimmunogenic cells include, but are not limited to, herpes simplex virus thymidine kinase and cytosine deaminase, or any gene that induces cell death that can be placed under the control of an inducible promoter / regulatory sequence (interchangeably referred to herein as "promoter / regulatory sequence" or "promoter"). The gene is transferred into hypoimmunogenic cells, the cells are selected under appropriate selection pressure, and the cells are transferred to a patient where they are allowed to engraft. The patient is then treated with an agent that induces promoter activity, thereby inducing expression of the gene whose product functions to kill the hypoimmunogenic cells. In the case of thymidine kinase, other agents that promote cell killing by this enzyme, such as ganciclovir, can also be used (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 useful for this purpose include the structural regulators of caspases 3, 8, and 9. Genetically active forms of bax, granzymes, diphtheria toxin, Pseudomonas A toxin, ricin, and other toxin genes disclosed elsewhere herein are included. The generation of suitable constructs for delivering such genes to humans is readily apparent to those skilled in the art and is 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).

[0059] It is important that the gene transferred into a hypoimmunogenic cell for the purpose of cell death be placed under the control of an appropriate promoter sequence so that induction of gene expression can be achieved when an appropriate inducer is added to the cell (administered to a mammal). Such inducible promoter sequences include, but are not limited to, promoters that are induced upon addition of metals to the cell, steroid-inducible promoters, and the like. In a preferred embodiment, an ecdysone promoter system can be used. In this embodiment, an ecdysone promoter is cloned upstream of an ecdysone receptor protein sequence, which is located upstream of a second promoter sequence that drives expression of a desired gene, for example, an ecdysone binding site operably linked to a desired toxin. Induction of the promoter induces expression of the toxin, thereby killing the cells in which the toxin gene is present.

[0060] When cells are transduced in an ex vivo manner, cells transduced with a gene for cell death can be selected (i.e., separated from cells that do not contain the gene) by providing the cells with a selectable marker in addition to the transduced gene. Selectable 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, NY).

[0061] Depletion of hypoimmunogenic cells can be further achieved by introducing oligonucleotides (e.g., but not limited to, antisense molecules) or ribozymes into the hypoimmunogenic cell population, which can induce the death or impair the function of hypoimmunogenic cells. Such oligonucleotides include those that target essential functions of hypoimmunogenic cells, defined herein as killing hypoimmunogenic cells or impairing their function with respect to T cell stimulation. Such functions of hypoimmunogenic cells include, but are not limited to, the costimulatory functions of B71 and B72, and CD40, among others. Thus, oligonucleotides and ribozymes useful in the methods of the present invention include, but are not limited to, those directed against these targets.

[0062] As described herein, depletion of hypoimmunogenic cells includes the impairment of hypoimmunogenic cell function. Impairment of hypoimmunogenic cell function includes all forms of hypoimmunogenic cell damage, with or without physical removal or depletion of hypoimmunogenic cells. Thus, impairment of hypoimmunogenic cell function includes the use of antibodies that block the function of hypoimmunogenic cell surface molecules important for hypoimmunogenic cell function.

[0063] Alternatively, peptides that block the function of hypoimmunogenic cell surface molecules, where blocking results in impaired hypoimmunogenic cell function, can be used to effectively deplete hypoimmunogenic cells in a host organism. Such peptides include, but are not limited to, those designed to specifically bind to receptor molecules on the surface of hypoimmunogenic cells and those designed to inhibit, for example, essential enzymatic functions of these cells.

[0064] Similarly, genes and oligonucleotides designed for the same purposes as described herein are also included as tools in the methods of the invention. Thus, peptides, oligonucleotides, and genes that impair the biological function of hypoimmunogenic cells, as that term is defined herein, are also contemplated for use in the methods of the invention disclosed herein.

[0065] The present invention further encompasses the use of pharmaceutical compositions of suitable low immunogenic cell-depleted compositions for practicing the methods of the present invention, the compositions comprising a suitable low immunogenic cell-depleted composition and a pharmaceutically acceptable carrier. In some embodiments, the cell-depleted composition comprises a chimeric antibody and a toxin. It is a composition.

[0066] As used herein, the term "pharmaceutically acceptable carrier" refers to a chemical composition that can be combined with a suitable low-immunogenic cell-depleted composition and, after combination, can be used to administer the suitable low-immunogenic cell-depleted composition to a mammal.

[0067] Pharmaceutical compositions useful in the methods of the present invention can be administered systemically in oral solid formulations, ophthalmic formulations, suppositories, aerosol formulations, topical formulations, or other similar formulations. In addition to the hypoimmunogenic cell-depleted composition, such pharmaceutical compositions may contain pharmaceutically acceptable carriers and other components known to promote and facilitate drug administration. Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically-based systems, can also be used to administer suitable hypoimmunogenic cell-depleted compositions according to the methods of the present invention.

[0068] Methods for introducing "suicide genes" into cells are disclosed in US Patent Application Publication No. 2006 / 0222633, which is incorporated herein by reference in its entirety.

[0069] The present invention includes a method of depleting hypoimmunogenic cells in a mammalian host, which method comprises contacting the hypoimmunogenic cells with a cell depletion composition after the hypoimmunogenic cells have been transplanted into the host, causing impairment of the hypoimmunogenic cell function or death of the hypoimmunogenic cells, thereby depleting the hypoimmunogenic cells in the mammalian host.

[0070] In another aspect, the hypoimmunogenic cell depletion composition is selected from the group consisting of a toxin, an antibody, a radioactive molecule, a nucleic acid, a peptide, a peptidomimetic, and a ribozyme.

[0071] In one embodiment, the toxin is an immunotoxin. The toxin is selected from the group consisting of ricin, diphtheria toxin, and Pseudomonas exotoxin A.

[0072] In another embodiment, the antibody is selected from the group consisting of an antibody specific for CD1a, an antibody specific for CD11c, an antibody specific for MHCII, 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 a type I lectin, and an antibody specific for a type II lectin.

[0073] In yet another embodiment, the nucleic acid molecule is selected from the group consisting of a gene and an oligonucleotide.

[0074] In a further embodiment, the radioactive molecule is a radiolabeled antibody.

[0075] In another embodiment, the antigen-depleted composition is a chimeric composition comprising an antibody and a toxin. The toxin may be selected from the group consisting of ricin, diphtheria toxin, and Pseudomonas exotoxin A.

[0076] In another embodiment, the antibody is selected from the group consisting of an antibody specific for CD1a, an antibody specific for CD11c, an antibody specific for MHCII, 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 lectin, and an antibody specific for type II lectin.

[0077] Combination Products The embodiments described herein also disclose combination products, which refer to devices loaded with hypoimmunogenic cells or therapeutic agents, i.e., each alone may be a potential medical device or cell product, but when used in combination they become a combination product. In the present application, the combination product refers to a perforated device loaded with low-immunogenic cells. This is referred to as a "combination product" or a "perforated combination product." The device (perforated or non-perforated) is disclosed in U.S. Patent Nos. 8,278,106 and 9,526,880, International Application No. PCT / US2016 / 0061442, and U.S. Design Patent Nos. D714956, D718472, D718467, D718466, D718468, D718469, D718470, D718471, D720469, D726306, D726307, and D728095. The macro cell delivery device may be any of the macro cell delivery devices described herein, including, but not limited to, cell encapsulation devices such as those described in US Pat. Nos. D734166, D734847, D747467, D747468, D747798, D750769, D750770, D755986, D760399, D761423, and D761424 (incorporated by reference in their entireties). The cells loaded into the device (perforated or non-perforated) can be any of the hypoimmunogenic cells discussed above, including, but not limited to, definitive endoderm, PDX1-positive endoderm, PDX1-positive foregut endoderm, pancreatic endoderm, pancreatic endoderm cells expressing PDX1 and NKX6.1, endocrine precursor cells, endocrine precursor cells 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.

[0078] A perforated delivery device loaded with pancreatic endoderm or hypoimmunogenic pancreatic progenitor cells that mature upon in vivo implantation is intended to reduce insulin dependence and / or reduce hypoglycemia in diabetic patients, including, but not limited to, high-risk type 1 diabetic patients who are hypoglycemic unaware, unstable (vulnerable), or have undergone organ transplantation and who can tolerate or are already undergoing immunosuppressive therapy. As substantially described in International Application PCT / US2016 / 0061442 (incorporated by reference in its entirety), the primary mode of action is via human pancreatic endoderm cells (PECs) or hypoimmunogenic pancreatic progenitor cells contained in a permeable, durable, implantable medical device that promotes direct host vascularization. Following implantation, the hypoimmunogenic PEC cells differentiate and mature into therapeutic, glucose-responsive, insulin-releasing hypoimmunogenic cells. Thus, the perforated combination product promotes human insulin secretion. The perforated combination product limits the distribution (egress) of PEC hypoimmunogenic cells in vivo. The perforated combination product is implanted in a location that maintains the therapeutic hypoimmunogenic cell population within the device and allows for sufficient vascularization to promote distribution of insulin and other pancreatic products into the bloodstream. The perforated combination product is implanted and explanted with conventional surgical tools and is intended to provide a therapeutic dose for two or more years. The device is intended to retain a sufficient dose of the PEC hypoimmunogenic cell product during prescription, storage, handling, and surgical implantation to achieve clinical efficacy and ensure that the cell product is placed within a tissue capsule to meet safety requirements.

[0079] Knock-in In certain embodiments, tolerogenic factors can be inserted or reinserted into the genome-edited stem cell line to create an immune-privileged universal donor stem cell line. In certain embodiments, the universal stem cells disclosed herein are further modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, but are not limited to, one or more of HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, and IL-35. Any method of gene editing can be used to facilitate the insertion of tolerogenic factors, such as those described above, into the AAVS1 locus to actively inhibit immune rejection.

[0080] Specifically, in certain embodiments, the invention disclosed herein comprises at least one M The present invention relates to stem cells whose genomes have been modified to reduce or delete key components of MHC class I genes and at least one NK-activating ligand gene, and further modified to increase expression of one or more tolerogenic factors. In certain embodiments, the invention disclosed herein relates to stem cells whose genomes have been modified to reduce or delete key components of at least one MHC class I gene and at least one NK-activating ligand gene, and further modified to increase expression of one or more of HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, and IL-35.

[0081] Embodiment Other embodiments of the present invention are described with reference to the following numbered items:

[0082] Compositions relating to blocking antibodies

[0083] Item 1: A composition comprising pluripotent-derived cells lacking at least one human leukocyte antigen (HLA) class I gene and at least one agent that binds to a natural killer (NK) cell-activating ligand.

[0084] Item 2: The composition of item 1, wherein the agent is an antibody.

[0085] Item 3: The composition of Item 1, wherein the HLA class I gene is B2M.

[0086] Item 4: The composition of items 1 to 3, wherein the NK cell-activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[0087] Item 5: The composition of Items 1 to 2, wherein the NK cell-activating ligand is ICAM-1 and CEACAM1.

[0088] Item 6: The composition of Items 1 to 2, wherein the NK cell-activating ligand is ICAM-1, CEACAM1, CADM1, MICA, or MICB.

[0089] Item 7: The composition of items 1 to 2, wherein the pluripotent-derived cells further express a protein that, when expressed in the presence of a cell death-inducing agent, enables the agent to kill the pluripotent cells.

[0090] Item 8: The composition of item 7, wherein the cell death inducer is ganciclovir.

[0091] Item 9: The composition of any one of items 1 to 8, wherein the pluripotent-derived cells further overexpress one or more tolerogenic factors.

[0092] Item 10: The composition of item 9, wherein the tolerogenic factor is HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, or IL-35.

[0093] Item 11: The composition of item 9, wherein the tolerogenic factors are HLA-C, HLA-E, and HLA-G.

[0094] Blocking antibodies: Methods

[0095] Item 1: A method for preventing cell graft rejection of human pluripotent-derived cells, comprising: a target cell population lacking at least one HLA class I gene; and an NK cell-activating ligand on the target cells. to a subject in need of treatment in an amount effective to suppress NK cell attack in the subject, thereby preventing cell graft rejection of human pluripotent-derived cells.

[0096] Item 2: The method of item 1, wherein the agent is an antibody.

[0097] Item 3: The method of item 1, wherein the subject's immune response to an NK cell-activating ligand is suppressed.

[0098] Item 4: The method of item 1, wherein the NK cell-activating ligand is ICAM-1, CEACAM1, CADM1, MICA, or MICB.

[0099] Item 5: The method of item 1, wherein the NK cell-activating ligand is ICAM-1, CEACAM1, CADM1, MICA, or MICB.

[0100] Item 6: The method of item 1, wherein the subject is a human.

[0101] Item 7: The method of item 2, wherein the antibody is a human antibody.

[0102] hES cell double knockout: composition

[0103] Item 1: An in vitro cell population comprising pluripotent-derived cells, wherein the pluripotent-derived cells lack at least one HLA class I gene and at least one natural killer (NK) cell-activating ligand gene.

[0104] Item 2: The in vitro cell population of Item 1, wherein the HLA class I gene is B2M.

[0105] Item 3: The in vitro cell population of items 1 to 2, wherein the NK cell-activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[0106] Item 4: The in vitro cell population of items 1 to 2, wherein the NK cell-activating ligands are ICAM-1 and CEACAM1.

[0107] Item 5: The in vitro cell population of items 1 to 2, wherein the NK cell-activating ligands are ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[0108] Item 6: The in vitro cell population of items 1 to 5, wherein the pluripotent-derived cells further express a protein that, when expressed in the presence of a cell death-inducing agent, is capable of killing the pluripotent-derived cells.

[0109] Item 7: The in vitro cell population of item 6, wherein the cell death inducer is ganciclovir.

[0110] Item 8: The in vitro cell population of any one of items 1 to 7, wherein the pluripotent-derived cells further overexpress one or more tolerogenic factors.

[0111] Item 9: The in vitro cell population of item 8, wherein the tolerogenic factor is HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, IL-35, or a combination thereof.

[0112] Item 10: The immune tolerogenic factors are HLA-C, HLA-E, and HLA-G. In vitro cell populations.

[0113] Pluripotent stem cell triple knockout: composition

[0114] A human pluripotent stem cell comprising a modified genome comprising: (a) a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell; (b) a second genomic modification in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell; and (c) a third genomic modification in which the CEACAM1 gene has been edited to reduce or eliminate CEACAM1 surface expression and / or activity in the cell.

[0115] Compositions for knockout of transcriptional regulators

[0116] Item 12: A pluripotent-derived cell comprising regulated expression of one or more MHC class I or MHC class II genes or protein complexes and one or more NK cell-activating ligands relative to wild-type pluripotent stem cells, wherein the pluripotent stem cell has one or more genes encoding one or more transcription factors of MHC class I or MHC class II and an NK cell-activating ligand gene deleted from at least one allele of the cell.

[0117] Item 13: A pluripotent stem cell comprising regulated expression of one or more NK cell-activating ligands relative to wild-type human pluripotent stem cells.

[0118] Item 14: Human pluripotent stem cells that do not express B2M or ICAM-1.

[0119] Item 15: Human pluripotent stem cells that do not express CIITA or ICAM-1.

[0120] Item 16: Human pluripotent stem cells that do not express LRC5 or ICAM-1.

[0121] Item 17: A human pluripotent stem cell that does not express one or more of NLRC5, CIITA, and B2M, and further does not express one or more of ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[0122] Item 18: Human pluripotent stem cells that do not express one or more of HLA-A, HLA-B, and HLA-C, and further do not express one or more of ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[0123] Item 19: A human that does not express one or more MHC class I antigens and one or more NK cell-activating ligands, and further has one or more tolerogenic factors inserted into a safe harbor locus of at least one allele of the cell. Pluripotent stem cells.

[0124] Item 20: A human pluripotent stem cell comprising a modified genome, comprising a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell, and the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell.

[0125] Item 21: A human pluripotent stem cell comprising a modified genome comprising: (a) a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell; and (b) a second genomic modification in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell.

[0126] Regarding hES double knockout cells: composition

[0127] Item 1: An in vitro cell population comprising pluripotent cells, wherein the pluripotent cells lack at least one functional MHC class I cell surface protein and at least one functional natural killer (NK) cell-activating ligand cell surface protein.

[0128] Item 2: The in vitro cell population of item 1, wherein the MHC class I cell surface protein is HLA-A, HLA-B, HLA-C, or a combination thereof.

[0129] Item 3: The in vitro cell population of items 1 to 2, wherein the MHC class I cell surface protein is B2M.

[0130] Item 4: The in vitro cell population of any one of items 1 to 3, wherein the pluripotent cells lack at least two functional NK cell-activating ligand cell surface proteins.

[0131] Item 5: The in vitro cell population of any one of items 1 to 4, wherein the pluripotent cells lack at least three functional NK cell-activating ligand cell surface proteins.

[0132] Item 6: The in vitro cell population of any one of items 1 to 5, wherein the NK cell-activating ligand cell surface protein is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[0133] Item 7: The in vitro cell population of any one of items 1 to 5, wherein the NK cell-activating ligand cell surface proteins are ICAM-1 and CEACAM1.

[0134] Item 8: The in vitro cell population of any one of items 1 to 7, wherein the pluripotent cells further express a protein that, when expressed in the presence of a cell death-inducing agent, enables the agent to kill the pluripotent cells.

[0135] Item 9: The in vitro cell population of any one of items 1 to 8, wherein the protein that, when expressed in the presence of a cell death-inducing agent, is capable of killing the pluripotent cells, is herpes simplex virus, thymidine kinase, or cytosine deaminase.

[0136] Item 10: The in vitro cell population of any one of items 8 to 9, wherein the cell death-inducing agent is ganciclovir.

[0137] Regarding hES double knockout cells: composition

[0138] Item 1: An in vitro cell population comprising pluripotent cells, wherein the 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 NK-activating ligand cell surface protein relative to the original genotype or wild-type human cells.

[0139] Item 2: An in vitro cell population comprising pluripotent cells, wherein the pluripotent cells have reduced expression of one or more of HLA-A, HLA-B, and HLA-C cell surface proteins and reduced function and / or expression of at least one NK-activating ligand cell surface protein relative to the original genotype or wild-type human cells.

[0140] Item 3: An in vitro cell population comprising pluripotent cells, wherein the pluripotent cells are functional HL An in vitro cell population that lacks expression of a cell surface protein, an NK cell activating ligand cell surface protein, and has a protein that, when expressed in pluripotent cells in the presence of a cell death-inducing agent, enables the agent to kill the pluripotent cells.

[0141] Item 4: Stem cells, in which the expression of one or more HLA class I cell surface proteins and one or more NK-activating ligand cell surface proteins is modulated relative to wild-type stem cells.

[0142] Item 5: A pluripotent cell, wherein the expression of one or more HLA class I cell surface proteins, one or more NK-activating ligand cell surface proteins, and one or more tolerogenic cell surface protein factors is modulated relative to a wild-type stem cell, and further expresses a protein that, when expressed in the presence of a cell death-inducing agent, enables the agent to kill the pluripotent cell.

[0143] Item 6: An in vitro cell population comprising pluripotent cells, wherein the pluripotent cells lack functional MHC class I genes and natural killer (NK) cell-activating ligand genes, the pluripotent cells overexpress a tolerogenic cell surface protein factor relative to wild-type pluripotent cells, and the pluripotent cells further express a protein that, when expressed in the presence of a cell death-inducing agent, enables the agent to kill the pluripotent cells.

[0144] Regarding PEC double knockout cells: composition

[0145] Item 1: An in vitro cell population comprising pancreatic endoderm (PEC) cells, wherein the PEC cells lack at least one functional HLA class I gene and at least one natural killer (NK) cell-activating ligand gene.

[0146] Item 2: The in vitro cell population of item 1, wherein at least one HLA class I gene is B2M.

[0147] Item 3: The in vitro cell population of items 1 to 2, wherein the at least one NK cell-activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[0148] Item 4: The in vitro cell population of items 1 to 2, wherein at least one NK cell-activating ligand is ICAM-1 and CEACAM1.

[0149] Item 5: The in vitro cell population of items 1 to 2, wherein at least one NK cell-activating ligand is ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[0150] Item 6: The in vitro cell population of items 1 to 5, wherein the PEC cells further express a protein that, when expressed in the presence of a cell death-inducing agent, enables the agent to kill the PEC cells.

[0151] Item 7: The in vitro cell population of item 6, wherein the cell death inducer is ganciclovir.

[0152] Item 8: The in vitro cell population of item 6, wherein the gene that, when expressed in the presence of a cell death-inducing agent, can cause the agent to kill PEC cells is herpes simplex virus, thymidine kinase, or cytosine deaminase.

[0153] Item 9: The in vitro cell population of any one of items 1 to 8, wherein the PEC cells further overexpress one or more tolerogenic cell surface proteins.

[0154] Item 10: The in vitro cell population of any one of items 1 to 9, wherein the tolerogenic factor is HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, IL-35, or a combination thereof.

[0155] Item 11: The in vitro cell population of any one of items 1 to 9, wherein the tolerogenic factors are HLA-C, HLA-E, and HLA-G.

[0156] Item 12: An in vitro cell population comprising pancreatic endoderm (PEC) cells, wherein the PEC cells lack at least one functional MHC class I gene, MHC class II gene, and natural killer (NK) cell-activating ligand gene.

[0157] Item 13: An in vitro cell population comprising pancreatic endoderm (PEC) cells, wherein the PEC cells lack at least one functional MHC class I gene and MHC class II gene and lack at least two natural killer (NK) cell-activating ligand genes.

[0158] Item 14: Pancreatic endoderm (PEC) cells, in which one or more HLA class I genes and one or more NK-activating ligand genes are modulated relative to wild-type PEC cells.

[0159] Item 15: A pancreatic endoderm (PEC) cell, wherein the expression of one or more HLA class I cell surface proteins, one or more NK-activating ligands, and one or more tolerogenic factors is modulated relative to a wild-type PEC cell, and further expresses a protein that, when expressed in the presence of a cell death-inducing agent, enables the agent to kill the PEC cell.

[0160] Item 16: Pancreatic endoderm (PEC) cells that do not express B2M or ICAM-1.

[0161] Item 17: Pancreatic endoderm (PEC) cells that do not express CIITA or ICAM-1.

[0162] Item 18: Pancreatic endoderm (PEC) cells that do not express LRC5 or ICAM-1.

[0163] Item 19: Pancreatic endoderm (PEC) cells that do not express one or more of NLRC5, CIITA, and B2M, and further do not express one or more of ICAM-1, CEACAM1, CADM1, MICa, and MICB.

[0164] Item 20: Pancreatic endoderm (PEC) cells that do not express one or more of HLA-A, HLA-B, and HLA-C, and further do not express one or more of ICAM-1, CEACAM1, CADM1, MICa, and MICB.

[0165] Item 21: A pancreatic endoderm (PEC) cell that does not express one or more MHC class I cell surface proteins and one or more NK cell-activating ligands, and further has one or more tolerogenic factors inserted into a safe harbor locus of at least one allele of the cell.

[0166] Item 22: A pancreatic endoderm (PEC) cell comprising a modified genome, comprising a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell, and the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell.

[0167] Item 23: (a) a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell; and (b) a second genomic modification in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell. , pancreatic endoderm (PEC) cells containing a modified genome.

[0168] Item 24: A pancreatic endoderm (PEC) cell comprising a modified genome comprising: (a) a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell; (b) a second genomic modification in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell; and (c) a third genomic modification in which the CEACAM1 gene has been edited to reduce or eliminate CEACAM1 surface expression and / or activity in the cell.

[0169] Regarding low immunogenic cells Item 1: An in vitro cell population comprising hypoimmunogenic cells, wherein the hypoimmunogenic cells lack at least one functional HLA class I cell surface protein and at least one functional NK cell-activating ligand cell surface protein.

[0170] Item 2: The in vitro cell population of item 1, wherein the HLA class I cell surface protein is B2M.

[0171] Item 3: The in vitro cell population of items 1 to 2, wherein the NK cell-activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[0172] Item 4: The in vitro cell population of items 1 to 2, wherein the NK cell-activating ligands are ICAM-1 and CEACAM1.

[0173] Item 5: The in vitro cell population of items 1 to 2, wherein the NK cell-activating ligands are ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[0174] Item 6: The in vitro cell population of items 1 to 5, wherein the hypoimmunogenic cells further express a protein that, when expressed in the presence of a cell death-inducing agent, enables the agent to kill the hypoimmunogenic cells.

[0175] Item 7: The in vitro cell population of any one of items 1 to 6, wherein the hypoimmunogenic cells further overexpress one or more tolerogenic factors.

[0176] Item 8: The in vitro cell population of items 1 to 7, wherein the tolerogenic factor is HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, IL-35, or a combination thereof.

[0177] Item 9: The in vitro cell population of items 1 to 7, wherein the tolerogenic factors are HLA-C, HLA-E, and HLA-G.

[0178] Item 10: An in vitro cell population comprising hypoimmunogenic cells, wherein the hypoimmunogenic cells lack at least one functional MHC class I gene and at least one NK cell-activating ligand gene.

[0179] Item 11: An in vitro cell population comprising hypoimmunogenic cells, wherein the hypoimmunogenic cells lack at least one functional MHC class I gene, MHC class II gene, and NK cell-activating ligand gene.

[0180] Item 12: The in vitro cell population of items 10 or 11, wherein the MHC class I gene is HLA-A, HLA-B, HLA-C, or a combination thereof.

[0181] Item 13: The in vitro cell population of items 10, 11, or 12, wherein the MHC class I gene is B2M.

[0182] Item 14: The in vitro cell population of any one of items 10 to 13, wherein the hypoimmunogenic cells lack at least two functional NK cell-activating ligand genes.

[0183] Item 15: The in vitro cell population of any one of items 10 to 13, wherein the hypoimmunogenic cells lack at least three functional NK cell-activating ligand genes.

[0184] Item 16: The in vitro cell population of any one of items 10 to 13, wherein the NK cell-activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[0185] Item 17: The in vitro cell population of any one of items 10 to 13, wherein the NK cell-activating ligands are ICAM-1 and CEACAM1.

[0186] Item 18: The in vitro cell population of any one of items 10 to 13, wherein the NK cell-activating ligands are ICAM-1, CEACAM1, CADM1, MICA, and MICB.

[0187] Item 19: The in vitro cell population of any one of items 10 to 18, wherein the hypoimmunogenic cells further express a protein that, when expressed in the presence of a cell death-inducing agent, enables the agent to kill the hypoimmunogenic cells.

[0188] Item 20: The in vitro cell population of any one of items 7 to 15, wherein the poorly immunogenic cells are hES cells or pancreatic lineage cells.

[0189] Item 21: A hypoimmunogenic cell in which the expression of one or more HLA class I cell surface proteins and one or more NK-activating ligand cell surface proteins is modulated relative to a wild-type hypoimmunogenic cell.

[0190] Item 22: A hypoimmunogenic cell in which the expression of one or more HLA class I cell surface proteins, one or more NK-activating ligands, and one or more tolerogenic cell surface protein factors is modulated relative to a wild-type hypoimmunogenic cell, and the pluripotent cell further expresses a protein that, when expressed in the presence of a cell death-inducing agent, enables the agent to kill the hypoimmunogenic cell.

[0191] Item 23: A hypoimmunogenic stem cell comprising regulated expression of one or more MHC class I or MHC class II cell surface proteins and one or more NK cell-activating ligands relative to wild-type pluripotent stem cells, wherein the pluripotent stem cell has one or more genes encoding one or more transcription factors of MHC class I or MHC class II and an NK cell-activating ligand gene deleted from at least one allele of the cell.

[0192] Item 24: A hypoimmunogenic cell comprising regulated expression of one or more NK cell-activating ligands relative to wild-type human hypoimmunogenic cells.

[0193] Item 25: Human hypoimmunogenic cells that do not express B2M or ICAM-1.

[0194] Item 26: Human low-immunogenic cells that do not express CIITA or ICAM-1.

[0195] Item 27: Human hypoimmunogenic cells that do not express LRC5 or ICAM-1.

[0196] Item 28: Human hypoimmunogenic cells that do not express one or more of NLRC5, CIITA, and B2M, and further do not express one or more of ICAM-1, CEACAM1, CADM1, MICa, and MICB.

[0197] Item 29: Human hypoimmunogenic cells that do not express one or more of HLA-A, HLA-B, and HLA-C, and further do not express one or more of ICAM-1, CEACAM1, CADM1, MICa, and MICB.

[0198] Item 30: A human hypoimmunogenic cell that does not express one or more MHC class I cell surface proteins and one or more NK cell-activating ligands, and further has one or more tolerogenic factors inserted into a safe harbor locus of at least one allele of the cell.

[0199] Item 31: A hypoimmunogenic cell comprising a modified genome, comprising a first genomic modification wherein the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell, and the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell.

[0200] Item 32: A hypoimmunogenic cell comprising a modified genome comprising: (a) a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell; and (b) a second genomic modification in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell.

[0201] Item 33: A hypoimmunogenic cell comprising a modified genome comprising: (a) a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell; (b) a second genomic modification in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell; and (c) a third genomic modification in which the CEACAM1 gene has been edited to reduce or eliminate CEACAM1 surface expression and / or activity in the cell.

[0202] How to perform a double knockout Item 1: a) A method for reducing graft rejection, comprising administering to a subject in need of a transplant an effective amount of a graft comprising a pancreatic endoderm cell population, wherein the function of at least one HLA class I cell surface protein and at least one NK cell activating ligand cell surface protein is disrupted.

[0203] Item 2: The method of Item 1, wherein the HLA class I cell surface protein is B2M.

[0204] Item 3: The method of Items 1 to 2, wherein the NK cell-activating ligand cell surface protein is ICAM-1, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[0205] Item 4: The method of Items 1 to 2, wherein the NK cell-activating ligand is ICAM-1 and CEACAM1.

[0206] Item 5: The method of items 1 to 2, wherein the NK cell-activating ligand is ICAM-1, CEACAM1, CADM1, MICA, or MICB.

[0207] Item 6: A method for depleting poorly immunogenic cells in a cell population, comprising: with a hypoimmunogenic cell-depleting composition to impair hypoimmunogenic cell function or kill said hypoimmunogenic cells, thereby depleting said hypoimmunogenic cells in said cell population.

[0208] Item 7: The method of claim 6, wherein the hypoimmunogenic cells lack expression of at least one functional HLA class I cell surface protein and at least one NK-activating ligand.

[0209] Item 8: A method for removing hypoimmunogenic cells from a host mammal, the method comprising: (a) transferring hypoimmunogenic cells into the host mammal; and (b) contacting the host with a hypoimmunogenic cell-depleting composition to cause impairment of hypoimmunogenic cell function or death of the hypoimmunogenic cells, thereby removing the hypoimmunogenic cells in the host mammal.

[0210] Item 9: The method of item 8, wherein the function of at least one HLA class I cell surface protein and at least one NK-activating ligand is diminished.

[0211] Item 10: A method for increasing an NK-activating ligand in a target cell population, comprising exposing the target cell population to IFN-γ stimulation, thereby increasing the NK-activating ligand in the target cells relative to wild-type.

[0212] Pluripotent cells in which the functions of both MHC class I genes and NK cell-activating ligand genes are disrupted or inhibited: Compositions Item 1: An in vitro cell population comprising pluripotent cells in which the function of at least one major histocompatibility complex (MHC) class I gene and at least one natural killer (NK) cell-activating ligand gene has been disrupted or inhibited.

[0213] Item 2: The in vitro cell population of item 1, wherein the MHC gene encodes beta-2 microglobulin (B2M).

[0214] Item 3: The in vitro cell population of item 1, wherein the NK cell-activating ligand is ICAM1, CD58, PVR, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[0215] Item 4: The in vitro cell population of item 1, wherein the NK cell activating ligands are ICAM1 and CD58.

[0216] Item 5: The in vitro cell population of item 1, wherein the NK cell-activating ligands are ICAM1, CD58, CD155, CEACAM1, CADM1, MICA, and MICB.

[0217] Item 6: The in vitro cell population of item 1, wherein the pluripotent cells are human embryonic stem cells.

[0218] Item 7: The in vitro cell population of item 1, wherein the pluripotent cells differentiate into pancreatic endoderm cells.

[0219] Item 8: The in vitro cell population of item 1, wherein the pluripotent cells further comprise a protein that, when expressed in the presence of a cell death-inducing agent, enables the agent to kill the cells.

[0220] Item 9: The in vitro cell population of item 1, wherein an MHC class I gene has been disrupted using genome editing applications.

[0221] Item 10: Genome editing applications rely on Zinc Finger Nucleases (ZFNs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / cDNA 10. The in vitro cell population of item 9, wherein the cell population is a transcription activator-like effector nuclease (TALEN) system.

[0222] Item 11: The in vitro cell population of item 1, wherein the NK cell-activating ligand is disrupted using genome editing applications.

[0223] Item 12: The in vitro cell population of item 1, wherein the NK cell-activating ligand is disrupted using an anti-NK cell-activating ligand agent.

[0224] Item 13: The in vitro cell population of item 8, wherein the agent is an antibody.

[0225] Compositions relating to pancreatic lineage cells in which the functions of both MHC class I genes and NK cell-activating ligand genes are disrupted or inhibited Item 14: An in vitro cell population comprising pancreatic lineage cells, in which the function of at least one major histocompatibility complex (MHC) class I gene and at least one natural killer (NK) cell-activating ligand is disrupted or inhibited.

[0226] Item 15: The in vitro cell population of Item 14, wherein the MHC class I gene encodes B2M.

[0227] Item 16: The in vitro cell population of item 14, wherein the NK cell-activating ligand is ICAM1, CD58, CD155, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[0228] Item 17: The in vitro cell population of item 14, wherein the NK cell activating ligands are ICAM1 and CD58.

[0229] Item 18: The in vitro cell population of item 14, wherein the NK cell-activating ligands are ICAM1, CD58, CD155, CEACAM1, CADM1, MICA, and MICB.

[0230] Item 19: The in vitro cell population of item 14, wherein the pancreatic lineage cells are definitive endoderm, foregut endoderm, pancreatic endoderm cells, endocrine precursors, or insulin-producing cells.

[0231] Item 20: The in vitro cell population of item 14, wherein the pancreatic lineage cells further express a protein that, when expressed in the presence of a cell death-inducing agent, enables the agent to kill the cells.

[0232] Methods for preventing pancreatic cell graft rejection - Patent Application 20070122999 Item 21: A method for preventing cell graft rejection of human pancreatic cells, comprising:

[0233] a. providing a population of human pancreatic cells that do not express at least one major histocompatibility complex (MHC) class I cell surface protein and do not express at least one natural killer (NK)-activating ligand; and b. Transplantation of a human pancreatic cell population into a mammalian subject and prevention of cell graft rejection due to the absence of MHC class I cell surface protein and NK activating ligand expression.

[0234] Item 22: The method of Item 21, wherein the major histocompatibility complex (MHC) class I cell surface protein is beta-2 microglobulin (B2M) or HLA-ABC cell surface protein.

[0235] Item 23: NK cell activating ligands include ICAM1, CD58, CD155, PVR, 22. The method of item 21, wherein the CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[0236] Item 24: The method of Item 21, wherein the human pancreatic cell population is pancreatic endoderm cells (PECs).

[0237] Item 25. The method of Item 21, wherein the human pancreatic cell population further comprises a protein that, when expressed in the human cell population in the presence of a cell death-inducing agent, enables the agent to kill the human pancreatic cell population.

[0238] How insulin is produced Item 26. Methods for producing insulin, including:

[0239] a) providing a human pancreatic endoderm cell population that does not express at least one major histocompatibility complex (MHC) class I cell surface protein and does not express at least one natural killer (NK)-activating ligand; and b) transplanting the human pancreatic endoderm cell population into a mammalian subject, wherein the pancreatic endoderm cells mature in the mammalian subject and produce insulin in response to glucose stimulation.

[0240] Item 27: The method of Item 26, wherein the major histocompatibility complex (MHC) class I cell surface protein is beta-2 microglobulin (B2M) or an HLA-ABC cell surface protein.

[0241] Item 28: The method of Item 26, wherein the NK cell-activating ligand is ICAM1, CD58, CD155, PVR, CEACAM1, CADM1, MICA, MICB, or a combination thereof.

[0242] Item 29: The method of Item 26, wherein the human pancreatic cell population further comprises a protein that, when expressed in the human pancreatic cell population in the presence of a cell death-inducing agent, enables the agent to kill the human pancreatic cell population.

[0243] Item 30: The method of item 29, wherein the protein is herpes simplex virus thymidine kinase and the agent is ganciclovir.

[0244] A method for preparing hypoimmunogenic stem cells, comprising regulating the expression of one or more MHC class I cell surface proteins and one or more NK activating ligands by hypoimmunogenic stem cells, thereby preparing hypoimmunogenic stem cells.

[0245] A method for preparing hypoimmunogenic stem cells, comprising regulating the expression of one or more MHC class I cell surface proteins, one or more NK-activating ligands, and regulating the expression of one or more immune tolerogenic factors on stem cells, thereby preparing hypoimmunogenic stem cells.

[0246] A method for modulating the expression of one or more MHC class I cell surface proteins and NK cell activating ligand cell surface proteins on stem cells, the method comprising deleting one or more genes encoding one or more transcriptional regulators of MHC class I genes and NK cell activating ligand genes from at least one allele of the cell, thereby modulating the expression of one or more MHC class I cell surface proteins and NK cell activating ligand cell surface proteins.

[0247] (a) injecting the cells into a cell culture medium containing a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 1 to 3 or a ribonucleic acid homologous to any one of SEQ ID NOs: 1 to 3, A hypoimmunogenic stem cell comprising a modified genome comprising: (a) a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell by contacting the cell with either a nucleic acid encoding an s protein or a Cas protein; and (b) a second genomic modification in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell by contacting the cell with either a Cas protein or a nucleic acid encoding a Cas protein in combination with either a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 4 to 6 or a ribonucleic acid homologous to any one of SEQ ID NOs: 4 to 6.

[0248] (a) a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cells by contacting the cells with either a Cas protein or a nucleic acid encoding a Cas protein in combination with either a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 1 to 3 or a ribonucleic acid homologous to any one of SEQ ID NOs: 1 to 3; and (b) a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cells by contacting the cells with either a Cas protein or a nucleic acid encoding a Cas protein in combination with either a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 4 to 6 or a ribonucleic acid homologous to any one of SEQ ID NOs: 4 to 6. and (c) a third genomic modification in which the CEACAM1 gene has been edited to reduce or eliminate CEACAM1 surface expression and / or activity in the cells by contacting the cells with either a Cas protein or a nucleic acid encoding a Cas protein in combination with either a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 7 to 9 or a ribonucleic acid homologous to any one of SEQ ID NOs: 7 to 9.

[0249] A pluripotent stem cell comprising a modified genome comprising: (a) a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell by contacting the cell with either a Cas protein or a nucleic acid encoding a Cas protein in combination with either a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 1 to 3 or a ribonucleic acid homologous to any one of SEQ ID NOs: 1 to 3; and (b) a second genomic modification in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell by contacting the cell with either a Cas protein or a nucleic acid encoding a Cas protein in combination with either a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 4 to 6 or a ribonucleic acid homologous to any one of SEQ ID NOs: 4 to 6.

[0250] The sequences of SEQ ID NOs: 1-9 are provided in Table 4 below, and these and additional sequences are described below.

[0251] SEQ ID NO: 1: Exon 1, minus strand.

[0252] SEQ ID NO: 2: Exon 2, minus strand.

[0253] SEQ ID NO: 3: Exon 1, minus strand.

[0254] SEQ ID NO: 4: Exon 2, plus strand.

[0255] SEQ ID NO: 5: Exon 2, minus strand.

[0256] SEQ ID NO: 6: Exon 1, plus strand.

[0257] SEQ ID NO: 7: Exon 1, minus strand.

[0258] SEQ ID NO: 8: Exon 1, plus strand.

[0259] SEQ ID NO: 9: Exon 1, plus strand.

[0260] SEQ ID NO: 10: Coding sequence of human ICAM1.

[0261] SEQ ID NO: 11: Coding sequence of human CEACAM1.

[0262] SEQ ID NO: 12: Coding sequence of human B2M.

[0263] SEQ ID NO: 13: Coding sequence of human CADM1.

[0264] SEQ ID NO: 14: Coding sequence of human CD58.

[0265] SEQ ID NO: 15: Coding sequence of human CD155.

[0266] Target sequences for CRISPR / Cas9 cleavage containing a PAM (NGG) are shown in the table below.

[0267] [Table 3]

[0268] (a) a first genomic modification in which the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell by contacting the cell with a Cas protein, a nucleic acid encoding a Cas protein, or a ribonucleic acid comprising any one of the sequences set forth in SEQ ID NOs: 1 to 3; (b) a second genomic modification in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell by contacting the cell with a Cas protein, a nucleic acid encoding a Cas protein, or a ribonucleic acid comprising any one of the sequences set forth in SEQ ID NOs: 4 to 6; and (c) a second genomic modification in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell by contacting the cell with a Cas protein, a nucleic acid encoding a Cas protein, or a ribonucleic acid comprising any one of the sequences set forth in SEQ ID NOs: 4 to 6. and a third genomic modification in which the CEACAM1 gene has been edited to reduce or eliminate CEACAM1 surface expression and / or activity in the cells by contacting the cells with a nucleic acid encoding the CEACAM1 gene or a ribonucleic acid comprising any one of the sequences of SEQ ID NOs: 7 to 9.

[0269] A method for alleviating hypoglycemia, comprising: a) administering to a subject in need of a transplant an effective amount of a graft comprising a pancreatic endoderm cell population, wherein the function of at least one HLA class I cell surface protein and at least one NK cell activating ligand cell surface protein is disrupted, and the pancreatic endoderm cell population matures in vivo and produces insulin in response to glucose stimulation in vivo, thereby alleviating hypoglycemia in the patient.

[0270] A method for reducing insulin dependence, comprising: a) administering to a subject in need of a transplant an effective amount of a graft comprising a pancreatic endoderm cell population, wherein the function of at least one HLA class I cell surface protein and at least one NK cell activating ligand cell surface protein is disrupted, and the pancreatic endoderm cell population matures in vivo and produces insulin in response to glucose stimulation in vivo, thereby reducing insulin dependence in the patient.

[0271] definition "Hypoimmunogenic" or "universal donor cells" or "mutated cells" or equivalents thereof refer to cells in which expression of at least one HLA class I cell surface protein and at least one NK-activating ligand has been reduced or eliminated. Such cells are expected to be less prone to immune rejection or graft rejection by a subject into which such cells or a graft is transplanted. For example, relative to unmodified wild-type cells, such hypoimmunogenic cells may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less prone to immune rejection by a subject into which such cells are transplanted.

[0272] The terms "treat" or "cure" or their equivalents refer to a therapeutic intervention that relieves (ameliorates) signs or symptoms.

[0273] The terms "patient" or "host" or "mammalian host" or "subject" or equivalents thereof refer to living multi-cellular vertebrates, a category that includes both human and non-human mammals. In some embodiments, the subject is a human subject. Preferred patients for treatment are humans. Target patient populations may change over time with clinical use / experience in a manner that is unrelated to the combination product itself, but rather is related to the nature of the immunosuppressive regimen, or lack thereof. For example, combination products may be used in T1D populations that achieve operational tolerance or use a hypoimmunogenic cell therapy in combination with an immunosuppressant drug (ISD) regimen with a low toxicity and side effect profile.

[0274] The term "blocking agent" as used herein refers to any agent that can bind to an NK-activating ligand on the surface of a target cell, including but not limited to, an antibody, or an agent that prevents or inhibits protein expression of an NK-activating ligand, including but not limited to, any protein, enzyme, or chemical now known or hereafter developed.

[0275] The term "antibody" as used herein is used in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological blocking activity.

[0276] The term "antibody fragment" as used herein refers to a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and the like. and multispecific antibodies formed from antibody fragments.

[0277] As used herein, the term "blocking antibody" refers to an antibody that, when bound to an NK cell-activating ligand on a target cell in vivo or in vitro, prevents or reduces the ability of an NK cell to lyse the target cell.

[0278] As used herein, the terms "syngenic" or "syngeneic" The term refers to cells, tissues, or organs that are genetically identical to, or derived from a source that is genetically identical to, the transplant recipient (e.g., an identical twin), particularly with respect to antigens or immunological responses. Such cells, tissues, or organs are called syngeneic transplants. As used herein, the terms "allogenic" or "allogeneic" refer to cells, tissues, or organs that are genetically identical to, or derived from a source that is genetically identical to, the transplant recipient (e.g., an identical twin), particularly with respect to antigens or immunological responses. Refers to cells, tissues, or organs derived from a source that is not genetically identical to, or genetically identical to, the transplant recipient (e.g., an unrelated donor) with respect to the immunological response. Such cells, tissues, or organs are called allografts, allogeneic transplants, homografts, or allotransplants. can be.

[0279] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be the core promoter sequence, and in other cases, this sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0280] The terms "effective amount" or "therapeutically effective amount" or their equivalents refer to an amount of an agent sufficient to achieve a desired effect in a treated subject or cell. For example, this may be the amount of cells necessary to inhibit or measurably reduce blood glucose levels, ultimately achieving homeostatic glycemic control. It may also refer to an effective amount of an agent to alter the function or structure of a cell or subject. A therapeutically effective amount of an agent may be administered in a single dose or in several doses. However, the effective amount depends on the particular agent applied, the subject being treated, the severity and type of affliction, and the method of administration.

[0281] As used herein, the terms "decrease," "destroyed," and "reduced" are used interchangeably. The terms "," "reduce," and "inhibit" are all used to mean a decrease generally, and specifically a decrease by a statistically significant amount. However, for the avoidance of doubt, "reduce," "reduced," "reduction," and "inhibition" include a decrease of at least 10% compared to a reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% (i.e., absent levels compared to a reference sample), including 100%, or any decrease from 10 to 100% compared to a reference level, or at least about a 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold or more decrease compared to a reference level.

[0282] As used herein, "increased," "increase," or " The terms "enhance," "enhance," or "activate" are all generally used to mean an increase by a statically significant amount. For the avoidance of doubt, the terms "increased," "increased" and "activate" are also used. The terms "activate," "enhance," or "activate" refer to an increase of at least 10% compared to a reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100%, including 100%, or any increase from 10 to 100% compared to a reference level, or at least about a 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase compared to a reference level, or any increase from 2-fold to 10-fold or more compared to a reference level.

[0283] The term "statistically significant" or "significantly" refers to statistical significance and generally means two standard deviations (2SD) below the concentration of a substandard reference. The term can also mean two standard deviations (2SD) or more above the concentration of a substandard reference. The term refers to statistical evidence of a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. This decision is often made using a p-value.

[0284] As used herein, "reduced hypoglycemia" or its equivalent means a reduction in the number of hypoglycemic episodes without worsening glycemic control, and a reduction in Hb levels of 0.2% or greater. Defined as an increase in A1c.

[0285] As used herein, "reduced insulin dependence" or its equivalent means a reduction in the number and / or dose of exogenous insulin injections without worsening glycemic control, defined as an increase in HbA1c of 0.2% or more.

[0286] As used herein, "tissue capsule" or its equivalent refers to a foreign body capsule that forms around an implant or graft. The cell-containing combination product and / or device or perforated device is intended to be retained within the capsule for the duration of implantation.

[0287] "Engraftment" or its equivalents refers to the differentiation of a progenitor or immature cell population into a mature cell type, for example, the engraftment of a PDX1-positive pancreatic endoderm cell population that matures into a pancreatic endocrine cell population.

[0288] "Graft" refers to a differentiated cell population encapsulated or delivered in a device herein, including, but not limited to, pancreatic endoderm, pancreatic progenitor cells, PDX-1 positive pancreatic endoderm, pancreatic endocrine precursors, pancreatic endocrine, single or multihormone endocrine, pre-beta, beta, and / or insulin secreting grafts.

[0289] The terms "essentially" or "substantially" or their equivalents refer to the majority, de minimus, or reduced amount of a component or cell present in any cell population or culture; for example, immature beta cell culture refers to "essentially or substantially immature beta cells that express INS, NKX6.1, and PDX1, and do not essentially or substantially express NGN3." Other examples include, but are not limited to, "essentially or substantially hES cells," "essentially or substantially definitive endoderm cells," "essentially or substantially foregut endoderm cells," "essentially or substantially PDX1-negative foregut endoderm cells," "essentially or substantially PDX1-positive pancreatic endoderm cells," "essentially or substantially pancreatic endocrine precursor cells," "essentially or substantially pancreatic endocrine cells," and the like.

[0290] With respect to cells in a cell culture or cell population, the term "substantially free" or its equivalent means that the cell culture or cell population is free of a particular cell type present in the cell culture or cell population. This means that the nucleoside analog is present in an amount of less than about 10%, 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.

[0291] The term "nonwoven" or its equivalents includes fabrics that are not made by weaving or knitting. These include, but are not limited to, bonded fabrics, formed fabrics, or engineered fabrics that are manufactured by other processes.

[0292] It is to be understood that the invention disclosed herein is not limited in its application to the details set forth or illustrated in the detailed description. The invention encompasses other embodiments and can be practiced or carried out in various ways. It is also to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0293] While certain compositions, methods, and assays of the present invention have been specifically described according to particular embodiments, the following examples are intended only to serve to illustrate, but not to limit, the methods and compositions of the present invention.

[0294] As used in the specification and claims, the articles "a" and "an" should be understood to include plural referents unless clearly indicated to the contrary. Where one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, a claim or detailed description including "or" between one or more group members is deemed satisfied unless clearly indicated to the contrary or otherwise apparent from the context. The invention includes embodiments in which exactly one group member is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one or all group members are present in, employed in, or otherwise relevant to a given product or process.

[0295] Furthermore, it should be understood that the present invention encompasses all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the enumerated claims, as introduced into another claim that depends from the same base claim (or other related claims), unless otherwise indicated or unless a contradiction or inconsistency would be apparent to one skilled in the art. Where elements are presented as enumerations (e.g., Markush groups or similar formats), it should be understood that each subgroup of elements is also disclosed, and that any element may be excluded from the group. Generally, when the invention or aspects of the invention are referred to as including certain elements, features, etc., it should be understood that a particular embodiment of the invention or aspect of the invention consists of, or consists essentially of, such elements, features, etc. For the sake of brevity, those embodiments have not been specifically described in numerous terms in all instances herein. It should also be understood that any embodiment or aspect of the invention may be explicitly excluded from the scope of the claims, regardless of whether a specific exclusion is set forth herein. Publications and other reference materials referred to herein to describe the background of the invention and to provide further details regarding its practice are incorporated herein by reference. The following non-limiting examples illustrate the present disclosure. [Example]

[0296] Example 1: Generation of B2M-deficient hES cells B2M-deficient hES cells were generated using the CyT49 cell line, although any human pluripotent stem cell line can be used. Targeted disruption of the B2M gene generated cells that do not express HLA class I proteins on the cell surface. Both alleles of the B2M locus in the CyT49 hESC line were transfected with CRISP technology using known techniques outlined in PCT Publication No. WO 2016183041A, which is incorporated herein in its entirety. It was disrupted using R / Cas9 technology. However, other nucleases, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), can be used to perform gene editing and conventional homologous recombination. Examples of published sequences of B2M are provided as SEQ ID NOs: 1, 2, and 3. A catalytically inactive Cas9 protein fused to a dual guide RNA and FokI nuclease NEXTGEN™ CRISPR (Transposagen Inc., Lexington, KY) was used to edit the gene. Plasmids containing guide RNA and Cas9 were electroporated into CyT49 hESCs, and the cells were seeded onto tissue culture plates. 12 days after electroporation, cells were sorted by fluorescence-activated cell sorting (FACS) for negative reactivity to B2M antibody (BioLegend catalog #316306). Sorted cells were plated at clonal density. Individual clones were picked and plated at approximately day 25. Clones were expanded and cryopreserved. Expanded clones that showed a normal karyotype by G-banding and negative for B2M protein expression and HLA class I protein surface expression by flow cytometry and / or immunofluorescence were selected for further experiments.

[0297] B2M surface expression in wild-type (WT) and knockout (KO) cells was assessed by flow cytometry under normal and inflammatory conditions (after exposure to interferon (IFN)-γ). See Figure 2A: normal: untreated growth medium (line B); inflammatory: exposure to 100 ng / mL IFN-γ for 18–24 hours (line A). The inflammatory response occurs in association with tissue trauma, resulting in the release of proinflammatory cytokines, some of which are IL-1-α, IL-1-β, TNF-α, IL-6, IL-8, and IFN-γ. While WT and B2M- / - ESCs and PECs were treated with IFN-γ, these observations can be extended to other cytokines as well.

[0298] Figure 2A shows B2M expression in WT hES cells without IFN-γ (line B) and after exposure of WT hES cells with IFN-γ (line A). The shift (increase) in fluorescence intensity of untreated WT hES cells (line B) compared to the background (shaded area) indicates that WT hES cells express B2M. The further shift (increase) in fluorescence intensity over WT B2M expression after exposure of WT hES cells to IFN-γ (line B) suggests that B2M expression increases in WT hES cells after exposure to IFN-γ (line A). Thus, HLA class I cell surface protein expression is upregulated by cellular stress and inflammation, at least that caused by IFN-γ treatment.

[0299] Figure 2B shows B2M expression in B2M knockout hES cells, which were generated using the CRISPR / Cas system. With or without exposure to IFN-γ, there was no substantial shift (increase) in fluorescence intensity compared to background (shaded area), suggesting that B2M knockout hES cells reduced or eliminated B2M surface expression and that B2M expression could not be induced by IFN-γ treatment. In such B2M knockouts, HLA class I cell surface protein expression is not upregulated by cellular stress and inflammation caused by IFN-γ treatment.

[0300] This example demonstrates that B2M knockout hES cells have reduced or eliminated B2M surface expression, as shown using a B2M antibody.

[0301] Example 2: Analysis of HLA class I cell surface protein expression in WT and B2M-deficient cells Wild-type and B2M knockout hES cells were then transfected with Pan-HLA-ABC monoclonal antibodies. Analysis using a monoclonal antibody (BD Pharmingen, catalog #560169) confirmed that these knockout cells do not express HLA class I proteins on the cell surface. Pan-HLA-ABC antibodies react with human major histocompatibility complex (MHC) class I proteins HLA-A, HLA-B, and HLA-C. Expression of pan-HLA-ABC antibodies was assessed by flow cytometry in wild-type and knockout cells under normal and inflammatory conditions after exposure to IFN-γ. Normal: no IFN-γ (line B); Inflammation: exposure to 100 ng / mL IFN-γ for 18–24 hours (line A).

[0302] Figure 3A shows Pan-HLA-ABC cell surface protein expression in WT hES cells (line B) and after treatment of WT hES cells with IFN-γ (line A). The change (increase) in fluorescence intensity of untreated WT hES cells (line B) compared to the background (shaded area) suggests that WT hES cells express Pan-HLA-ABC. The shift (increase) in fluorescence intensity over WT hES expression after exposure to IFN-γ suggests increased Pan-HLA-ABC expression in WT hES cells after exposure to IFN-γ.

[0303] Figure 3B shows Pan-HLA-ABC cell surface protein expression in B2M knockout hES cells using the CRISPR / Cas system. There was no shift (increase) in fluorescence intensity compared to background (shaded area) with or without exposure to IFN-γ, suggesting that the knockout reduced or eliminated HLA class I cell surface expression and that HLA class I protein expression was not induced by IFN-γ treatment.

[0304] This example demonstrates that B2M knockout hES cells have reduced or eliminated HLA class I cell surface expression, as shown using a Pan-HLA-ABC antibody.

[0305] Example 3: Differentiation of B2M-deficient cells into pancreatic lineage cells B2M knockout hES cells were grown using a scalable system for production of B2M knockout hES cells. The cells were cultured, passaged, and expanded under the same conditions as WT hES cells, as described in "Functional Pancreatic Progenitors from Human Embryonic Stem Cells PLoS One 7:5 1-17 (2012)" and U.S. Patent No. 8,895,300, both of which are incorporated by reference in their entireties. Specifically, Schulz et al. describe the expansion and suspension-based differentiation of adherent hESCs.

[0306] Briefly, WT hES cells and B2M - / - hES cells were differentiated in suspension aggregates over a period of approximately 2 weeks (or 14 days) using a four-stage procedure to generate a population of pancreatic cell types, including pancreatic progenitor cells, endocrine precursor cells, and hormone-expressing cells, collectively referred to as pancreatic endoderm cells (PECs). Accutase was used to dissociate human ES cells, and single cells were aggregated in roller bottles. To initiate differentiation, aggregates were pooled into conical tubes, allowed to settle by gravity, and subsequently washed with growth factor-free Stage 1 medium (RPMI + 0.2% vol / vol FBS containing a 1:5000 dilution of insulin-transferrin-selenium (ITS)). Aggregates were again pelleted and then resuspended in day 1 medium consisting of RPMI + 0.2% vol / vol FBS containing a 1:5000 dilution of insulin-transferrin-selenium (ITS), activin A (100 ng / mL), and wnt3a (50 ng / mL) and dispensed at a density of 2 μL / mL into roller bottles. The roller bottles were then placed on a FlexiRoll digital cell roller (Argos Technologies). During the remainder of the differentiation process, the cultures were rotated at approximately 3 1 / 2 rpm and rotated every 1 / 2 hour as described in Table 2 below, adapted from Schulz et al. (2012) supra. A medium change was performed on day 1. Growth, passaging, and expansion of hES cells were essentially as described in U.S. Patent Nos. 7,964,402, 8,211,699, 8,334,138, 8,008,07, and 8,153,429. The standard production method used to generate human embryonic stem cell-derived pancreatic endoderm cells (PECs) is provided in Table 2 below.

[0307] [Table 4]

[0308] hESC Agg.: hESC aggregates; XF HA: DMEM / F12 with GlutaMAX supplemented with 10% v / v Xeno-free Knock Out 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); r0.2 FBS: RPMI1640 (Mediatech); 0.2% FBS (HyClone), 1× GlutaMAX-1 (Life Technologies), 1% v / v penicillin / streptomycin; ITS: Insulin-Transferrin-Selenium (Life Technologies) diluted 1:5000 or 1:1000; A100: 100 ng / mL recombinant human activin A (R&D Systems). 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).

[0309] Differentiated B2M− / − and WT PECs were analyzed using flow cytometry to determine the relative abundance of endocrine cells and pancreatic progenitor cells in the stage 4 population, as shown in Table 3.

[0310] [Table 5]

[0311] In all three clones, the relative levels of pancreatic endocrine cells, progenitor cells, PDX-1-only cells, and triple-negative cells in B2M- / - differentiated cells are substantially similar to those observed in WT cells (top row).

[0312] This example is for B2M - / - This indicates that hES cells are capable of differentiating along pancreatic lineages in a manner similar to that of WT hES cells.

[0313] Example 4: Analysis of B2M expression in WT and B2M-deficient pancreatic endoderm cells Wild-type and B2M knockout pancreatic endoderm cells (PECs) from Example 3 were then analyzed using flow cytometry without or with IFN-γ: no IFN-γ (line B); exposed to 100 ng / mL IFN-γ for 18-24 hours (line A).

[0314] Figure 4A shows B2M expression in WT PEC cells without IFN-γ (line B) and after treatment with IFN-γ (line A). The shift (increase) in fluorescence intensity of untreated WT PEC cells (line B) compared to the background (shaded area) indicates that WT PEC cells express B2M. The shift (increase) in fluorescence intensity over WT expression after exposure of WT PEC to IFN-γ (line A) indicates increased B2M expression in WT PEC cells after exposure to IFN-γ. That is, exposure to IFN-γ increases B2M expression in WT PEC.

[0315] Figure 4B shows B2M expression in PEC cells derived from B2M knockout hES cells. With or without exposure to IFN-γ, there was no shift (increase) in fluorescence intensity compared to background (shaded area), indicating that B2M knockout PECs had reduced or eliminated B2M cell surface expression, and that B2M expression could not be induced by IFN-γ treatment in PECs derived from B2M knockout hES cells.

[0316] This example demonstrates that PECs derived from hES cells in which B2M expression was modulated / eliminated had reduced or eliminated B2M surface expression.

[0317] Example 5: Analysis of HLA class I cell surface protein expression in WT and B2M knockout pancreatic endoderm cells Wild-type and B2M knockout PECs were analyzed for HLA class I cell surface expression using a Pan-HLA-ABC monoclonal antibody (BD Pharmingen, catalog #560169) as in Example 2. Expression was assessed 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).

[0318] Figure 5A shows HLA class I cell surface protein expression in WT PEC under untreated conditions (line B) and expression after treatment with IFN-γ (line A). The shift (increase) in fluorescence intensity of untreated WT PEC (line B) compared to the background (shaded area) indicates that WT PEC express HLA class I on the cell surface. The change (increase) in fluorescence intensity over WT PEC expression after exposure to IFN-γ suggests increased HLA class I cell surface expression in WT PEC cells after exposure to IFN-γ.

[0319] Figure 5B shows HLA class I cell surface protein expression in PECs derived from B2M knockout hES cells. There was no shift (increase) in fluorescence intensity compared to the background (shaded area) of PEC cells with or without exposure to IFN-γ, indicating that the knockout reduced or eliminated HLA surface expression and that HLA class I expression in B2M- / - PECs could not be induced by IFN-γ treatment.

[0320] Thus, in PEC cells in which B2M expression was modulated / eliminated, reduced or eliminated HLA class I cell surface expression was observed.

[0321] Example 6: Analysis of ICAM-1 cell surface protein expression in WT and B2M knockout HES cells To further define the effect of IFN-γ treatment on target cells, ICAM-1 expression in WT and B2M knockout hES cells was assessed by flow cytometry under two conditions: (1) untreated (line B) and (2) treated with 100 ng / mL IFN-γ for 18 to 24 hours (line A). ICAM-1 is required for several immunological functions, including antigen presentation on target cells, and is a known NK-activating ligand. In vivo, disruption of intercellular ICAM / LFA binding interactions by application of specific monoclonal antibodies ("mAbs"), i.e., anti-ICAM-1 or anti-LFA-1, can provide immunological benefits. 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 is a licensee of Milteny Biotec Inc. The ICAM-1 antibody obtained from Biosciences, Inc., catalog number 130-103-909, was used.

[0322] Figure 6A shows ICAM-1 protein expression on the cell surface of WT hES cells untreated (line B) and after treatment with IFN-γ (line A). The shift (increase) in fluorescence intensity of untreated WT hES cells (line B) compared to the background (shaded area) indicates that WT hES cells express ICAM-1 protein on the cell surface. The shift (increase) in fluorescence intensity over WT expression after exposure of WT hES cells to IFN-γ suggests that ICAM-1 expression is increased after exposure to IFN-γ.

[0323] Figure 6B shows ICAM-1 cell surface protein expression in B2M knockout hES cells untreated (line B) and after treatment with IFN-γ (line A). Figure 6B shows that ICAM-1 cell surface protein expression in B2M knockout hES cells was similar to that in WT hES cells.

[0324] This example demonstrates that treatment of WT and B2M knockout hES cells with IFN-γ increases cell surface protein expression of ICAM-1.

[0325] Example 7: Analysis of ICAM-1 cell surface protein expression in WT and B2M knockout pancreatic endoderm cells Wild-type and B2M knockout PECs were analyzed using an antibody against a known NK-activating ligand, e.g., ICAM-1, as in Examples 4 and 5. Cell surface protein expression of ICAM-1 was assessed by flow cytometry in WT and knockout PECs under two conditions: (1) untreated and (2) treated with 100 ng / mL IFN-γ for 18 to 24 hours.

[0326] Figure 7A shows ICAM-1 cell surface protein expression on WT PEC (line B) and after treatment of WT PEC with IFN-γ (line A). The shift (increase) in fluorescence intensity of untreated WT PEC (line B) compared to the background (shaded area) indicates that WT PEC express ICAM-1 protein on the cell surface. The further shift (increase) in fluorescence intensity over that of WT PEC after exposure to IFN-γ suggests that ICAM-1 expression is increased in WT PEC after exposure to IFN-γ. This indicates that NK-activating ligands, particularly ICAM-1, are highly inducible by IFN-γ stimulation.

[0327] Figure 7B shows ICAM-1 cell surface protein expression in B2M knockout PECs. ICAM-1 cell surface protein expression in B2M knockout PECs was similar to that in WT PECs, with or without IFN-γ exposure, and fluorescence was slightly reduced. Thus, treating B2M knockout PECs with IFN-γ increases ICAM-1 cell surface protein expression.

[0328] Figure 8 shows RNA expression array data (Affymetrix) demonstrating that exposure to IFN-γ increases ICAM-1 expression in WT hESCs, B2M- / - hESCs, WT PECs, and B2M- / - PECs at the mRNA level. Applicants discovered that cell surface protein expression of the NK-activating ligand ICAM-1 increased in differentiated cell types (WT PECs and B2M- / - PECs) after exposure to IFN-γ. Thus, ICAM-1 is highly inducible in PECs by IFN-γ stimulation.

[0329] Example 8: Effect of IFN-γ treatment on additional NK activating ligands To further characterize the effect of IFN-γ treatment on target cells, WT PECs were Cell surface protein expression of other known NK-activating ligands was analyzed by flow cytometry under two conditions: (1) untreated (line B) and (2) treated with 100 ng / mL IFN-γ for 18–24 hours (line A).

[0330] Figure 9 shows CD58 (also known as LFA-3) cell surface protein expression in WT PECs untreated (line B) and after treatment with IFN-γ (line A) using antibody from BioLegend, catalog #330909. The large shift (increase) in fluorescence intensity of untreated WT hES cells (line B) compared to background (line C) indicates that the majority of cells express CD58 protein on their surface. After exposure to IFN-γ, there was a further small shift (increase) in fluorescence intensity.

[0331] Figure 10 shows CD155 (also known as PVR, NECL-5, and HVED) cell surface protein expression in WT PECs untreated (line B) and after treatment with IFN-γ (line A) using antibody obtained from Milteneyi Biotech Inc., catalog number 130-105-905. The shift (increase) in fluorescence intensity of untreated WT PECs (line B) compared to background (line C) indicates that WT PECs express CD155. There was no further increase (increase) in fluorescence intensity above the untreated condition after exposure to IFN-γ, suggesting that CD155 expression is not increased in WT PECs after exposure to IFN-γ.

[0332] Figure 11 shows CEACAM1 (also known as CD66a, BGP, and BGP1) cell surface protein expression in WT PECs untreated (line B) and after treatment with IFN-γ (line A) using antibody, catalog #130-098-858, obtained from Milteneyi Biotech Inc. The shift (increase) in fluorescence intensity of untreated WT PECs (line B) compared to background (line C) indicates that WT PECs express CEACAM1 protein on the cell surface. The shift (increase) in fluorescence intensity over the untreated condition after exposure of WT PECs to IFN-γ suggests that CEACAM1 protein expression on the cell surface is increased in WT PECs after exposure to IFN-γ.

[0333] Figure 12 shows the BAT3 antibody obtained from Abcam Inc., catalog #ab210838, in untreated (line B) and WT PECs after treatment with IFNγ (line A). (Also known as BAG6) cell surface protein expression is shown. The change (increase) in fluorescence intensity of untreated PEC (line B) compared to background (line C) suggests that the cells express CEACAM1. There was no further shift (increase) in fluorescence intensity above the untreated condition after exposure of WT PEC to IFN-γ, suggesting that BAT3 expression is not increased in WT PEC after exposure to IFN-γ.

[0334] Figure 13 shows CADM1 (also known as NECL2, TSLC1, IGSF4, and RA175) cell surface protein expression in WT PECs untreated (line B) and after treatment with IFN-γ (line A) using antibody, catalog #CM004-4, obtained from MBL International Corporation. The shift (increase) in fluorescence intensity in the untreated condition (line B) compared to background (line C) suggests that WT PECs express CADM1 protein on the cell surface. There was no additional shift (increase) in fluorescence intensity above the untreated condition after exposure of PECs to IFN-γ, suggesting that CADM1 expression is not increased after exposure to IFN-γ.

[0335] Figure 14 shows the immunoreactivity of CD112 (also known as nectin-2, PVRR2, HVEB) in WT PECs untreated (line B) and after treatment with IFN-γ (line A) using antibody obtained from Milteneyi Biotech Inc., catalog #130-109-056. The graph shows the expression of CD112 protein. The shift (increase) in fluorescence intensity in the untreated condition (line B) compared to the background (line C) suggests that WT PECs express CD112 protein on the cell surface. There was no further shift (increase) in fluorescence intensity above the untreated condition after exposure of PECs to IFN-γ, suggesting that CD112 expression is not increased after exposure to IFN-γ.

[0336] Example 9: MHC class I-deficient, NK cell-activating ligand-deficient cells prevent NK cell-mediated lysis To test whether the combination of reduced or eliminated HLA class I expression and reduced or eliminated NK cell-activating ligand expression was sufficient to prevent NK-mediated cytolysis, ICAM-1 expression was blocked on target cells using an ICAM-1-blocking antibody at concentrations of 5 μg / mL and 10 μg / mL. Addition of the ICAM-1 antibody to WT or B2M- / - ES cells or PECs reduced NK lysis of target cells after IFN-γ treatment (Figure 15).

[0337] Staining of target cells with calcein-AM The calcein release assay is a non-radioactive alternative for studying NK cell cytotoxicity. Target cells take up a fluorescent dye (calcein AM) and convert it in the cytoplasm to an active fluorescent dye, which is released from the cells only upon lysis. Lysed cells release the fluorescent dye into the supernatant, which is then harvested and the amount of fluorescence quantified using a fluorometer. The percentage of cytolysis is calculated from the amount of fluorescence present in the supernatant after incubation in the presence or absence of NK cells (effectors), blocking antibodies, or both.

[0338] Target cells were WT ESCs, B2M, or IFN-γ-treated or not treated with 100 ng / mL IFN-γ before labeling. - / - ESC, WT PEC, or B2M - / - To prepare target cells, target cell populations were stained with 2 μg / ml calcein AM staining medium (Enzo biosciences 1 mg / mL stock solution (catalog #C3100MP)). Target cells were incubated for 1 hour at 37°C in an 8% CO2 incubator with intermittent mixing. Target cells were washed twice to remove any free calcein AM and then resuspended in RPMI complete medium (RPMI, 10% heat-inactivated FBS, and 1% antibiotics) at 1 x 10 5 cells / ml.

[0339] Co-culture target NK cells and blocking antibodies The calcein AM-labeled target cells were then incubated with NK cells (effector cells) at an effector-to-target ratio (E:T ratio) of 10:1. Specifically, 100 μL of NK cells (1 × 10) were added per well. 6 96-well V-bottom (density of 1000 cells / mL) Add 100 μL of calcein-stained ESC or PEC cells to the plate. (1×10 5Where indicated, a blocking antibody against the human ICAM-1 surface antigen (R&D Systems, Inc., catalog #BBA3) at concentrations of 5 μg / mL and 10 μg / mL was added to the wells to determine whether it could reduce NK-mediated cell lysis.

[0340] The plates were incubated for 4 hours at 37°C in an 8% CO2 incubator. After the incubation period, the plates were centrifuged at 200 x g for 2 minutes. 100 μL of supernatant was carefully removed and transferred to a black-colored 96-well plate, and fluorescence was measured using a Molecular Devices plate reader (excitation filter: 485 nm / emission filter: 530 nm). Specific lysis was calculated using the formula: % Lysis = 100 x [(mean fluorescence with antibody - mean spontaneous fluorescence) / (mean maximum fluorescence - mean spontaneous fluorescence)]. Maximum fluorescence was determined by lysis of cells incubated with detergent (1% Triton X-100), while spontaneous lysis was determined by lysis of target cells without antibody or effector cells. The fluorescence obtained was taken as

[0341] result As shown in Figure 1, the goal is to transition from scenario C (NK cells attacking target cells) to scenario A (no or reduced response). In Figure 15, this scenario is depicted in conditions 4 and 8. In conditions 4 and 8, target cells (B2M- / - hES cells or PECs) lack functional HLA class I surface expression and are exposed to IFN-γ. As discussed above and seen in conditions 4 and 8, exposure to IFN-γ causes an increase in the NK-activating ligand (ICAM-1), which results in greater cytolysis compared to HLA class I knockouts without IFN-γ exposure (compare the first bars in conditions 3 vs. 4 and 7 vs. 8). However, upon treatment with an ICAM-1-blocking antibody, which acts to block the expression of NK-activating signals on target cells, NK-mediated cytolysis is reduced from 83% to 73% in B2M- / - hES cells and from 72% to 61% in B2M- / - PECs treated with IFN-γ. The rate of NK-mediated cytolysis does not decrease to zero because ICAM-1 cell surface protein expression cannot be completely blocked using blocking antibodies, and as discussed above, target cells express more than one NK-activating ligand. The rate of NK-mediated cytolysis is expected to decrease more significantly if ICAM-1 expression is further inhibited and other NK-activating ligands are blocked on target cells. Therefore, ICAM-1 inhibitory antibodies can be combined with additional NK-activating ligand-inhibiting antibodies, including inhibitory antibodies against any of the ligands listed in Table 1, preferably category 1 (known activating ligands) and category 2 (potential candidate activating ligands identified from gene chip data, which are upregulated in PECs and / or ESCs after IFNγ). In one embodiment, the ICAM1 gene and other NK-activating ligand genes are disrupted to completely block their activity.

[0342] Scenario D in Figure 1 is shown in the first bar of conditions 2 and 6 in Figure 15. In conditions 2 and 6, WT hES cells and PECs upregulated cell surface protein expression of both HLA class I antigens and NK-activating ligands as a result of exposure to IFN-γ. When cells were incubated with an ICAM-1-blocking antibody, this represented a transition from scenario D to scenario B in Figure 1. When incubated with an ICAM-1-blocking antibody, cell death decreased from 79 to 60% in WT hES cells and from 53 to 27% in WT PECs exposed to IFN-γ. Indeed, when WT PECs were exposed to IFN-γ and incubated with an ICAM-1-blocking antibody, NK cell lysis was below that of untreated WT PECs: 27% in the case of WT PECs, IFN-γ, and an ICAM-1-blocking antibody, compared to 37% in the case of WT PECs and an ICAM-1-blocking antibody without IFN-γ.

[0343] Scenario B in Figure 1 is exemplified by the ICAM-1 antibody-treated bars in conditions 1, 2, 5, and 6 in Figure 15. Here, the target hES cells and PECs have HLA class I and NK-activating ligands, but when the target cells are not exposed to IFN-γ, there is no increase in cell surface protein expression of ICAM-1. As a result, the ICAM-1 blocking antibody is less effective. Thus, cell lysis remains roughly the same: 58% to 57% for hES cells and 33% to 37% for PEC cells.

[0344] Scenario C in Figure 1 corresponds to the first bar in conditions 3, 4, 7, and 8 in Figure 15. In these conditions, the cells lack HLA class I cell surface expression as a result of B2M- / -. Therefore, when NK-activating ligands are not activated by IFN-γ exposure of the cells, ICAM-1-blocking antibodies have little effect, 71% to 70% for hES cells and 54% to 57% for PECs.

[0345] As a general observation, NK-mediated cytolysis was observed in differentiated cell populations (WT PEC and B2M - / -In PEC, undifferentiated cell populations (WT hES and B2M) - / - NK-mediated cytolysis is significantly increased when cells (WT or B2M- / - knockout hES or PEC) are activated with IFN-γ.

[0346] Example 10: Generation of NK-activating ligand-deficient B2M− / − knockout hES cells Example 9 describes that inhibition or suppression of ICAM-1 expression protects IFN-γ-treated B2M- / - PECs from NK-mediated cell killing activity. Therefore, to protect B2M- / - PECs from NK cell-mediated killing after transplantation, it is desirable to disrupt the NK cell-activating ligand gene. For example, based on the above example, ICAM-1, a known NK-activating ligand gene, can be disrupted or "knocked out." Preferably, both alleles of the ICAM-1 locus in the B2M- / - CyT49 hESC line can be disrupted using CRISPR / Cas9 or other gene editing techniques now known or to become known in the future. See PCT Publication No. WO 2016183041A, which is incorporated herein by reference in its entirety. Exemplary published sequences of ICAM-1 are provided as SEQ ID NOS: 4, 5, and 6. For example, in one embodiment of the present invention, NEXTGEN™ CRISPR (Transposagen Inc., Lexington, KY), which introduces a catalytically inactive Cas9 protein fused to dual guide RNA and Fokl nuclease, is used to gene edit cells.

[0347] Plasmids containing guide RNA and Cas9 can be electroporated into B2M- / - CyT49 hESCs and plated onto tissue culture plates. After electroporation, cells can be sorted by flow cytometry for negative reactivity to ICAM-1 antibodies. Sorted cells can be plated at clonal density. Individual clones can be picked and replated. Clones can be expanded and cryopreserved. Expanded clones that show a normal karyotype by G-banding and negative for ICAM-1 protein expression and surface expression by flow cytometry and / or immunofluorescence can be selected for further experiments.

[0348] As described above, cells lacking ICAM1 and B2M are unable to express at least one NK-activating ligand and at least one or all MHC class I proteins on their cell surface, and therefore are unable to bind NK cell-activating receptors and are protected from NK-mediated cell death.

[0349] Example 11: Liver function of B2M- / - knockout hES cells lacking multiple NK-activating ligands Growth Examples 9 and 10 demonstrate that genetic disruption of an NK cell-activating ligand (e.g., ICAM1- / -) combined with inhibition of functional cell surface expression (anti-NK activating ligand) and disruption of MHC class I cell surface expression (e.g., B2M- / -) can protect target cells from NK-mediated cell death. Transplantation of cells deficient in two or more NK cell-activating ligands can be performed to further protect from NK-mediated cell death.

[0350] The CD58 gene is chosen as the NK-activating ligand gene to knock out. Both alleles of the CD58 locus can be disrupted in the B2M- / -:ICAM- / - double knockout CyT49 hESC line using CRISPR / Cas9 technology using known techniques outlined in International Publication No. 2016183041A. Examples of published sequences for CEACAM1 are provided as SEQ ID NOs: 7, 8, and 9. Again, edited versions can be generated using NEXTGEN™ CRISPR (Transpeptide-binding protein). osagen Inc., Lexington, KY).

[0351] Plasmids containing guide RNA and Cas9 can be electroporated into B2M- / -, ICAM- / - knockout CyT49 hESCs and plated onto tissue culture plates. After electroporation, cells can be sorted by flow cytometry for negative reactivity to CD58 antibodies. Sorted cells can be plated at clonal density. Individual clones can be picked and replated. Clones can be expanded and cryopreserved. Expanded clones that show a normal karyotype by G-banding and negative for CD58 protein expression and surface expression by flow cytometry and / or immunofluorescence can be selected for further experiments.

[0352] As described above, cells with disrupted, deleted, or altered ICAM1, CD58, and B2M cannot express ICAM1, CD58, or MHC class I proteins on the cell surface and therefore cannot bind to NK cell activating receptors and are protected from NK-mediated cell death.

[0353] The CD155 (also known as PVR) gene can also be selected as the NK-activating ligand gene to be knocked out. CD155 can be selected in addition to or instead of CD58. As described above, the CD28 gene can be disrupted using CRISPR / Cas9 technology. The CD155 gene can also be selected as the NK-activating ligand gene to be knocked out. CD155 can be selected in addition to or instead of CD58. The CAECAM1 gene can also be selected as the NK-activating ligand gene to be knocked out. CAECAM1 can be selected in addition to or instead of CD58 and / or CD155.

[0354] Example 12: Inactivation of NK activating ligands ICAM1 and CD58

[0355] To completely eliminate NK activation, it may be necessary to eliminate / reduce multiple NK-activating ligands by gene knockout in target cells (e.g., hES cell-derived cell therapy) or by using blocking antibodies or other strategies currently known or developed in the future. To determine whether NK cytotoxicity can be reduced by inhibiting the effects of NK-activating ligands on target cells, the expression of NK-activating ligands in WT and B2M- / - hES cells and PECs can be blocked using ICAM1 and CD58-blocking antibodies to block ICAM1 and CD58 proteins on the target cell surface. Cytolysis of target cells can be expected to be reduced (similar to Figure 15). Therefore, cytolysis by NK cells can be reduced by blocking NK-activating ligands such as ICAM1 and CD58. Blocking the expression of ICAM1 and CD58 in B2M- / - cells using antibodies against NK-activating ligands is a proof-of-concept for generating cells with triple knockouts (HLA class I gene knockout and NK-activating ligand gene knockout). In doing so, the target cells (e.g., hES and / or pancreatic lineage cells) transition from scenario C to scenario A in Figure 1. Specifically, the cells, tissues, and organs of the present invention have inhibited or absent HLA class I cell surface protein expression (B2M- / -) and inhibited or absent NK activating ligand cell surface protein expression (e.g., ICAM1- / - and CD58- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or by using antibodies to block protein expression. Other strategies for interfering with cell surface protein expression include antisense RNA, RNA decoys, ribozymes, RNA aptamers, siRNA, shRNA / miRNA, transdominant These include the use of tumor negative proteins (TNPs), chimeric / fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFv), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors. Multiple gene knockouts effectively prevent both cytotoxic T cell (CTL)- and NK cell-mediated killing because there is little or no HLA class I expressed on the cell surface and little or no NK activating ligand protein for CTL or NK cells to bind.

[0356] Example 13: Inactivation of NK-activating ligands CADM1 and CD58 To completely eliminate NK activation, it may be necessary to eliminate / reduce multiple NK-activating ligands by gene knockout in target cells (e.g., hES cell-derived cell therapy) or by using blocking antibodies or other strategies currently known or developed in the future. To determine whether NK cytotoxicity can be reduced by inhibiting the effects of NK-activating ligands on target cells, we can block the expression of NK-activating ligands in WT and B2M- / - hES cells and PECs using CADM1 and CD58-blocking antibodies to block CADM1 and CD58 proteins on the target cell surface. Cytolysis of target cells can be expected to be reduced (similar to Figure 15). Therefore, cytolysis by NK cells can be reduced by blocking NK-activating ligands such as CADM1 and CD58. Blocking the expression of CADM1 and CD58 in B2M- / - cells using antibodies against NK-activating ligands is a proof-of-concept for generating cells with triple knockouts (HLA class I gene knockout and NK-activating ligand gene knockout). In doing so, the target cells (e.g., hES and / or pancreatic lineage cells) transition from scenario C to scenario A in Figure 1. Specifically, the cells, tissues, and organs of the present invention have inhibited or absent HLA class I cell surface protein expression (B2M- / -) and inhibited or absent NK-activating ligand cell surface protein expression (e.g., CADM1- / - and CD58- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or by using antibodies to block protein expression. Other strategies to interfere with cell surface protein expression include the use of antisense RNA, RNA decoys, ribozymes, RNA aptamers, siRNA, shRNA / miRNA, transdominant negative proteins (TNPs), chimeric / fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFv), nucleoside analogues (NRTIs), non-nucleoside analogues (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors.The multiple gene knockout effectively prevents both cytotoxic T cell (CTL)- and NK cell-mediated killing because there is little or no HLA class I expressed on the cell surface and little or no NK activating ligand protein for CTL or NK cells to bind.

[0357] Example 14: Inactivation of NK-activating ligands CD155 and CD58 To completely eliminate NK activation, it may be necessary to eliminate / reduce multiple NK-activating ligands by gene knockout in target cells (e.g., hES cell-derived cell therapy) or by using blocking antibodies or other strategies currently known or developed in the future. To determine whether NK cytotoxicity can be reduced by inhibiting the effects of NK-activating ligands on target cells, the expression of NK-activating ligands in WT and B2M- / - hES cells and PECs can be blocked using CD155 and CD58 blocking antibodies to block CD155 and CD58 proteins on the target cell surface. Target cell cytolysis can be expected to be reduced (similar to Figure 15). Therefore, cytolysis by NK cells is dependent on NK-activating ligands such as CD155 and CD58. Blocking expression of CD155 and CD58 using antibodies against NK-activating ligands in B2M- / - cells is a proof-of-concept for generating cells with a triple knockout (HLA class I gene knockout and NK-activating ligand gene knockout). In doing so, the target cells (e.g., hES and / or pancreatic lineage cells) transition from scenario C to scenario A in Figure 1. Specifically, the cells, tissues, and organs of the present invention have inhibited or absent HLA class I cell surface protein expression (B2M- / -) and inhibited or absent NK-activating ligand cell surface protein expression (e.g., CD155- / - and CD58- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or by using antibodies to block protein expression. Other strategies to disrupt cell surface protein expression include the use of antisense RNA, RNA decoys, ribozymes, RNA aptamers, siRNA, shRNA / miRNA, transdominant-negative proteins (TNPs), chimeric / fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFv), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors. Multiple gene knockouts effectively prevent both cytotoxic T cell (CTL)- and NK cell-mediated damage because there is little or no HLA class I expressed on the cell surface and little or no NK activating ligand protein for CTL or NK cells to bind.

[0358] Example 15: Inactivation of NK-activating ligands ICAM1, CD155, and CD58 To completely eliminate NK activation, it may be necessary to eliminate / reduce multiple NK-activating ligands by gene knockout in target cells (e.g., hES cell-derived cell therapy) or by using blocking antibodies or other strategies currently known or developed in the future. To determine whether NK cytotoxicity can be reduced by inhibiting the effects of NK-activating ligands on target cells, the expression of NK-activating ligands in WT and B2M- / - hES cells and PECs can be blocked using ICAM1, CD155, and CD58-blocking antibodies to block ICAM1, CD155, and CD58 proteins on the target cell surface. Cytolysis of target cells can be expected to be reduced (similar to Figure 15). Therefore, cytolysis by NK cells can be reduced by blocking NK-activating ligands such as ICAM1, CD58, and CD155. Blocking the expression of ICAM1, CD58, and CD155 in B2M- / - cells using antibodies against NK-activating ligands is a proof-of-concept for generating cells with four knockouts (HLA class I gene knockout and NK-activating ligand gene knockout). In doing so, the target cells (e.g., hES and / or pancreatic lineage cells) transition from scenario C to scenario A in Figure 1. Specifically, the cells, tissues, and organs of the present invention have inhibited or absent HLA class I cell surface protein expression (B2M- / -) and inhibited or absent NK activating ligand cell surface protein expression (e.g., ICAM1- / -, CD58- / -, and CD155- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or by using antibodies to block protein expression.Other strategies to disrupt cell surface protein expression include the use of antisense RNA, RNA decoys, ribozymes, RNA aptamers, siRNA, shRNA / miRNA, transdominant-negative proteins (TNPs), chimeric / fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFv), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors. Multiple gene knockouts effectively prevent both cytotoxic T cell (CTL)- and NK cell-mediated damage, but require the expression of proteins on the cell surface for CTL or NK cell binding. This is because they have little or no HLA class I receptors and little or no NK activating ligand proteins.

[0359] Example 16: Inactivation of NK-activating ligands ICAM1, CD155, CD58, and CADM1 To completely eliminate NK activation, it may be necessary to eliminate / reduce multiple NK-activating ligands by gene knockout in target cells (e.g., hES cell-derived cell therapy) or by using blocking antibodies or other strategies currently known or developed in the future. To determine whether NK cytotoxicity can be reduced by inhibiting the effects of NK-activating ligands on target cells, the expression of NK-activating ligands in WT and B2M- / - hES cells and PECs can be blocked using ICAM1, CD155, CD58, and CADM1 blocking antibodies to block ICAM1, CD155, CD58, and CADM1 proteins on the target cell surface. Cytolysis of target cells can be expected to be reduced (similar to Figure 15). Therefore, cytolysis 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 in B2M- / - cells using antibodies against NK-activating ligands is a proof-of-concept for generating cells with five knockouts (HLA class I gene knockout and NK-activating ligand gene knockout). In doing so, the target cells (e.g., hES and / or pancreatic lineage cells) transition from scenario C to scenario A in Figure 1. Specifically, the cells, tissues, and organs of the present invention have inhibited or absent HLA class I cell surface protein expression (B2M- / -) and inhibited or absent NK-activating ligand cell surface protein expression (e.g., ICAM1- / -, CD58- / -, CD155- / -, and CADM1- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or by blocking protein expression using antibodies.Other strategies to disrupt cell surface protein expression include the use of antisense RNA, RNA decoys, ribozymes, RNA aptamers, siRNA, shRNA / miRNA, transdominant-negative proteins (TNPs), chimeric / fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFv), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors. Multiple gene knockouts effectively prevent both cytotoxic T cell (CTL)- and NK cell-mediated damage because there is little or no HLA class I expressed on the cell surface and little or no NK activating ligand protein for CTL or NK cells to bind.

[0360] Example 17: Inactivation of NK activating ligands ICAM1, CD155, and CADM1 To completely eliminate NK activation, it may be necessary to eliminate / reduce multiple NK-activating ligands by gene knockout in target cells (e.g., hES cell-derived cell therapy) or by using blocking antibodies or other strategies currently known or developed in the future. To determine whether NK cytotoxicity can be reduced by inhibiting the effects of NK-activating ligands on target cells, the expression of NK-activating ligands in WT and B2M- / - hES cells and PECs can be blocked using ICAM1, CD155, and CADM1 blocking antibodies to block ICAM1, CD155, and CADM1 proteins on the target cell surface. Cytolysis of target cells can be expected to be reduced (similar to Figure 15). Therefore, cytolysis by NK cells can be reduced by blocking NK-activating ligands such as ICAM1, CD155, and CADM1. Using antibodies against NK-activating ligands in B2M- / - cells, Blocking the expression of ICAM1, CD155, and CADM1 is a proof-of-concept for generating cells with four knockouts (HLA class I gene knockout and NK-activating ligand gene knockout). In doing so, target cells (e.g., hES and / or pancreatic lineage cells) transition from scenario C to scenario A in Figure 1. Specifically, the cells, tissues, and organs of the present invention have inhibited or absent HLA class I cell surface protein expression (B2M- / -) and inhibited or absent NK-activating ligand cell surface protein expression (e.g., ICAM1- / -, CD155- / -, and CADM1- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or by blocking protein expression using antibodies. Other strategies to disrupt cell surface protein expression include the use of antisense RNA, RNA decoys, ribozymes, RNA aptamers, siRNA, shRNA / miRNA, transdominant-negative proteins (TNPs), chimeric / fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFv), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors. Multiple gene knockouts effectively prevent both cytotoxic T cell (CTL)- and NK cell-mediated damage because there is little or no HLA class I expressed on the cell surface and little or no NK activating ligand protein for CTL or NK cells to bind.

[0361] Blocking antibodies useful in the present invention are as follows: CADM1 antibody, clone 9D2, Creative Biolabs, catalog #BRD-0007MZ; CD155 antibody, clone SKII.4, Biolegend, catalog #337602; and CD58 antibody, clone TS2 / 9, Biolegend, catalog #330911.

Claims

1. A human in vitro endocrine cell population, wherein the function of at least one major histocompatibility complex (MHC) class I gene and at least one natural killer (NK) cell-activating ligand has been disrupted or inhibited, and binding of the NK cell-activating ligand to an NK cell-activating receptor in the human in vitro endocrine cell population is reduced, and the NK cell-activating ligand comprises intercellular adhesion molecule 1 (ICAM1), CD58, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), or a combination thereof; and the human in vitro endocrine cell population is an insulin-producing cell population.

2. 2. The human in vitro endocrine cell population of claim 1, wherein the MHC class I gene encodes beta-2 microglobulin (B2M) or human leukocyte antigen (HLA)-ABC cell surface protein.

3. The human in vitro endocrine cell population of claim 1 or 2, wherein the NK cell activating ligand further comprises CD155, cell adhesion molecule 1 (CADM1), major histocompatibility class I-related chain protein A (MICA), major histocompatibility class I-related chain protein B (MICB), or a combination thereof.

4. The human in vitro endocrine cell population according to claim 1 or 2, wherein the NK cell activating ligands are ICAM1 and CD58.

5. 3. The human in vitro endocrine cell population of claim 1 or 2, wherein the NK cell activating ligands are ICAM1, CD58, CD155, CEACAM1, CADM1, MICA, and MICB.

6. The human in vitro endocrine cell population of claim 1 or 2, wherein the NK cell activating ligands are ICAM1, CD58, and CD155.

7. The human in vitro endocrine cell population of claim 1 or 2, wherein the NK cell activating ligands are ICAM1, CD58, CD155, and CADM1.

8. The human in vitro endocrine cell population of claim 1 or 2, wherein the NK cell activating ligands are CD58 and CADM1.

9. The human in vitro endocrine cell population of claim 1 or 2, wherein the NK cell activating ligands are ICAM1, CADM1, and CD155.

10. The human in vitro endocrine cell population of claim 1 or 2, wherein the NK cell activating ligand is CD58.

11. The human in vitro endocrine cell population according to claim 1 or 2, wherein the NK cell activating ligand is CEACAM1.

12. The human in vitro endocrine cell population of claim 1 or 2, wherein the NK cell activating ligand is ICAM1.

13. The human in vitro endocrine cell population of any one of claims 1 to 12, further comprising a protein that, when expressed in the presence of a cell death-inducing agent, enables the agent to kill cells in the cell population.

14. 14. The human in vitro endocrine cell population of claim 13, wherein the protein is herpes simplex virus thymidine kinase and the cell death inducer is ganciclovir.

15. A composition comprising a human in vitro endocrine cell population according to any one of claims 1 to 14, further comprising an anti-NK cell activating ligand agent.

16. The composition of claim 15 , wherein the anti-NK cell activating ligand agent is an antibody or an antibody fragment.

17. The human in vitro endocrine cell population according to any one of claims 1 to 14, wherein the human in vitro endocrine cell population has been further modified to increase the expression of one or more immune tolerance factors.

18. 18. The human in vitro endocrine cell population of claim 17, wherein the immune tolerance factor is selected from the group consisting of HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, and IL-35.

19. The human in vitro endocrine cell population of any one of claims 1 to 14, 17 and 18, wherein the human in vitro endocrine cell population is contained in a perforated cell delivery device.

20. Use of the human in vitro endocrine cell population according to any one of claims 1 to 14, 17 and 18, or a perforated cell delivery device comprising said human in vitro endocrine cell population, in the manufacture of a composition for treating type I diabetes.

21. Use of a human in vitro endocrine cell population according to any one of claims 1 to 14, 17 and 18, or a perforated cell delivery device comprising said human in vitro endocrine cell population, in the manufacture of a composition for treating type I diabetes comprising a human insulin-secreting cell population.

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