Universal donor cells and related methods

Abstract

Disclosed herein are universal donor stem cells and cells derived therefrom and related methods of use and production thereof. The universal donor stem cells disclosed herein can be used to overcome alloimmune rejection in cell transplantation-based therapies. 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, the expression of at least one NK activating ligand of which is disrupted or inhibited.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. utility model application No. 15 / 648,337, filed on July 12, 2017, entitled UNIVERSAL DONOR CELLS AND RELATEDMETHODS, which is incorporated herein by reference in its entirety.

[0003] field

[0004] This article relates to the fields of gene expression, genome engineering, and gene / cell therapy. background

[0005] Human pluripotent stem cells (hPSC) are a useful tool for generating any type of adult cell for transplantation into a patient. In principle, hPSC-based cell therapy has the potential to treat most, if not all, degenerative diseases, however, the success of such therapies may be limited by the subject's immune response. The immune system protects organisms from infection with layered defenses that increase specificity. In short, physical barriers prevent pathogens such as bacteria and viruses from entering the organism. If the pathogen breaks through these barriers, the innate immune system provides an immediate but non-specific response. If the pathogen successfully escapes the innate response, the vertebrate has a second layer of protection, the adaptive immune system activated by the innate response. The adaptive immune system produces a more specific response. At this point, the immune system adapts its response to improve its recognition of pathogens during infection. After the pathogen is eliminated, this improved response is retained in the form of immune memory and enables the adaptive immune system to launch faster and stronger attacks 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 the generation of responses adjusted to specific pathogens or pathogen-infected cells. Interferon gamma (IFN-γ) plays a vital role in fighting infectious and non-infectious diseases. The main source of IFN-γ in human immune response is T cells. NK cells, macrophages and IFN- play an important role in both innate and acquired immunity.

[0006] The major histocompatibility complex (MHC) is a group of cell surface proteins that are essential for regulating the immune system. The main function of MHC molecules is to bind to antigens derived from pathogens and display 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 known as the HLA (human leukocyte antigen) complex (often shortened to HLA). Natural killer (NK) cells are lymphocytes that function at the interface between innate and adaptive immunity. NK cells contribute directly to immune defense through their effector functions, such as cytotoxicity and cytokine secretion, and by regulating both innate and adaptive immune responses. When a target or host cell encounters a NK cell, several outcomes are possible. The extent of the NK response is determined by the number and type of activating and inhibitory receptors on the NK cell, as well as the amount and type of activating and inhibitory ligands on the target cell. See also Figure 1 . In case A, when the target cell has no human leukocyte antigen (HLA) class I antigen and no NK activation ligand, NK cells expressing MHC-I class inhibitory receptors and activating ligand receptors do not attack the target cell (no response, or without permission). In case B, when the target cell expresses HLA-I class antigen but has no activating ligand, NK cells expressing inhibitory receptors and activating receptors cannot attack the target. In case C, when the target cell downregulates HLA-I class antigen or has no HLA-I class antigen and expresses NK activation ligand, NK cells expressing inhibitory receptors and activating receptors attack the target cell. In case D, when the target cell expresses both its own HLA-I class antigen and NK activation ligand, the response level of NK cells expressing inhibitory receptors and activating receptors is determined by the balance of inhibitory and activating signals to NK cells. Haynes et al., THE IMMUNE SYSTEM IN HEALTH AND DISEASE, PART 15: Immune-Mediated, Inflammatory, and Rheumatologic Disorders, 372e Introduction to the ImmuneSystem.

[0007] In the past, efforts to overcome the host's immune response to allogeneic cells have focused on adaptive immune responses, that is, interfering with the adhesion between T cells and MHC-I class antigens presented on foreign cells. In this way, CRISPR and TALEN systems have been used to generate loss-of-function genetic modifications, and thus prepare stem cells that do not express one or more classical MHC / HLA genes. However, these cells and cells derived from them are still vulnerable to the host's innate immune response (NK cells). See, e.g., Parham et al. (2005) Nat Rev Immunol.5(3):201-214. In order to overcome the host's innate immune response, others have attempted to redirect tolerogenic factors back into target cells; the focus is on "losing oneself". See WO2016183041A2, the disclosure of which is incorporated by reference as a whole. The applicant was surprised to find that the key to escaping the host's NK-mediated immune response is not "losing oneself", but the expression and magnitude of NK cell activating ligands on target cells.

[0008] Thus, there remains a need for compositions and methods for developing target cells that lack some or all classical HLA expression but are not lysed by NK cell attack.

[0009] Overview

[0010] Disclosed herein is a strategy for overcoming transplant rejection, particularly allogeneic immune transplant rejection in cell-based transplantation therapy, by providing a universal donor cell line. In one embodiment, human pluripotent stem cells lacking some or all typical HLA-I class cell surface protein expression and NK activation ligand expression are provided. In one embodiment, cells derived from human pluripotent stem cells lacking some or all typical HLA-I class cell surface protein expression and NK activation ligand expression, such as pancreatic cells, are provided. In one embodiment, a method for preventing cell transplant rejection by providing transplanted pancreatic cells is provided, wherein at least one MHC gene (such as β-2-microglobulin (B2M)) and at least one NK activation ligand gene (such as intracellular adhesion molecule 1 (ICAM-1)) have been destroyed, deleted, modified or suppressed. In another embodiment, a method for preventing cell transplant rejection by providing transplanted pancreatic cells is provided, wherein at least one MHC gene (such as B2M) and at least one NK activation ligand protein (such as ICAM-1) have been destroyed, deleted, modified or suppressed. Disruption, deletion, modification, or inhibition of B2M results in defects in surface expression and function of all HLA class I. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1Figure 372e-4 is a reproduction of Haynes et al., supra, in which Figures 372e-4 (incorporated herein in their entirety) show different scenarios (A) NK-mediated responses to target cells. In the absence of MHC-I class and NK activating ligands on the target cell, inhibitory and activating receptors on the NK cell do not engage, and the NK cell remains unresponsive (scenario A). In the presence of MHC-I class but no NK activating ligands on the target cell, inhibitory receptors on the NK cell engage, but the NK cell remains unresponsive (scenario B). In the absence of self-MHC-I class on the target cell but the presence of NK activating ligands, inhibitory receptors on the NK cell do not engage but activating receptors on the NK cell engage, and the NK cell attacks (scenario C). In the presence of MHC-I class and NK activating ligands on the target cell, inhibitory and activating receptors on the NK cell engage, and the outcome is determined by the balance of signals.

[0012] Figure 2A Representative flow cytometric analysis of B2M cell surface protein expression on wild-type (WT) hES cells in the absence of IFN-γ (B line) and after exposure to IFN-γ (A line) is shown. The shaded area is background expression without antibody staining. Exposure to IFN-γ increased B2M cell surface protein expression in WT hES cells.

[0013] Figure 2B Representative flow cytometry analysis of B2M cell surface protein expression on B2M knockout (B2M- / -) hES cells in the absence of IFN-γ (B lines) and after exposure to IFN-γ (A lines) is shown. B2M- / - hES cells have very little B2M cell surface protein expression, which is not significantly changed after exposure to IFN-γ.

[0014] Figure 3A Representative flow cytometric analysis of HLA-ABC cell surface protein expression on WT hES cells using a pan-HLA class I antibody in the absence of IFN-γ (B line) and after exposure to IFN-γ (A line) is shown. The shaded area is background expression without antibody staining.

[0015] Figure 3B Representative flow cytometric analysis of HLA-ABC cell surface protein expression on B2M knockout hES cells in the absence of IFN-γ (line B) and after exposure to IFN-γ (line A) are shown. B2M- / -hES cells have no detectable expression of HLA-ABC cell surface proteins.

[0016] Figure 4ARepresentative flow cytometric analysis of B2M cell surface protein expression on WT pancreatic endoderm cells (PECs) in the absence of IFN-γ (B lines) and after exposure to IFN-γ (A lines) is shown. The shaded area is background expression without antibody staining. Exposure to IFN-γ increases B2M cell surface protein expression in WT PECs.

[0017] Figure 4B Representative flow cytometric analysis of B2M cell surface protein expression on B2M knockout PECs in the absence of IFN-γ (line B) and after exposure to IFN-γ (line A) are shown. B2M- / - PECs had no detectable expression of B2M cell surface protein.

[0018] Figure 5A Representative flow cytometric analysis of HLA-ABC cell surface protein expression on WT PECs in the absence of IFN-γ (B line) and after exposure to IFN-γ (A line) is shown. The shaded area is background expression without antibody staining. Exposure to IFN-γ increased HLA-ABC cell surface protein expression in WT PECs.

[0019] Figure 5B Representative flow cytometric analysis of HLA-ABC cell surface protein expression on B2M knockout PECs in the absence of IFN-γ (line B) and after exposure to IFN-γ (line A) are shown. B2M- / - PECs had no detectable expression of HLA-ABC cell surface proteins.

[0020] Fig. 6A Representative flow cytometric analysis of ICAM-1 cell surface protein expression on WT hES cells in the absence of IFN-γ (B line) and after exposure to IFN-γ (A line) is shown. The shaded area is background expression without antibody staining. Exposure to IFN-γ increases ICAM-1 cell surface protein expression in WT hES cells.

[0021] Figure 6B Representative flow cytometric analysis of ICAM-1 cell surface protein expression on B2M knockout hES cells in the absence of IFN-γ (B line) and after exposure to IFN-γ (A line) is shown. B2M- / -hES cells have similar ICAM-1 cell surface protein expression to WT hES cells before and after IFN-γ exposure.

[0022] Fig. 7ARepresentative flow cytometric analysis of ICAM-1 cell surface protein expression on WT PECs in the absence of IFN-γ (B line) and after exposure to IFN-γ (A line) is shown. The shaded area is background expression without antibody staining. Exposure to IFN-γ increases ICAM-1 cell surface protein expression in WT PECs.

[0023] Figure 7B Representative flow cytometric analysis of ICAM-1 cell surface protein expression on B2M knockout PECs in the absence of IFN-γ (B line) and after exposure to IFN-γ (A line) is shown. After exposure to IFN-γ, B2M- / - PECs have similar ICAM-1 cell surface protein expression to WT PECs, which is greater than background ICAM-1 cell surface protein expression (shaded area).

[0024] Figure 8 is a bar graph showing mRNA expression data (Affymetrix expression array) of ICAM-1 in WT hES cells, B2M(- / -) hES cells, WT PECs, and B2M(- / -) PECs, respectively, not exposed to IFN-γ (control) or 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 matured into insulin-producing cells in vivo, human islet cells, and two different samples of peripheral blood mononuclear cells (PBMCs) (not exposed to IFN-γ). ICAM-1 mRNA expression increased following exposure of hES cells (WT or B2M- / -) or PECs (WT or B2M- / -) to IFN-γ.

[0025] Fig. 9 Representative flow cytometric analysis of CD58 (alias: LFA-3) cell surface protein expression on WT PECs without exposure to IFN-γ (B line) and after exposure to IFN-γ (A line) is shown. C line is background expression without antibody staining. Exposure to IFN-γ only slightly increased CD58 cell surface protein expression in WT PECs compared to untreated controls. Antibodies were from BioLegend, catalog number 330909.

[0026] Fig.10Representative flow cytometric analysis of CD155 cell surface protein expression on WT PECs without exposure to IFN-γ (B line) and after exposure to IFN-γ (A line) is shown. C line is background expression without antibody staining. After exposure to IFN-γ, WT PECs have similar CD155 cell surface protein expression as WT treated PEC controls. Gene symbol PVR (alias: CD155, NECL-5, HVED). Antibodies are from Milteneyi Biotech Inc., catalog number 130-105-905.

[0027] Fig.11 Representative flow cytometric analysis of CEACAM1 (alias: CD66a, BGP, BGP1) cell surface protein expression on WT PECs without exposure to IFN-γ (B line) and after exposure to IFN-γ (A line) is shown. C line is background expression without antibody staining. Exposure to IFN-γ slightly increased CEACAM1 cell surface protein expression in WT PECs compared to untreated controls. Antibodies were from Milteneyi Biotech Inc., catalog number 130-098-858.

[0028] Fig.12 Representative flow cytometry analysis of BAT3 cell surface protein expression on WT PECs without exposure to IFN-γ (B lines) and after exposure to IFN-γ (A lines) is shown. Line C is background expression without antibody staining. WT PECs in the untreated control have similar BAT3 cell surface protein expression as WT PECs exposed to IFN-γ. Gene symbol BAG6 (alias: BAT3, HLA-B-associated transcript 3). Antibodies are from Abcam, Inc., catalog number ab210838.

[0029] Fig.13 Representative flow cytometric analysis of CADM1 (alias: NECL2, TSLC1, IGSF4, RA175) cell surface protein expression on WT PECs without exposure to IFN-γ (line B) and after exposure to IFN-γ (line A) is shown. 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. Antibodies are from MBL International Corp., catalog number CM004-4.

[0030] Fig.14Representative flow cytometric analysis of CD112 cell surface protein expression on WT PECs without exposure to IFN-γ (B line) and after exposure to IFN-γ (A line) is shown. C line 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 (alias: CD112, Nectin-2, PVRR2, HVEB). Antibodies are from Milteneyi Biotech Inc., catalog number 130-109-056.

[0031] Fig.15 Is a bar graph showing the reduction of NK cell lysis of target cells after blocking ICAM-1 expression in target cells with anti-ICAM1 antibody (from left to right cells used for NK cytotoxicity assay: WT hESC, WT hESC exposed to IFN-γ, B2M- / -hESC, B2M- / -hESC exposed to IFN-γ, WT PEC, WT PEC exposed to IFN-γ, B2M- / -PEC, B2M- / -PEC exposed to IFN-γ, K562-control cell line used for NK cytotoxicity assay). From left to right, three bars in each group represent: NK-062216; NK-062216+ICAM1 AB (5μg / ml); NK-062216+ICAM1 AB (10μg / ml).

[0032] Sequence Listing

[0033] As defined in 37 C.FR 1.822, the nucleic acid and amino acid sequences listed in the attached sequence table are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids. Each nucleic acid sequence shows only one strand, but the complementary strand is understood to be included by any reference to the displayed strand. The sequence table was submitted on July 3, 2018 in the form of an ASCII text file Sequence_Listing (11KB), which is incorporated herein by reference.

[0034] Details

[0035] MHC-I class molecules are one of the two main categories of major histocompatibility complex (MHC) molecules (the other is MHC-II class). Their function is to display peptide fragments of non-self proteins from within the cell to cytotoxic T cells; this will trigger the immune system to respond immediately to specific non-self antigens displayed by MHC-I class proteins. In humans, the HLA corresponding to the MHC-I class is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G. Human HLA-E, HLA-F and HLA-G are atypical MHC class I molecules, characterized by limited polymorphism and lower cell surface expression than typical paralogs (HLA-A, -B and -C). All MHC class I proteins must associate with β2-microglobulin (B2M) to produce a functional heterodimeric MHC class I protein complex, which is then functionally expressed on the cell surface. MHC-I class molecules can also be used as inhibitory ligands for NK cells. The reduction of normal levels of cell surface MHC class I activates the killing effect of NK cells.

[0036] In the past, it was thought that target cells bearing MHC class I inhibitory ligands would evade attack when exposed to NK cells due to the presumed dominant nature of MHC class I inhibitory signals (see Figure 1 , Case B, reproduced from Harrison's Principles of Internal Medicine 19E (Volumes 1 and 2) A Major Histocompatibility Complex, Part 15, Figure 372e-4 therein). However, the applicant surprisingly found that the opposite is true.

[0037] It has been shown that exposure of cells to IFN-γ increases the mRNA expression of MHC-I class molecules and also the expression of MHC class I protein complexes on the cell surface. This increase in MHC-I class expression is expected to inhibit NK cells. Applicants have found that wild-type (WT) hES cells (which have been shown to increase the MHC-I class molecules on the cell surface of hES cells, Figure 2A and 3A ) has increased NK cell-mediated cytotoxicity. Fig.15 , showing an increase in NK cell-mediated toxicity (lysis) from 58% to 79% (compare Fig.15 The same was true when WT PEC cells were exposed to IFN-γ, where the expression of B2M and HLA-ABC by the cells also increased, see Figure 4A and 5A , NK-mediated toxicity (lysis) increased from 33% to 53% (compared Fig.15This data suggests that the key to overcoming the host's NK cell immune response is not overexpression of inhibitory MHC-I signals, but blocking NK activating ligand signals.

[0038] To further test this hypothesis in the context of emphasized NK cell-mediated cytotoxicity, the Applicants generated B2M- / - (knockout) hES cells (similar to Figure 1 Case C). As expected, B2M- / - eliminated hES cells ( Figure 2B and 3B ) and PEC( Figure 4B and 5B ) cell surface expression of MHC class I molecules on WT cells. Also as expected, B2M- / - cells exhibited increased NK cell-mediated lysis relative to WT cells, ranging from 58% to 71% for hES cells and 33% to 54% for PECs (compare Fig.15 The applicant found that exposure of B2M- / -hES cells or PEC to IFN-γ further increased the percentage of NK cell-mediated toxicity (lysis) (compare Fig.15 4 and 6 vs. 8 in the first straight line). Accordingly, the applicant found that NK cell activation ligand cell surface expression ( Figure 6B and 7B ) and mRNA expression ( Figure 8 ) is increased upon exposure to IFN-γ. This data suggests that NK cell activating ligands on target cells play a key role in NK cell cytotoxicity and leads to the hypothesis that in the context of reduced MCH class I expression, such as in the context of B2M- / -, inhibition of NK cell activating ligand expression can prevent NK cell-mediated cytotoxicity.

[0039] To determine whether the toxicity of NK cells can be reduced by inhibiting the action of NK activating ligands on target cells, the applicant blocked the expression of NK activating ligands in WT and B2M- / -hES cells and PECs, for example, using ICAM1 blocking antibodies to block ICAM1 proteins on the surface of target cells. The applicant surprisingly found that the cell lysis of target cells was reduced (compared to Fig.15 The first bar and the second and third bars of conditions 2, 4, 6 and 8 in the above table). Therefore, the applicant has found that NK cell lysis can be reduced by blocking NK activating ligands. Blocking ICAM1 expression in B2M- / - cells using antibodies against NK activating ligands is a proof of concept for generating cells with double knockouts (HLA-I class gene knockout and NK activating ligand gene knockout). In this way, the applicant can remove target cells (such as hES and / or pancreatic lineage cells) from Figure 1Situation C in is converted to A. Specifically, cells, tissues and organs of the present invention suppress or do not have HLA-I class cell surface protein expression (B2M- / -), and suppress or do not have NK activation ligand cell surface protein expression (such as 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 baits, ribozymes, RNA aptamers, siRNA, shRNA / miRNA, trans-dominant negative proteins (TNP), chimeric / fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFvs), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides and chemicals) and protease inhibitors. Double or multiple gene knockouts will effectively prevent cytotoxic T cell (CTL)-mediated and NK cell-mediated toxicity, because there is almost no HLA-I class, and there is almost no NK activation ligand protein expressed on the cell surface for CTL or NK cell combination. Furthermore, to completely eliminate NK activation, Applicants anticipate that it will be necessary to eliminate or reduce the expression of multiple NK activating ligands by gene knockout in target cells (e.g., hES cell-derived cell therapy) or by using blocking antibodies or other currently known or future developed strategies.

[0040] NK cell activating ligand blockers

[0041] According to one aspect of the present invention, a method of treating NK cell function is provided. According to another aspect of the present invention, a method of treating at least one immune response is provided. Each method involves administering an agent that inhibits NK cell function to a subject in need of treatment. In some embodiments, the agent is an antibody. In some embodiments, the antibody selectively binds to an NK cell activating ligand on a target cell.

[0042] It is contemplated that various types of agents, including antibodies and blocking proteins, may be used to interfere with the adhesion of NK cells to the NK activating ligands of target cells.

[0043] In certain embodiments, such NK activating ligands are selected from Table 1.

[0044] Table 1: Natural Killer (NK) Activating Ligands

[0045]

[0046]

[0047] NK activating ligands are further described 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.

[0048] Low immunogenicity hES cells and cells derived therefrom

[0049] HLA is a cell surface molecule encoded by a large gene family and can be divided into class I and class II molecules. HLA-I class molecules are present on the surface of each nucleated cell and are the focus of the invention described herein. HLA mismatches between donor (target) cells and recipient immune cells (such as T cells) during transplantation usually lead to immune rejection or graft rejection. The HLA-I class complex is structurally composed of polymorphic heavy chains and light chain beta-2-microglobulin (β2m or B2M) composed of HLA-I class peptides (such as HLA-A, HLA-B and HLA-C). In the absence of B2M, class I HLA cannot be assembled correctly and will not be expressed on the cell surface or cell membrane. In the invention described herein, the applicant produces hES cell lines and cells derived therefrom by destroying the B2M gene (adding or removing some base pairs, thereby generating frameshift and loss-of-function mutations in mRNA / protein) and thereby depleting HLA-1 class expression from the cell surface in hESC.

[0050] By additionally destroying genes encoding NK activation ligands (such as ICAM1), the above method can also be used to produce or generate hES cell lines and cells derived therefrom. Therefore, in one embodiment of the present invention, compositions and methods are provided to prepare target cells lacking at least one HLA-I class antigen and at least one NK activation ligand, and thereby produce low immunogenic cells. Such low immunogenic cells are expected to be less susceptible to immune rejection by subjects transplanted with such cells. During transplantation, such low immunogenic cells should be implanted (not rejected). In one embodiment, such target cells can be transplanted and survive with little need for immunosuppression of the recipient.

[0051] In one embodiment, the inhibition, reduction and / or deletion of HLA-I class expression and NK activation ligand expression in hESC cells and cells derived therefrom (or HLA-I class deficiency and NK activation ligand deficiency) can be used as a universal donor cell source for transplantation therapy. These double knockouts (HLA-I class deficiency and NK activation ligand deficiency) can be universally transplanted without the need for minor histocompatibility complex (MiHC) matching, human leukocyte antigen (HLA) matching or immunosuppression.

[0052] Disclosed herein are novel in vitro derived low immunogenic compositions and cells. In particular, in certain embodiments, the present invention disclosed herein relates to stem cells, whose genome has been changed (modified) to reduce or lack one or more MHC-I class genes and key components of one or more NK activating ligand genes. In certain embodiments, the invention disclosed herein relates to pancreatic lineage cells, such as pancreatic endoderm cells, pancreatic epithelial cells, pancreatic progenitor cells, pancreatic precursor endocrine cells, pancreatic endocrine cells, pancreatic pre-β cells or pancreatic β cells, whose genome has been changed (modified) to reduce or lack one or more MHC-I class genes and key components of one or more NK activating ligand genes, thereby producing low immunogenic pancreatic-lineage type cells. Natural killer activating ligands include, but are not limited to, ligands from categories 1, 2, 3, or combinations thereof listed in Table 1. Natural killer activating ligands include, for example, ICAM-1, CEACAM1, CADM1, MICA, and MICB. MHC-I class genes include HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G and B2M. In certain aspects, such reduced expression or knockout of MHC-I class and / or MHC-II class genes is accomplished by directly and / or indirectly targeting NLRC5, B2M and CIITA genes and other components of the MHC enhanceosome (enhanceosomes are higher-order protein complexes that assemble at enhancers and regulate the expression of target genes, such as transcriptional regulators of MHC-I class or MHC-II class).

[0053] Also disclosed herein is a method for preparing low immunogenic cells, the method comprising regulating the expression of one or more NK activation ligands expressed by cells and regulating the expression of one or more MHC-I and / or MHC-II classes of cells, thereby preparing low immunogenic cells. In some aspects, regulating the cell surface protein expression of one or more MHC-I and / or MHC-II complexes includes reducing, inhibiting and / or interfering with the expression of one or more MHC-I and / or MHC-II genes or proteins. In some embodiments, regulating the expression of one or more MHC-I and / or MHC-II complexes includes deleting one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II from at least one allele of the cell. For example, in some embodiments, such methods include deleting one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II genes selected from LRC5, CIITA, B2M and combinations thereof. In some aspects, regulating the expression of one or more NK activation ligands includes deleting, inhibiting or reducing the expression of one or more genes encoding NK activation ligands. In some embodiments, such NK activation ligands are selected from Table 1. In certain embodiments, such NK activating ligands are selected from categories 1, 2, 3 or a combination thereof in Table 1. In certain embodiments, such NK activating ligands are selected from categories 1, 2 and 3 in Table 1. In certain embodiments, such NK activating ligands are selected from categories 1 and 3 in Table 1. In certain embodiments, such NK activating ligands are selected from categories 1 and 2 in Table 1. In certain embodiments, such NK activating ligands are selected from categories 2 and 3 in Table 1. In certain embodiments, such NK activating ligands are selected from ICAM-1, CEACAM1, CADM1MICA, MICB and combinations thereof.

[0054] In certain embodiments, the transplanted low immunogenic cells are in a culture medium that is free of animal-derived products, such as xenofree products.

[0055] The present invention encompasses changing the target polynucleotide sequence in any manner available to the skilled person (e.g., using any of a large range of nucleases or endodeoxyribonucleases, zinc finger nucleases (ZFNs or ZNFs), nucleases based on transcription activator-like effectors (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR / Cas or CRISPR / Cas9) systems or traditional homologous recombination techniques). Such CRISPR / Cas systems can employ 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 class I system. In some embodiments, the CRISPR / Cas system is a CRISPR class II system. In some embodiments, the CRISPR / Cas system is a CRISPR class V system. NEXTGEN incorporating dual guide RNA and a catalytically inactive Cas9 protein fused to a FokI nuclease TM CRISPR (Transposagen Inc., Lexington Kentucky) can also be used to change the target nucleotide sequence.Other methods of targeting polynucleotide sequences now known or later discovered by those skilled in the art to reduce or eliminate expression in target cells can be used to generate the low immunogenic cells described herein.

[0056] In some embodiments, the change results in a decrease in expression of the target nucleotide sequence. In some embodiments, the change is a homozygous change. In some embodiments, the change is a heterozygous change.

[0057] In some embodiments, the target nucleotide sequence is a genomic sequence. In some embodiments, the target nucleotide sequence is a human genomic sequence. In some embodiments, the target nucleotide sequence is a mammalian genomic sequence. In some embodiments, the target nucleotide sequence is a vertebrate genomic sequence.

[0058] In some embodiments, low immunogenic cells are embryonic stem cells. In certain embodiments, low immunogenic cells are pluripotent stem cells. In certain embodiments, low immunogenic cells are induced pluripotent stem cells, reprogrammed cells, dedifferentiation or transdifferentiation cells. In certain embodiments, low immunogenic cells are pluripotent pancreatic progenitor cells. In certain embodiments, low immunogenic cells are single hormone cells or multi-hormone cells. In certain embodiments, low immunogenic cells are mesendoderm cells, definitive endoderm cells, PDX1-negative foregut endoderm cells, PDX1-positive foregut endoderm cells, pancreatic endoderm cells, endocrine progenitor cells / precursor cells, endocrine cells, appropriately specified endocrine cells, immature endocrine cells or functional β-cells. In some embodiments, low immunogenic cells can be homogeneous or heterogeneous cell groups. In some embodiments, low immunogenic cells are cells that produce one or more target biologically active substances. Low immunogenic cells may initially have no therapeutic activity when first transplanted, such as pancreatic progenitor cells or PDX1-positive pancreatic endoderm, but once transplanted they further develop and mature and have therapeutic effects.

[0059] In some embodiments, the low immunogenic cells can be any cell that can be derived from human pluripotent stem cells including, but not limited to, any cell, tissue, or organ, and may include skin cells, beta cells (i.e., cells located in the pancreas of the islets of Langerhans), parathyroid cells, intestinal cells, endocrine cells heart cells, brain cells, kidney cells, liver cells, cells of the digestive tract and accessory digestive organs, 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.

[0060] In some embodiments, low immunogenic cells can be separate (single) cells in a suspension or cell aggregate. In some embodiments, low immunogenic cells include omnipotent cells. In one embodiment, low immunogenic cells include pluripotent cells. In one embodiment, low immunogenic cells include unipotent cells.

[0061] In some embodiments, low immunogenic cells are derived from a multipotent cell population lacking functional HLA-I class expression and NK activating ligand expression. Derived cells may be selected from any cell, tissue or organ, and may include skin cells, β cells (i.e. cells in the pancreas of the islets of Langerhans), parathyroid cells, intestinal cells, endocrine cells, heart cells, brain cells, kidney cells, liver cells, cells of the digestive tract and accessory digestive organs, salivary cells, adrenal 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.

[0062] The low immunogenic cells, tissues and / or organs to be transplanted may be syngeneic or allogeneic to the subject receiving the transplant.

[0063] In one embodiment, low immunogenic cells are human pluripotent cells. In one embodiment, low immunogenic cells are human pancreatic-lineage cells. In one embodiment, low immunogenic cells are human pancreatic endoderm cells. In one embodiment, low immunogenic cells are human pancreatic precursor cells. In one embodiment, low immunogenic cells are human pancreatic progenitor cells. In one embodiment, low immunogenic cells are human pancreatic endocrine cells. In one embodiment, low immunogenic cells are human pancreatic endocrine precursor cells. In one embodiment, low immunogenic cells are human pancreatic endocrine pre-β cells. In one embodiment, low immunogenic cells are human pancreatic β cells. In one embodiment, low immunogenic cells are human pancreatic single hormone or multi-hormone cells. In one embodiment, low immunogenic cells are human insulin expressing cells.

[0064] In one embodiment, low immunogenic cells are well-known publicly available pluripotent cell lines. The invention described herein is useful in all hES cells and iPSC lines, and at least hESCs such as CyT49, CyT25, CyT203, and CyT212. Pluripotent cell lines include those cells commercially available from WiCell (www.wicell.org / home / stem-cell-lines / order-stem-cell-lines / obtain-stem-cell-lines.cmsx), and particularly include BG01, BG02, and BG03.

[0065] In one embodiment, the low immunogenic cells are substantially similar to the cells 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 (which is incorporated herein by reference in its entirety). D'Amour et al. describe a 5-step differentiation protocol: Stage 1 (leading primarily to definitive endoderm), Stage 2 (leading primarily to PDX1-negative foregut endoderm), Stage 3 (leading primarily to PDX1-positive foregut endoderm), Stage 4 (leading primarily to pancreatic endoderm also known as multipotent pancreatic progenitor cells or pancreatic endocrine progenitor cells), and Stage 5 (leading primarily to hormone-expressing endocrine cells). In one embodiment, the low immunogenic cells are substantially similar to the low immunogenic cells described in U.S. Patent Nos. 7,510,876, 7,695,965, 7,985,585, 8,586,357, 8,633,024, and 8,129,182 (which are herein incorporated by reference in their entireties).

[0066] In one embodiment, the low immunogenic cells are substantially similar to the cells described in Schulz et al. A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells, PLoS One 7: 51-17 (2012), which is incorporated herein by reference in its entirety. Schulz et al. described a hESC expansion and stacking method and a suspension-based differentiation system. Specifically, undifferentiated pluripotent cells are aggregated into clusters in a dynamic rotating suspension culture, and then differentiated en masse for two weeks through a four-stage protocol. In brief, from the hES cell aggregate suspension, the hESC monolayer is dissociated with Accutase (Innovative Cell Technologies), collected and cultured at 1x10 6Cells / mL were resuspended in StemPro hESC SFM (Life Technologies; combined DMEM / F12, containing Glutamax, StemPro hESC supplement, BSA and 1% (v / v) penicillin / streptomycin; FGF-2 and 2-mercaptoethanol were omitted). The single cell suspension was dispensed into a 6-well plate (5.5 mL / well) without TC treatment and spun at 95 rpm on an Innova 2000 rotator (New Brunswick Scientific), or dispensed into a 500 mL Nalgene filter receiver storage bottle (150 mL / bottle) and spun at 65 rpm on a Sartorius Certomat RM-50 rotator (equipped with a 5 cm rotating shaft). The cells were rotated overnight in a 37°C / 8% CO2 incubator and aggregates of approximately 100-200 μm were formed. For aggregates of 100-200 μm in diameter, a 6-well culture dish can use a rotation speed of 60-140 rpm; a 500 mL bottle can use a rotation speed of 5-20 rpm. Differentiation of suspended polymers involves few modifications from D'Amour. TGF-βRI kinase inhibitor IV is included during stage 2, and retinoic acid is replaced with a more stable retinoid analog TTNPB (3 nM) during stage 3. Growth factors KGF (50 ng / mL) and EGF (50 ng / mL) are added to stage-4 to retain cell clumps. Noggin (50 ng / mL) is also included in stage 4. In one embodiment, the low immunogenic cells are substantially similar to those described in U.S. Patent Nos. 8,008,075 and 8,895,300 (which are incorporated herein by reference in their entirety).

[0067] In one embodiment, the low immunogenic 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 VivoStem Cells Translationalmedicine 4: 1-9 (2015), which is incorporated herein by reference in its entirety. Agulnick et al. describe a modified method for preparing pancreatic progenitor cells such that 73%-80% of the cell population consists of PDX1-positive (PDX1+) and NKX6.1+ pancreatic progenitor cells. Pancreatic progenitor cells are further differentiated into islet-like cells (ICs) that reproducibly contain 73%-89% endocrine cells, of which about 40%-50% express insulin. A large portion of these insulin-positive cells are single hormone-positive and express transcription factors PDX1 and NKX6.1. Agulnick et al. describe a protocol in which the 2012 protocol of Schulz et al. is modified by additional treatment with Activin A, Wnt3A, and Heregulin β1 in stage 3 (days 5-7) and Activin A and Heregulin β1 in stage 4 (days 7-13). In one embodiment, the low immunogenic cells are substantially similar to the cells described in U.S. Pat. No. 8,859,286 (which is incorporated herein by reference in its entirety).

[0068] Growth, passaging and proliferation of human embryonic stem cells can be performed essentially as described in US Pat. Nos. 7,964,402; 8,211,699; 8,334,138; 8,008,07; and 8,153,429.

[0069] Standard preparation protocol

[0070] Table 2 below discloses a standard preparation method for generating hESC-derived pancreatic endoderm cells (PECs).

[0071]

[0072]

[0073] 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 LifeTechnologies), 10 ng / mL heregulin-1β (Peprotech), and 10 ng / mL activin A (R&D Systems); SP: hESC SFM (Life Technologies); r0.2FBS: RPMI 1640 (Mediatech); 0.2% FBS (HyClone), 1x GlutaMAX-1 (Life Technologies), 1% v / v penicillin / streptomycin; ITS: 1:5000 or 1:1000 dilution of insulin-transferrin-selenium (Life Technologies); 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 0.5x 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).

[0074] Calcein release assay

[0075] The calcein release assay is a non-radioactive alternative method for studying NK cell cytotoxicity. Target cells take up a fluorescent dye (Calcein AM) and will be converted into an active fluorescent dye in the cytoplasm, which is released from the cells only after lysis. The lysed cells release the fluorescent dye into the supernatant, which is then collected and the amount of fluorescence is quantified in a fluorimeter. The percentage of cell lysis is calculated based on the amount of fluorescence present in the supernatant after incubation in the presence or absence of NK cells (effectors), blocking antibodies, or both.

[0076] Specific lysis can be calculated by using the following formula: % lysis = 100 x [(mean fluorescence with antibody - mean autofluorescence) / (mean maximum fluorescence - mean autofluorescence)]. Maximum fluorescence is determined by lysis of cells incubated with detergent (1% Triton X-100), and spontaneous lysis is the fluorescence obtained with target cells in the absence of any antibody or effector cells.

[0077] Various cell compositions derived from pluripotent stem cells and methods thereof are described herein and can be found in the following U.S. patent applications of the applicant: 10 / 486,408, entitled METHODS FOR CULTURE OF HESC ON FEEDERCELLS, filed August 6, 2002; 11 / 021,618, entitled DEFINITIVE ENDODERM, filed December 23, 2004; 11 / 115,868, entitled PDX1 EXPRESSING ENDODERM, filed April 26, 2005; 11 / 165,305, entitled METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIATING DEFINITIVE ENDODERM, filed June 23, 2005; 11 / 573,662, entitled METHODS FOR INCREASING DEFINITIVE ENDODERM DIFFERENTIATION OF PLURIPOTENT HUMAN EMBRYONIC STEM CELLS WITH PI-3KINASE INHIBITORS, filed August 15, 2005; 12 / 729,084, entitled PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM, filed October 27, 2005; 12 / 093,590, entitled MARKERS OF DEFINITIVE ENDODERM, filed November 14, 2005; 11 / 993,399, entitled EMBRYONIC STEM CELL CULTURE COMPOSITIONS AND METHODS OF USE THEREOF, filed June 20, 2006; 11 / 588,693, entitled PDX1-EXPRESSING DORSAL AND VENTRAL FOREGUT ENDODERM, filed October 27, 2006; 11 / 681,687, entitled ENDOCRINE PROGENITOR / PRECURSOR CELLS, PANCREATIC HORMONE-EXPRESSING CELLS AND METHODS OF PRODUCTION, filed March 2, 2007; 11 / 807,223, entitled METHODS FOR CULTURE AND PRODUCTION OF SINGLE CELL POPULATIONS OF HESC, filed May 24, 2007;11 / 773,944, entitled METHODS OF PRODUCING PANCREATIC HORMONES, filed July 5, 2007; 11 / 860,494, entitled METHODS FOR INCREASING DEFINITIVE ENDODERM PRODUCTION, filed December 24, 2007; 12 / 099,759, entitled METHODS OF PRODUCING PANCREATIC HORMONES, filed April 8, 2008; 12 / 107,020, entitled METHODS FOR PURIFYING ENDODERM AND PANCREATIC ENDODERM CELLS DERIVED FORM HUMAN EMBRYONIC STEMCELLS, filed April 21, 2008; 12 / 618,659, entitled ENCAPSULATION OF PANCREATIC LINEAGE CELLS DERIVED FROM HUMAN PLURIPOTENT STEM CELLS, filed November 13, 2009; 12 / 765,714 and 13 / 761,078, both entitled CELL COMPOSITIONS FROM DEDIFFERENTIATED REPROGRAMMEDCELLS, filed April 22, 2010 and February 6, 2013; 11 / 838,054, entitled COMPOSITIONS AND METHODS USEFUL FOR CULTURING DIFFERENTIABLE CELLS, filed August 13, 2007; 12 / 264,760, entitled STEM CELL AGGREGATE SUSPENSION COMPOSITIONS AND METHODS OF DIFFERENTIATION THEREOF, filed November 4, 2008; 13 / 259,15, entitled SMALL MOLECULESS UPPORTING PLURIPOTENT CELL GROWTH, filed April 27, 2010; PCT / US11 / 25628, entitled LOADING SYSTEM FOR AN ENCAPSULATION DEVICE, filed February 21, 2011; 13 / 992,931, entitled AGENTS AND METHODS FOR INHIBITING PLURIPOTENT STEM CELLS, filed December 28, 2010;and the following U.S. Design Applications: 29 / 408,366, filed December 12, 2011; 29 / 408,368, filed December 12, 2011; 29 / 423,365, filed May 31, 2012; and 29 / 447,944, filed March 13, 2013; U.S. Application No. 14 / 201,630, entitled 3-DIMENSIONAL LARGE CAPACITY CELL ENCAPSULATION DEVICE ASSEMBLY, filed March 7, 2014; and U.S. Application No. 14 / 106,330, entitled IN VITRODIFFERENTIATION OF PLURIPOTENT STEM CELLS TO PANCREATIC ENDODERM CELLS (PEC) AND ENDOCRINE CELLS, filed December 13, 2013; PCT / US2016 / 061442, entitled PDX1 PANCREATICENDODERM CELLS IN CELL DELIVERY DEVICES AND METHODS THEREOF, filed November 10, 2016; all of which are incorporated herein by reference in their entirety. ;

[0078] Various cell compositions and methods derived from pluripotent stem cells are described herein and can be found in applicant's exclusively licensed applications: U.S. Patent Publication No. 2009 / 0269845, entitled Pluripotent cells, filed April 24, 2008; U.S. Patent Publication No. 2011 / 0014703, entitled Differentiation of Human Embryonic Stem Cells, filed July 20, 2010; U.S. Patent Publication No. 2011 / 0014702, entitled Differentiation of Human Embryonic Stem Cells, filed July 19, 2010; U.S. Patent Publication No. 2011 / 0151561, entitled Differentiation of Human Embryonic Stem Cells, filed December 16, 2010; U.S. Patent Publication No. 2010 / 0112692, entitled Differentiation of Human Embryonic Stem Cells Cells, filed October 22, 2009; U.S. Patent Publication No. 2012 / 0052576, entitled Differentiation of Pluripotent Stem Cells, filed August 17, 2011; U.S. Patent Publication No. 2010 / 0112693, entitled Differentiation of human pluripotent stem cells, filed October 23, 2009; U.S. Patent Publication No. 2011 / 0151560, entitled Differentiation of human embryonic stem cells, filed December 16, 2010; U.S. Patent Publication No. 2010 / 0015100, entitled Differentiation of human embryonic stem cells, filed July 31, 2008; U.S. Patent Publication No. 2009 / 0170198, entitled Differentiation of human embryonic stem cells, filed November 25, 2008; U.S. Patent Publication No. 2015 / 0329828, entitled Use of Small Molecules to Enhance MAFA Expression in Pancreatic Endocrine Cells, filed May 7, 2015; U.S. Patent Publication No. US2013 / 0330823, entitled Differentiation of Human Embryonic Stem Cells into Pancreatic Endocrine Cells, filed on June 6, 2013; International Patent Publication No. WO 2013 / 192005, entitled Differentiation of human embryonic stem cells into pancreatic endocrine cells, filed on June 13, 2013; U.S. Patent Publication No. US2014 / 0242693, entitled Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells, filed on December 30, 2013; U.S. Patent Publication No. US2014 / 0295552, entitled Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells, filed on June 17, 2014; International Patent Publication No. WO 2015 / 065524, entitled Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells, filed May 21, 2014; U.S. Patent Publication No. US2013 / 0330823, entitled Differentiation of Human Embryonic Stem Cells into Pancreatic Endocrine Cells, filed June 6, 2013; U.S. Patent Publication No. US2014 / 0186953, entitled Differentiation of Human Embryonic Stem Cells Into Pancreatic Endocrine Cells Using HB9 Regulators, filed December 18, 2013; U.S. Application No. 14 / 963730, filed December 9, 2015; U.S. Application No. 14 / 898,015, filed December 11, 2015, all of which are incorporated herein by reference in their entirety. .

[0079] In one embodiment, the low immunogenic cells are encapsulated in a cell delivery device. The cell delivery device may include a nonwoven fabric. The cell delivery device includes multiple layers, each of which serves one or more functions. In some embodiments, the cell delivery device includes both a cell-exclusion membrane and a nonwoven fabric. In another embodiment, the delivery device is a TheraCyte (formerly Baxter) device (Irvine, Calif.). The TheraCyte cell delivery device is also described in the following U.S. patents: 6,773,458; 6,156,305; 6,060,640; 5,964,804; 5,964,261; 5,882,354; 5,807,406; 5,800,529; 5,782,912; 5,741,330; 5,733,336; 5,713,888 ; 5,653,756; 5,593,440; 5,569,462; 5,549,675; 5,545,223; 5,453,278; 5,421,923; 5,344,454; 5,314,471; 5,324,518; 5,219,361; 5,100,392; and 5,011,494, all of which are incorporated herein by reference in their entirety.

[0080] In another embodiment, the delivery device is a device substantially as described in U.S. Patent No. 8,278,106 and as described in U.S. Application No. 14 / 201,630 filed on March 7, 2014 and PCT Application No. PCT / US2016 / 061442 filed on November 10, 2016, and as described in the following U.S. Designations: 29 / 447,944, 29 / 509,102, 29 / 484,363, 29 / 484,360, 29 / 484,3 59, 29 / 484,357, 29 / 484,356, 29 / 484,355, 29 / 484,362, 29 / 484,358, 29 / 408,366, 29 / 517,319, 29 / 408,368, 29 / 518,513, 29 / 518,516, 29 / 408,370, 29 / 517,144, 29 / 423,365, 29 / 530,325, 29 / 584,046, all of which are incorporated herein by reference in their entirety. In other embodiments, the cell delivery device or large volume component is composed of one or two or more seals, i.e., separation seals, that further separate the lumen of the cell delivery device. See, for example, applicant's U.S. Design Applications 29 / 408366, 29 / 408368, 29 / 408370, 29 / 423,365, and 29 / 584,046.

[0081] In one embodiment, low immunogenic cells are transplanted into a perforated cell delivery device that can provide direct cell-to-cell contact between the host vasculature and the encapsulated cells. Perforation means a hole or hole in the device. In some embodiments, not all layers of the device are perforated. For example, see PCT application PCT / US2016 / 0061442, which is incorporated herein by reference in its entirety, which discusses a perforated cell delivery device with perforations only in one layer, such as a cell-exclusion membrane; or only in a cell-exclusion membrane and a nonwoven layer. In one embodiment, low immunogenic cells are encapsulated in a perforated device surrounded by a nonwoven. In these embodiments, the nonwoven is outside the cell delivery device. The nonwoven does not affect the implanted cells, but enhances the host vascularization around the cell shell. For example, see PCT / US2016 / 0061442 and U.S. Patent No. 8,278,106 (both of which are incorporated herein by reference in their entirety), which describes perforated devices and device polymers.

[0082] In one embodiment, the holes / perforations are smaller than the cell aggregates contained in the device, such as hPSC-derived aggregates, such as aggregates of definitive endoderm lineage cells contained therein. In one embodiment, the perforated cell delivery device implanted in a rat or human contains perforations just in the cell-exclusion membrane (the other layers of the device are not perforated), and the holes therein are spaced about 2 mm or more apart and the pore diameter provided therein is less than about 100 microns.

[0083] Depletion of hypoimmunogenic cells (“suicide genes”)

[0084] The versatility of embryonic stem cells and induced pluripotent stem (iPS) cells to replace and repair any tissue in the body goes hand in hand with an increased risk of cancer, which has also been 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 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 from other pluripotent or progenitor cells, as well as from cells treated with gene therapy vectors, present safety concerns (Knoepfler, P.S. Deconstructing Stem Cell Tumorigenicity: A Roadmap to Safe Regenerative Medicine. Stem Cells 27, 1050-1056, (2009), which is incorporated herein by reference in its entirety). For example, iPS cells transplanted at the same time cause teratomas, and iPS chimeric animals develop primary malignant tumors with a high incidence rate (Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676, (2006); Knoepfler, PS Deconstructing Stem Cell Tumorigenicity: A Roadmap to Safe Regenerative Medicine. Stem Cell 27, 1050-1056, (2009)). Although benign teratomas can be easily removed by surgery, invasive cancers still remain a risk of cell therapy.

[0085] Strategies for overcoming stem cell tumorigenicity have been considered, including suicide gene strategies (Knoepfler, PS Deconstructing Stem Cell Tumorigenicity: A Roadmap to Safe Regenerative Medicine. Stem Cell 27, 1050-1056, (2009)). In particular, genes can be selectively introduced into transplanted cells, which encode enzymes that metabolize systemically available prodrugs into locally active antitumor agents. For example, ganciclovir treatment is used, and ganciclovir is converted into a toxic compound after cell kinase triphosphorylation by thymidine kinase, resulting in in vitro destruction of tumor cells. Therefore, implanted cells can be modified to artificially produce available biochemical differences between host tissues and implanted cells. By selecting a vector for delivering suicide genes and the biology of the suicide gene / prodrug system used, targeting of implanted cells is achieved. As a result, high-dose drugs produced only in the environment of implanted cells limit side effects in other tissues.

[0086] Low immunogenic cell depletion can be achieved by selectively introducing genes into low immunogenic cells, the expression of which directly causes low immunogenic cell death or makes low immunogenic cells particularly sensitive to other pharmacological agents. According to the method, low immunogenic cells can be depleted in vivo or in vitro by using a viral gene delivery system (such as but not limited to retrovirus, adenovirus or adeno-associated virus gene delivery system) to deliver the desired gene to low immunogenic cells. The required viral delivery system may include a virus, and its genome encodes a protein that directly causes cell death, such as by inducing apoptosis of low immunogenic cells. Alternatively, the viral delivery system may contain a virus whose genome encodes a herpes simplex virus thymidine kinase gene. The expression of herpes simplex virus thymidine kinase gene in low immunogenic cells makes low immunogenic cells sensitive to pharmacological doses of ganciclovir. Therefore, the subsequent contact of low immunogenic cells transduced by viruses with ganciclovir can cause low immunogenic cell death. Low immunogenic cell depletion can be achieved by introducing so-called "suicide genes" such as ZFN, CRISPR / cas and TALEN systems via genome editing.

[0087] Agents such as ganciclovir that mediate cell killing upon expression of genes such as thymidine kinase are referred to herein as "cell death inducers."

[0088] Genes that can be used to kill low immunogenic cells include, but are not limited to, herpes simplex virus thymidine kinase and cytosine deaminase, or any gene that induces cell death, which 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 to low immunogenic cells, the cells are selected under appropriate selective pressure, the cells are transferred to a patient and implanted therein. The patient is then treated with an agent that induces promoter activity, thereby inducing expression of a gene whose product acts to kill low immunogenic cells. In the case of thymidine kinase, other agents that promote cell killing through this enzyme, such as, for example, ganciclovir (Bonini et al., 1997, Science 276: 1719-1724; Bordignon et al., 1995, Human Gene Therapy 6: 813-819; Minasi et al., 1993, J. Exp. Med. 177: 1451-1459; Braun et al., 1990, Biology of Reproduction 43: 684-693) can also be used. Other genes that can be used for this purpose include, but are not limited to, constitutively active forms of caspases 3, 8, and 9, bax, granzymes, diphtheria toxin, Pseudomonas A toxin, ricin, and other toxin genes are disclosed elsewhere herein. The generation of appropriate constructs for delivering such genes to humans is readily apparent to the skilled artisan 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).

[0089] In order to kill cells, it is important to place the gene transferred into the low immunogenic cells under the control of an appropriate promoter sequence so that induction of the gene expression can be achieved after adding an appropriate inducer to the cells (administered to a mammal). Such inducible promoter sequences include, but are not limited to, promoters induced after adding metals to the cells, steroid inducible promoters, and the like. In a preferred embodiment, an ecdysone promoter system can be used. In this embodiment, the ecdysone promoter is cloned upstream of an ecdysone receptor protein sequence, which is located upstream of a second promoter sequence, which drives the expression of an ecdysone binding site operably linked to a desired gene, such as a desired toxin. The induction of the promoter induces toxin expression, thereby achieving killing of the cells where the toxin gene is located.

[0090] When cells are transduced in vitro, cells in which a gene for cell killing has been transduced 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).

[0091] Low immunogenic cell depletion can be further achieved by introducing oligonucleotides (such as but not limited to antisense molecules) or ribozymes into the low immunogenic cell population, which can induce low immunogenic cell death or induce low immunogenic cell function impairment. Such oligonucleotides include those targeting the basic functions of low immunogenic cells, which are defined herein as oligonucleotides that kill low immunogenic cells or impair the function of low immunogenic cells in stimulating T cells. Such functions of low immunogenic cells include but are not limited to the costimulatory functions of B71 and B72, CD40, etc. Therefore, oligonucleotides and ribozymes that can be used in the methods of the present invention include but are not limited to those directed to these targets.

[0092] As described herein, depletion of low immunogenic cells includes impairment of low immunogenic cell function. Impairment of low immunogenic cell function includes all forms of low immunogenic cell impairment with or without physical removal or depletion of low immunogenic cells. Therefore, impairment of low immunogenic cell function includes the use of antibodies that block the function of low immunogenic cell surface molecules that are critical for low immunogenic cell function.

[0093] Alternatively, peptides that block the function of low immunogenic cell surface molecules (the blocking leads to low immunogenic cell function impairment) can be used to effectively deplete low immunogenic cells in the host organism. Such peptides include but are not limited to those designed to specifically bind to receptor molecules on the surface of low immunogenic cells, and those designed to, for example, inhibit the enzymatic functions necessary in these cells.

[0094] Similarly, genes and oligonucleotides designed for the same purposes as described herein are also included as tools in the methods of the present invention. Therefore, peptides, oligonucleotides and genes that impair the biological functions of low immunogenic cells, as such terms are defined herein, are also considered for use in the methods of the present invention disclosed herein.

[0095] The present invention also encompasses the use of a pharmaceutical composition of an appropriate low immunogenic cell depleting component to practice the methods of the present invention, the composition comprising an appropriate low immunogenic cell depleting component and a pharmaceutically acceptable carrier. In some embodiments, the cell depleting component is a chimeric component comprising an antibody and a toxin.

[0096] As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition that can be combined with an appropriate low immunogenic cell-depleting component and, after combination, can be used to administer the appropriate low immunogenic cell-depleting component to a mammal.

[0097] The pharmaceutical compositions useful in the methods of the present invention can be administered systemically in the form of oral solid dosage forms, ophthalmic, suppositories, aerosols, topical or other similar formulations. In addition to the low immunogenic cell depleting components, such pharmaceutical compositions may also contain pharmaceutically acceptable carriers and other known components that enhance and facilitate drug administration. Other possible formulations such as nanoparticles, liposomes, resealed red blood cells and immunologically based systems may also be used to administer appropriate low immunogenic cell depleting components according to the methods of the present invention.

[0098] Methods of introducing "suicide genes" into cells are disclosed in US20060222633, which is incorporated herein by reference in its entirety.

[0099] The present invention includes a method for depleting low immunogenic cells in a mammalian host. After transplanting low immunogenic cells into the host, the method includes contacting the low immunogenic cells with a cell depletion component to achieve functional impairment of the low immunogenic cells or killing of the low immunogenic cells, thereby depleting the low immunogenic cells in the mammalian host.

[0100] In another aspect, the poorly immunogenic cell-depleting component is selected from the group consisting of a toxin, an antibody, a radioactive molecule, a nucleic acid, a peptide, a peptide mimetic, and a ribozyme.

[0101] In one aspect, the toxin is an immunotoxin. The toxin is selected from ricin, diphtheria toxin and Pseudomonas exotoxin A.

[0102] 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.

[0103] In yet another embodiment, the nucleic acid molecule is selected from the group consisting of a gene and an oligonucleotide.

[0104] In another embodiment, the radioactive molecule is a radiolabeled antibody.

[0105] In another embodiment, the antigen depleting component is a chimeric component comprising an antibody and a toxin. The toxin may be selected from ricin, diphtheria toxin and Pseudomonas exotoxin A.

[0106] In another embodiment, the antibody is selected from 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.

[0107] Combination Products

[0108] The embodiments described herein also disclose a combination product, which refers to a device loaded with low immunogenic cells or therapeutic agents, that is, each can be a candidate medical device or cell product alone, but using them together can make a combination product. In one embodiment, the combination product refers to a perforated device loaded with low immunogenic cells. This is referred to as a "combination product" or "perforated combination product". The device (perforated or not) can be any large cell delivery device described herein, including but not limited to those cell encapsulation devices as described in the following: U.S. Patent Nos. 8,278,106 and 9,526,880, PCT Application No. PCT / US2016 / 0061442 and U.S. Design Patent Nos. D714956, D718472, D718467, D718466, D718468, D718473, D718474, D718475, D718476, D718477, D718478, D718479, D718470, D718471 ...2, D718473, D718474, D718475, D718476, D718477, D718478, D718479, D7 70, D755986, D760399, D761423, D761424 (which are hereby incorporated by reference in their entireties). The cells loaded into the device (perforated or not) can be any of the low immunogenic 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 progenitor cells, endocrine progenitor cells expressing NKX6.1 and INS, immature β cells, immature β cells expressing NKX6.1, INS and MAFB, mature endocrine cells, mature endocrine cells and mature β cells expressing INS, GCG, SST and PP, and mature β cells expressing INS and MAFA.

[0109] The perforated delivery device loaded with mature pancreatic endoderm or pancreatic low immunogenic progenitor cells when implanted in vivo is intended to reduce insulin dependence and / or reduce hypoglycemia in diabetic patients. This includes but is not limited to high-risk type I diabetes patients whose hypoglycemia is not perceived, unstable (fragile) or has received organ transplantation and which can tolerate or have received immunosuppressive therapy. As described in PCT application number PCT / US2016 / 0061442 (incorporated by reference as a whole), the main method of action is to directly promote the human pancreatic endoderm cells (PEC) or pancreatic low immunogenic progenitor cells contained in a permeable, durable, implantable medical device of host vascularization. PEC low immunogenic cells differentiate and mature into therapeutic glucose responsiveness, low immunogenic cells that release insulin after implantation. In this way, the perforated combination product supports the secretion of human insulin. The perforated combination product limits the distribution (outgoing) of PEC low immunogenic cells in vivo. The perforated combination product will be implanted in a location that allows adequate vascular access to maintain the therapeutic low immunogenic cell population within the device and promote distribution of insulin and other pancreatic products to the bloodstream. The perforated combination product is intended to be implanted and explanted with conventional surgical tools and provide therapeutic doses for two years or more. The device is designed to retain an adequate dose of the PEC low immunogenic cell product during formulation, shelf life, handling, and surgical implantation to achieve clinical efficacy and ensure that the cell product is located within the tissue capsule to meet safety requirements.

[0110] Knock in

[0111] In certain embodiments, tolerogenic factors can be inserted or reinserted into a genome-edited stem cell line to produce a universal donor stem cell line for immune immunity. In certain embodiments, the universal stem cells disclosed herein have been further modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, but are not limited to, one or more of the following: HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitors, and IL-35. Any gene editing method can be used to facilitate the insertion of tolerogenic factors (such as the above-mentioned tolerogenic factors) into the AAVS1 locus to actively suppress immune rejection.

[0112] Specifically, in certain embodiments, the invention disclosed herein relates to stem cells whose genome has been altered to reduce or delete key components of at least one MHC-I class gene and at least one NK activating ligand gene and which have been further altered to increase the expression of one or more tolerogenic factors. In certain embodiments, the invention disclosed herein relates to stem cells whose genome has been altered to reduce or delete key components of at least one MHC-I class gene and at least one NK activating ligand gene and which have been further altered to increase the 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.

[0113] Implementation

[0114] Other embodiments of the invention are described with reference to the following numbered paragraphs.

[0115] Blocking antibodies: Composition

[0116] Paragraph 1: A composition comprising cells of pluripotent origin 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.

[0117] Paragraph 2: The composition of Paragraph 1, wherein the agent is an antibody.

[0118] Paragraph 3: The method described in Paragraph 1, wherein the HLA-I class gene is B2M.

[0119] Paragraph 4: The composition of paragraphs 1-3, wherein the NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB or a combination thereof.

[0120] Paragraph 5: The composition of Paragraphs 1-2, wherein the NK cell activating ligands are ICAM-1 and CEACAM1.

[0121] Paragraph 6: The composition of Paragraphs 1-2, wherein the NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA and MICB.

[0122] Paragraph 7: The composition of Paragraphs 1-2, wherein the cell of pluripotent origin further expresses a protein that, when expressed in the presence of a cell death inducer, is capable of killing the pluripotent cell.

[0123] Paragraph 8: The composition of Paragraph 7, wherein the cell death inducing agent is ganciclovir.

[0124] Paragraph 9: The composition of any of Paragraphs 1-8, wherein the cells of pluripotent origin further overexpress one or more tolerogenic factors.

[0125] Paragraph 10: The composition of Paragraph 9, wherein the tolerogenic factor is HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor or IL-35.

[0126] Paragraph 11: The composition as described in Paragraph 9, wherein the tolerogenic factors are HLA-C, HLA-E and HLA-G.

[0127] Involving blocking antibodies: Methods

[0128] Paragraph 1: A method for preventing rejection of a cell transplant of human pluripotent cells, comprising administering to a subject in need of treatment a composition comprising a target cell population lacking at least one HLA-I class gene and at least one agent that binds to an NK cell activating ligand on the target cells in an amount effective to inhibit NK cell attack by the subject, thereby preventing rejection of a cell transplant of human pluripotent cells.

[0129] Paragraph 2: The method of Paragraph 1, wherein the agent is an antibody.

[0130] Paragraph 3: The method of Paragraph 1, wherein the subject's immune response to a NK cell activating ligand is suppressed.

[0131] Paragraph 4: The method of Paragraph 1, wherein the NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA or MICB.

[0132] Paragraph 5: The method as described in Paragraph 1, wherein the NK cell activating ligands are ICAM-1, CEACAM1, CADM1, MICA and MICB.

[0133] Paragraph 6: The method of Paragraph 1, wherein the subject is a human.

[0134] Paragraph 7: The method of Paragraph 2, wherein the antibody is a human antibody.

[0135] Double knockout in hES cells: Composition

[0136] Paragraph 1: An in vitro cell population comprising cells of pluripotent origin, wherein the cells of pluripotent origin lack at least one HLA-I class gene and at least one natural killer (NK) cell activating ligand gene.

[0137] Paragraph 2: The in vitro cell population as described in Paragraph 1, wherein the HLA-I class gene is B2M.

[0138] Paragraph 3: The in vitro cell population as described in paragraphs 1-2, wherein the NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB or a combination thereof.

[0139] Paragraph 4: The in vitro cell population as described in Paragraphs 1-2, wherein the NK cell activating ligands are ICAM-1 and CEACAM1.

[0140] Paragraph 5: The in vitro cell population as described in Paragraphs 1-2, wherein the NK cell activating ligands are ICAM-1, CEACAM1, CADM1, MICA and MICB.

[0141] Paragraph 6: The in vitro cell population of Paragraphs 1-5, wherein the cells of pluripotent origin further express a protein that, when expressed in the presence of a cell death inducer, is capable of killing the cells of pluripotent origin.

[0142] Paragraph 7: The in vitro cell population as described in Paragraph 6, wherein the cell death inducing agent is ganciclovir.

[0143] Paragraph 8: The in vitro cell population of any of Paragraphs 1-7, wherein the cells of pluripotent origin further overexpress one or more tolerogenic factors.

[0144] Paragraph 9: The in vitro cell population as described in paragraph 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.

[0145] Paragraph 10: The in vitro cell population as described in Paragraph 9, wherein the tolerogenic factors are HLA-C, HLA-E and HLA-G.

[0146] Triple knockout involving pluripotent stem cells: composition

[0147] A human pluripotent stem cell comprising a modified genome, comprising: a first genome modification, wherein the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell; (b) a second genome modification, wherein 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 genome modification, wherein the CEACAM1 gene has been edited to reduce or eliminate CEACAM1 surface expression and / or activity in the cell.

[0148] Compositions involving knocking out transcriptional regulators

[0149] Paragraph 12: A cell of pluripotent origin comprising regulated expression of one or more MHC-I or MHC-II class genes or protein complexes and one or more NK cell activating ligands relative to wild-type pluripotent stem cells, wherein one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II class and NK cell activating ligand genes of the pluripotent stem cells are deleted from at least one allele of the cell.

[0150] Paragraph 13: A pluripotent stem cell comprising regulated expression of one or more NK cell activating ligands relative to wild-type human pluripotent stem cells.

[0151] Paragraph 14: A human pluripotent stem cell that does not express B2M or ICAM-1.

[0152] Paragraph 15: A human pluripotent stem cell that does not express CIITA or ICAM-1.

[0153] Paragraph 16: A human pluripotent stem cell that does not express LRC5 or ICAM-1.

[0154] Paragraph 17: A human pluripotent stem cell that does not express one or more of NLRC5, CIITA and B2M, and also does not express one or more of ICAM-1, CEACAM1, CADM1, MICA and MICB.

[0155] Paragraph 18: A human pluripotent stem cell that does not express one or more of HLA-A, HLA-B and HLA-C, and also does not express one or more of ICAM-1, CEACAM1, CADM1, MICA and MICB.

[0156] Paragraph 19: A human pluripotent stem cell that does not express one or more of one or more MHC-I class antigens and one or more NK cell activating ligands, and also has one or more tolerogenic factors inserted into the safe harbor locus of at least one allele of the cell.

[0157] Paragraph 20: A human pluripotent stem cell comprising a modified genome, comprising a first genome modification, wherein the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell and wherein the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell.

[0158] Paragraph 21: A human pluripotent stem cell comprising a modified genome, comprising: a first genome modification, wherein the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell; and (b) a second genome modification, wherein the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell.

[0159] hES double knockout cells: composition

[0160] Paragraph 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.

[0161] Paragraph 2: The in vitro cell population as described in Paragraph 1, wherein the MHC-I class cell surface protein is HLA-A, HLA-B, HLA-C or a combination thereof.

[0162] Paragraph 3: The in vitro cell population as described in Paragraphs 1-2, wherein the MHC-I class cell surface protein is B2M.

[0163] Paragraph 4: The in vitro cell population of any of Paragraphs 1-3, wherein the pluripotent cells lack at least two functional NK cell activating ligand cell surface proteins.

[0164] Paragraph 5: The in vitro cell population of any one of Paragraphs 1-4, wherein the pluripotent cells lack at least three functional NK cell activating ligand cell surface proteins.

[0165] Paragraph 6: The in vitro cell population as described in any of Paragraphs 1-5, wherein the NK cell activating ligand cell surface protein is ICAM-1, CEACAM1, CADM1, MICA, MICB or a combination thereof.

[0166] Paragraph 7: The in vitro cell population as described in any of Paragraphs 1-5, wherein the NK cell activating ligand cell surface proteins are ICAM-1 and CEACAM1.

[0167] Paragraph 8: The in vitro cell population of any of Paragraphs 1-7, wherein the pluripotent cells further express a protein that, when expressed in the presence of a cell death inducer, is capable of killing the pluripotent cells.

[0168] Paragraph 9: The in vitro cell population as described in any of paragraphs 1-8, wherein the protein is herpes simplex virus, thymidine kinase or cytosine deaminase, which is capable of killing the pluripotent cells when expressed in the presence of a cell death inducer.

[0169] Paragraph 10: The in vitro cell population as described in any of Paragraphs 8-9, wherein the cell death inducing agent is ganciclovir.

[0170] hES double knockout cells: composition

[0171] Paragraph 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 relative to wild-type human cells.

[0172] Paragraph 2: An in vitro cell population comprising pluripotent cells, wherein the pluripotent cells have reduced expression of one or more 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 relative to wild-type human cells.

[0173] Paragraph 3: An in vitro cell population comprising pluripotent cells, wherein the pluripotent cells lack functional HLA cell surface protein expression, NK activating ligand cell surface protein expression and have the following protein: when it is expressed in the pluripotent cells in the presence of a cell death inducer, the inducer is capable of killing the pluripotent cells.

[0174] Paragraph 4: A stem cell, wherein the expression of one or more HLA-I class cell surface proteins and one or more NK activating ligand cell surface proteins is modulated relative to wild-type stem cells.

[0175] Paragraph 5: A pluripotent cell, wherein the expression of one or more HLA-I class 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 pluripotent cell, and wherein the pluripotent cell also expresses a protein that, when expressed in the presence of a cell death inducer, the inducer is capable of killing the pluripotent cell.

[0176] Paragraph 6: An in vitro cell population comprising pluripotent cells, wherein the pluripotent cells lack functional MHC-I class genes and natural killer (NK) cell activating ligand genes relative to wild-type pluripotent cells and wherein the pluripotent cells overexpress tolerogenic cell surface protein factors, and wherein the pluripotent cells also express the following protein: when it is expressed in the presence of a cell death inducer, the inducer is capable of killing the pluripotent cells.

[0177] PEC double knockout cells: composition

[0178] Paragraph 1: An in vitro cell population comprising pancreatic endoderm (PEC) cells, wherein the PEC cells lack at least one functional HLA-I class gene and at least one natural killer (NK) cell activating ligand gene.

[0179] Paragraph 2: An in vitro cell population as described in Paragraph 1, wherein the at least one HLA-I class gene is B2M.

[0180] Paragraph 3: The in vitro cell population as described in paragraphs 1-2, wherein the at least one NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB or a combination thereof.

[0181] Paragraph 4: The in vitro cell population as described in Paragraphs 1-2, wherein the at least one NK cell activating ligand is ICAM-1 and CEACAM1.

[0182] Paragraph 5: The in vitro cell population as described in Paragraphs 1-2, wherein the at least one NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA and MICB.

[0183] Paragraph 6: The in vitro cell population of Paragraphs 1-5, wherein the PEC cells further express a protein that, when expressed in the presence of a cell death inducer, is capable of killing the PEC cells.

[0184] Paragraph 7: The in vitro cell population as described in Paragraph 6, wherein the cell death inducing agent is ganciclovir.

[0185] Paragraph 8: The in vitro cell population as described in Paragraph 6, wherein the gene is herpes simplex virus, thymidine kinase or cytosine deaminase, which when expressed in the presence of a cell death inducer is capable of killing PEC cells.

[0186] Paragraph 9: The in vitro cell population of any of Paragraphs 1-8, wherein the PEC cells further overexpress one or more tolerogenic cell surface proteins.

[0187] Paragraph 10: The in vitro cell population as described in paragraphs 1-9, 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.

[0188] Paragraph 11: The in vitro cell population of Paragraphs 1-9, wherein the tolerogenic factors are HLA-C, HLA-E and HLA-G.

[0189] Paragraph 12: An in vitro cell population comprising pancreatic endoderm (PEC) cells, wherein the PEC cells lack at least one functional MHC-I class gene, MHC-II class gene and natural killer (NK) cell activating ligand gene.

[0190] Paragraph 13: An in vitro cell population comprising pancreatic endoderm (PEC) cells, wherein the PEC cells lack at least one functional MHC-I class gene and MHC-II class gene and lack at least two natural killer (NK) cell activating ligand genes.

[0191] Paragraph 14: A pancreatic endoderm (PEC) cell, wherein one or more HLA class I genes and one or more NK activating ligand genes are regulated relative to wild-type PEC cells.

[0192] Paragraph 15: A pancreatic endoderm (PEC) cell, wherein the expression of one or more HLA-I class cell surface proteins, one or more NK activating ligands and one or more tolerogenic factors is modulated relative to wild-type PEC cells, and wherein the pancreatic endoderm cell also expresses a protein that, when expressed in the presence of a cell death inducer, is capable of killing PEC cells.

[0193] Paragraph 16: A pancreatic endoderm (PEC) cell that does not express B2M or ICAM-1.

[0194] Paragraph 17: A pancreatic endoderm (PEC) cell that does not express CIITA or ICAM-1.

[0195] Paragraph 18: A pancreatic endoderm (PEC) cell that does not express LRC5 or ICAM-1.

[0196] Paragraph 19: A pancreatic endoderm (PEC) cell that does not express one or more of NLRC5, CIITA and B2M, and also does not express one or more of ICAM-1, CEACAM1, CADM1, MICa and MICB.

[0197] Paragraph 20: A pancreatic endoderm (PEC) cell that does not express one or more of HLA-A, HLA-B and HLA-C, and also does not express one or more of ICAM-1, CEACAM1, CADM1, MICa and MICB.

[0198] Paragraph 21: A pancreatic endoderm (PEC) cell that does not express one or more MHC-I class cell surface proteins and one or more NK cell activating ligands, and also has one or more tolerogenic factors inserted into a safe harbor locus of at least one allele of the cell.

[0199] Paragraph 22: A pancreatic endoderm (PEC) cell comprising a modified genome, comprising a first genome modification, wherein the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell and wherein the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell.

[0200] Paragraph 23: A pancreatic endoderm (PEC) cell comprising a modified genome, comprising: a first genome modification, wherein the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell; and (b) a second genome modification, wherein the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell.

[0201] Paragraph 24: A pancreatic endoderm (PEC) cell comprising a modified genome, comprising: a first genome modification, wherein the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell; (b) a second genome modification, wherein 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 genome modification, wherein the CEACAM1 gene has been edited to reduce or eliminate CEACAM1 surface expression and / or activity in the cell.

[0202] Involvement of low immunogenic cells

[0203] Paragraph 1: An in vitro cell population comprising low immunogenic cells, wherein the low immunogenic cells lack at least one functional HLA-I class cell surface protein and at least one functional NK cell activating ligand cell surface protein.

[0204] Paragraph 2: The in vitro cell population as described in Paragraph 1, wherein the HLA-I class cell surface protein is B2M.

[0205] Paragraph 3: The in vitro cell population as described in paragraphs 1-2, wherein the NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB or a combination thereof.

[0206] Paragraph 4: The in vitro cell population as described in Paragraphs 1-2, wherein the NK cell activating ligands are ICAM-1 and CEACAM1.

[0207] Paragraph 5: The in vitro cell population as described in Paragraphs 1-2, wherein the NK cell activating ligands are ICAM-1, CEACAM1, CADM1, MICA and MICB.

[0208] Paragraph 6: An in vitro cell population as described in paragraphs 1-5, wherein the low immunogenicity cells further express a protein that, when expressed in the presence of a cell death inducer, is capable of killing the low immunogenicity cells.

[0209] Paragraph 7: The in vitro cell population of any of Paragraphs 1-6, wherein the low immunogenic cells also overexpress one or more tolerogenic factors.

[0210] Paragraph 8: An in vitro cell population as described in paragraphs 1-7, wherein the tolerogenic factor is HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, IL-35 and combinations thereof.

[0211] Paragraph 9: The in vitro cell population described in Paragraphs 1-7, wherein the tolerogenic factors are HLA-C, HLA-E and HLA-G.

[0212] Paragraph 10: An in vitro cell population comprising low immunogenic cells, wherein the low immunogenic cells lack at least one functional MHC-I class gene and at least one NK cell activating ligand gene.

[0213] Paragraph 11: An in vitro cell population comprising low immunogenic cells, wherein the low immunogenic cells lack at least one functional MHC-I class gene, MHC-II class gene and NK cell activating ligand gene.

[0214] Paragraph 12: The in vitro cell population as described in paragraph 10 or 11, wherein the MHC-I class gene is HLA-A, HLA-B, HLA-C or a combination thereof.

[0215] Paragraph 13: The in vitro cell population as described in Paragraph 10, 11 or 12, wherein the MHC class I gene is B2M.

[0216] Paragraph 14: An in vitro cell population as described in any of paragraphs 10-13, wherein the low immunogenic cells lack at least two functional NK cell activating ligand genes.

[0217] Paragraph 15: An in vitro cell population as described in any of paragraphs 10-13, wherein the low immunogenic cells lack at least three functional NK cell activating ligand genes.

[0218] Paragraph 16: The in vitro cell population as described in any of paragraphs 10-13, wherein the NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA, MICB or a combination thereof.

[0219] Paragraph 17: The in vitro cell population of any of Paragraphs 10-13, wherein the NK cell activating ligands are ICAM-1 and CEACAM1.

[0220] Paragraph 18: The in vitro cell population of any of paragraphs 10-13, wherein the NK cell activating ligands are ICAM-1, CEACAM1, CADM1, MICA and MICB.

[0221] Paragraph 19: The in vitro cell population of any of paragraphs 10-18, wherein the low immunogenicity cells further express a protein that, when expressed in the presence of a cell death inducer, is capable of killing the low immunogenicity cells.

[0222] Paragraph 20: The in vitro cell population of any of paragraphs 7-15, wherein the low immunogenic cells are hES cells or pancreatic lineage cells.

[0223] Paragraph 21: A low immunogenic cell, wherein the expression of one or more HLA-I class cell surface proteins and one or more NK activating ligand cell surface proteins is modulated relative to wild-type low immunogenic cells.

[0224] Paragraph 22: A low immunogenic cell, wherein the expression of one or more HLA-I class cell surface proteins, one or more NK activating ligands and one or more tolerogenic cell surface protein factors is regulated relative to wild-type low immunogenic cells, and wherein the pluripotent cell also expresses the following protein: when it is expressed in the presence of a cell death inducer, the inducer is capable of killing the low immunogenic cell.

[0225] Paragraph 23: A low immunogenic stem cell comprising regulated expression of one or more MHC-I or MHC-II cell surface proteins and one or more NK cell activating ligands relative to a wild-type pluripotent stem cell, wherein the pluripotent stem cell has one or more genes encoding one or more transcriptional regulators of MHC-I or MHC-II and the NK cell activating ligand gene is deleted from at least one allele of the cell.

[0226] Paragraph 24: A low immunogenic cell comprising regulated expression of one or more NK cell activating ligands relative to wild-type human low immunogenic cells.

[0227] Paragraph 25: A human low immunogenic cell that does not express B2M or ICAM-1.

[0228] Paragraph 26: A human low immunogenic cell that does not express CIITA or ICAM-1.

[0229] Paragraph 27: A human low immunogenic cell that does not express LRC5 or ICAM-1.

[0230] Paragraph 28: A human low immunogenic cell that does not express one or more of NLRC5, CIITA and B2M and also does not express one or more of ICAM-1, CEACAM1, CADM1, MICa and MICB.

[0231] Paragraph 29: A human low immunogenic cell that does not express one or more of HLA-A, HLA-B and HLA-C and does not express one or more of ICAM-1, CEACAM1, CADM1, MICa and MICB.

[0232] Paragraph 30: A human low immunogenic cell that does not express one or more of one or more MHC-I class cell surface proteins and one or more NK cell activating ligands, and also has one or more tolerogenic factors inserted into a safe harbor locus of at least one allele of the cell.

[0233] Paragraph 31: A low immunogenic cell comprising a modified genome, comprising a first genome modification, wherein the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell and wherein the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell.

[0234] Paragraph 32: A low immunogenic stem cell comprising a modified genome, comprising: a first genome 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 genome modification, in which the ICAM-1 gene has been edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell.

[0235] Paragraph 33: A low immunogenic stem cell comprising a modified genome, comprising: a first genome modification, wherein the B2M gene has been edited to reduce or eliminate B2M surface expression and / or activity in the cell; (b) a second genome modification, wherein 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 genome modification, wherein the CEACAM1 gene has been edited to reduce or eliminate CEACAM1 surface expression and / or activity in the cell.

[0236] Methods for double knockout

[0237] Paragraph 1: A method of reducing transplant rejection comprising:

[0238] a) administering to a subject in need of a transplant an effective amount of a transplant comprising a population of pancreatic endoderm cells, 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.

[0239] Paragraph 2: The method as described in Paragraph 1, wherein the HLA-I class cell surface protein is B2M.

[0240] Paragraph 3: The method as described in Paragraphs 1-2, wherein the NK cell activating ligand cell surface protein is ICAM-1, CEACAM1, CADM1, MICA, MICB or a combination thereof.

[0241] Paragraph 4: The method as described in Paragraphs 1-2, wherein the NK cell activating ligands are ICAM-1 and CEACAM1.

[0242] Paragraph 5: The method as described in Paragraphs 1-2, wherein the NK cell activating ligand is ICAM-1, CEACAM1, CADM1, MICA and MICB.

[0243] Paragraph 6: A method for depleting low immunogenic cells in a cell population, the method comprising contacting the low immunogenic cells with a low immunogenic cell depletion component to achieve functional impairment of the low immunogenic cells or killing of the low immunogenic cells, thereby depleting the low immunogenic cells in the cell population.

[0244] Paragraph 7: The method of claim 6, wherein the low immunogenic cells lack expression of at least one functional HLA-I class cell surface protein and at least one NK activating ligand.

[0245] Paragraph 8: A method for removing low immunogenic cells from a host mammal, the method comprising: (a) transferring low immunogenic cells to the host mammal; and (b) contacting the host with a low immunogenic cell depletion component to achieve impaired function of the low immunogenic cells or killing of the low immunogenic cells, thereby removing the low immunogenic cells in the host mammal.

[0246] Paragraph 9: The method of Paragraph 8, wherein the function of at least one HLA-I class cell surface protein and at least one NK activating ligand is impaired.

[0247] Paragraph 10: A method for increasing NK activating ligands in a target cell population, comprising exposing the target cell population to IFN-γ stimulation, thereby increasing NK activating ligands in the target cells compared to wild type.

[0248] Multipotent cells with disrupted or inhibited functions involving MHC class I genes and NK cell activating ligand genes: a combination thing

[0249] Paragraph 1: An in vitro cell population comprising pluripotent cells, wherein the function of at least one major histocompatibility complex (MHC) class I gene and at least one natural killer (NK) cell activating ligand gene is disrupted or inhibited.

[0250] Paragraph 2: The in vitro cell population as described in Paragraph 1, wherein the MHC gene encodes beta-2 microglobulin (B2M).

[0251] Paragraph 3: The in vitro cell population as described in Paragraph 1, wherein the NK cell activating ligand is ICAM1, CD58, PVR, CEACAM1, CADM1, MICA, MICB or a combination thereof.

[0252] Paragraph 4: The in vitro cell population as described in Paragraph 1, wherein the NK cell activating ligands are ICAM1 and CD58.

[0253] Paragraph 5: The in vitro cell population as described in Paragraph 1, wherein the NK cell activating ligands are ICAM1, CD58, CD155, CEACAM1, CADM1, MICA and MICB.

[0254] Paragraph 6: The in vitro cell population as described in Paragraph 1, wherein the pluripotent cells are human embryonic stem cells.

[0255] Paragraph 7: The in vitro cell population as described in Paragraph 1, wherein the pluripotent cells differentiate into pancreatic endoderm cells.

[0256] Paragraph 8: The in vitro cell population of Paragraph 1, wherein the pluripotent cells further comprise a protein that, when expressed in the presence of a cell death inducer, is capable of killing the cell.

[0257] Paragraph 9: An in vitro cell population as described in Paragraph 1, wherein the MHC-I class gene is disrupted using genome editing.

[0258] Paragraph 10: An in vitro cell population as described in paragraph 9, wherein the genome editing application is zinc finger nuclease (ZFN), clustered regularly interspaced short palindromic repeats (CRISPR) / cas or transcription activator-like effector nuclease (TALEN) system.

[0259] Paragraph 11: An in vitro cell population as described in Paragraph 1, wherein the NK cell activating ligand is destroyed using genome editing.

[0260] Paragraph 12: The in vitro cell population as described in Paragraph 1, wherein the NK cell activating ligand is destroyed using an anti-NK cell activating ligand agent.

[0261] Paragraph 13: The in vitro cell population as described in Paragraph 8, wherein the inducing agent is an antibody.

[0262] Pancreatic lineage cells are involved in the disruption or suppression of the function of MHC class I genes and NK cell activating ligand genes. Cell: Composition

[0263] Paragraph 14: An in vitro cell population comprising pancreatic lineage cells, wherein 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.

[0264] Paragraph 15: The in vitro cell population as described in Paragraph 14, wherein the MHC class I gene encodes B2M.

[0265] Paragraph 16: The in vitro cell population as described in Paragraph 14, wherein the NK cell activating ligand is ICAM1, CD58, CD155, CEACAM1, CADM1, MICA, MICB or a combination thereof.

[0266] Paragraph 17: The in vitro cell population as described in Paragraph 14, wherein the NK cell activating ligands are ICAM1 and CD58.

[0267] Paragraph 18: The in vitro cell population as described in Paragraph 14, wherein the NK cell activating ligands are ICAM1, CD58, CD155, CEACAM1, CADM1, MICA and MICB.

[0268] Paragraph 19: The in vitro cell population of Paragraph 14, wherein the pancreatic lineage cell is a definitive endoderm, foregut endoderm, pancreatic endoderm cell, an endocrine precursor, or an insulin-producing cell.

[0269] Paragraph 20: The in vitro cell population of Paragraph 14, wherein the pancreatic lineage cells further express a protein that when expressed in the presence of a cell death inducer is capable of killing the cell.

[0270] Method for preventing rejection of cell transplants of pancreatic cells

[0271] Paragraph 21: A method for preventing cell transplant rejection of human pancreatic cells, comprising:

[0272] 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

[0273] b. Transplanting a human pancreatic cell population into a mammalian subject, wherein the absence of MHC class I cell surface proteins and NK activating ligand expression prevents cell transplant rejection.

[0274] Paragraph 22: The method of Paragraph 21, wherein the major histocompatibility complex (MHC) class I cell surface protein is beta-2 microglobulin (B2M) or HLA-ABC cell surface protein.

[0275] Paragraph 23: The method of Paragraph 21, wherein the NK cell activating ligand is ICAM1, CD58, CD155, PVR, CEACAM1, CADM1, MICA, MICB or a combination thereof.

[0276] Paragraph 24: The method of Paragraph 21, wherein the human pancreatic cell population is pancreatic endoderm cells (PECs).

[0277] Paragraph 25. The method of paragraph 21, wherein the human pancreatic cell population further comprises a protein that, when expressed by the human cell population in the presence of a cell death inducer, is capable of killing the human pancreatic cell population.

[0278] How insulin is produced

[0279] Paragraph 26. A method of producing insulin, comprising:

[0280] a) providing a population of human pancreatic endoderm 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

[0281] b) transplanting a population of human pancreatic endoderm cells into a mammalian subject, wherein the pancreatic endoderm cells mature in the mammalian subject and produce insulin in response to glucose stimulation.

[0282] Paragraph 27: The method of Paragraph 26, wherein the major histocompatibility complex (MHC) class I cell surface protein is beta-2 microglobulin (B2M) or HLA-ABC cell surface protein.

[0283] Paragraph 28. The method of paragraph 26, wherein the NK cell activating ligand is ICAM1, CD58, CD155, PVR, CEACAM1, CADM1, MICA, MICB or a combination thereof.

[0284] Paragraph 29: The method of Paragraph 26, wherein the human pancreatic cell population further comprises a protein that, when expressed by the human pancreatic cell population in the presence of a cell death inducer, the inducer is capable of killing the human pancreatic cell population.

[0285] Paragraph 30. The method of paragraph 29, wherein the protein is herpes simplex virus thymidine kinase and the agent is ganciclovir.

[0286] A method for preparing low-immunogenic stem cells, the method comprising regulating the expression of one or more MHC-I class cell surface proteins and one or more NK activation ligands of low-immunogenic stem cells, thereby preparing low-immunogenic stem cells.

[0287] A method for preparing low-immunogenic stem cells, the method comprising regulating the expression of one or more MHC-I class cell surface proteins, one or more NK activation ligands and one or more tolerogenic factors on stem cells, thereby preparing low-immunogenic stem cells.

[0288] A method for regulating the expression of one or more MHC-I class cell surface proteins and NK cell activating ligand cell surface proteins on stem cells, comprising deleting one or more transcriptional regulators encoding MHC-I class genes and one or more genes encoding NK cell activating ligands from at least one allele of the cell, thereby regulating the expression of one or more MHC-I class cell surface proteins and NK cell activating ligand cell surface proteins.

[0289] A low immunogenic stem cell comprising a modified genome, comprising (a) a first genome modification, wherein the B2M gene is edited to reduce or eliminate B2M surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 1-3 or a combination of ribonucleic acids homologous to any one of SEQ ID NOs: 1-3; and (b) a second genome modification, wherein the ICAM-1 gene is edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 4-6 or a combination of ribonucleic acids homologous to any one of SEQ ID NOs: 4-6.

[0290] A low immunogenic stem cell comprising a modified genome, comprising (a) a first genome modification, wherein the B2M gene is edited by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein and a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 1-3 or a combination of ribonucleic acids homologous to any one of SEQ ID NOs: 1-3 to reduce or eliminate B2M surface expression and / or activity in the cell; (b) a second genome modification, wherein the ICAM-1 gene is edited by contacting the cell with a Cas protein or a nucleic acid encoding a Cas protein and a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 4-6 or a combination of ribonucleic acids homologous to any one of SEQ ID NOs: 4-6 to reduce or eliminate ICAM-1 surface expression and / or activity in the cell; and (c) a third genome modification, wherein the cell is contacted with a Cas protein or a nucleic acid encoding a Cas protein and a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 7-9 or a combination of ribonucleic acids homologous to SEQ ID NOs: 8-10. The method comprises contacting the CEACAM1 gene with a combination of RNAs homologous to any one of Nos. 7-9 to edit the CEACAM1 gene to reduce or eliminate the surface expression and / or activity of CEACAM1 in cells.

[0291] A pluripotent stem cell comprising a modified genome, comprising (a) a first genome modification, wherein the B2M gene is edited to reduce or eliminate B2M surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 1-3 or a combination of ribonucleic acids homologous to any one of SEQ ID NOs: 1-3; and (b) a second genome modification, wherein the ICAM-1 gene is edited to reduce or eliminate ICAM-1 surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a plasmid encoding a ribonucleic acid homologous to any one of SEQ ID NOs: 4-6 or a combination of ribonucleic acids homologous to any one of SEQ ID NOs: 4-6.

[0292] The sequences of SEQ ID Nos: 1-9 are provided in Table 4 below, and these and additional sequences are described below.

[0293] SEQ ID NO. 1: Exon 1, negative strand.

[0294] SEQ ID NO. 2: Exon 2, negative strand.

[0295] SEQ ID NO.3: Exon 1, negative strand.

[0296] SEQ ID NO.4: Exon 2, positive strand.

[0297] SEQ ID NO.5: Exon 2, negative strand.

[0298] SEQ ID NO.6: Exon 1, positive strand.

[0299] SEQ ID NO.7: Exon 1, negative strand.

[0300] SEQ ID NO.8: Exon 1, positive strand.

[0301] SEQ ID NO.9: Exon 1, positive strand.

[0302] SEQ ID NO. 10: Coding sequence of human ICAM1.

[0303] SEQ ID NO.11: Coding sequence of human CEACAM1.

[0304] SEQ ID NO.12: Coding sequence of human B2M.

[0305] SEQ ID NO. 13: Coding sequence of human CADM1.

[0306] SEQ ID NO. 14: Coding sequence of human CD58.

[0307] SEQ ID NO.15: Coding sequence of human CD155.

[0308] The table below provides target sequences for CRISPR / Cas9 cleavage, including the PAM (NGG).

[0309]

[0310]

[0311] A pancreatic endoderm (PEC) cell comprising a modified genome, comprising (a) a first genome modification, wherein the B2M gene is edited by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a ribonucleic acid comprising the sequence of any one of SEQ ID NOs: 1-3 to reduce or eliminate B2M surface expression and / or activity in the cell; (b) a second genome modification, wherein the ICAM-1 gene is edited by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a ribonucleic acid comprising the sequence of any one of SEQ ID NOs: 4-6 to reduce or eliminate ICAM-1 surface expression and / or activity in the cell; and (c) a third genome modification, wherein the CEACAM1 gene is edited by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a ribonucleic acid comprising the sequence of any one of SEQ ID NOs: 7-9 to reduce or eliminate CEACAM1 surface expression and / or activity in the cell.

[0312] A method for alleviating hypoglycemia, comprising: a) administering to a subject in need of a transplant an effective amount of a transplant comprising a pancreatic endoderm cell population, wherein the function of at least one HLA-I class cell surface protein and at least one NK cell activating ligand cell surface protein is disrupted, wherein the pancreatic endoderm cell population matures in vivo and produces insulin in response to glucose stimulation in vivo, thereby alleviating hypoglycemia in the patient.

[0313] A method for reducing insulin dependence, comprising: a) administering to a subject in need of a transplant an effective amount of a transplant comprising a pancreatic endoderm cell population, wherein the function of at least one HLA-I class cell surface protein and at least one NK cell activating ligand cell surface protein is disrupted, wherein 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.

[0314] definition

[0315] "Low immunogenicity" or "universal donor cells" or "mutated cells" or their equivalents means cells with reduced or eliminated expression of at least one HLA-I class cell surface protein and at least one NK activating ligand. Such cells are expected to be less susceptible to immune rejection or graft rejection by subjects transplanted with such cells or transplants. For example, such low immunogenic cells may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less susceptible to immune rejection by subjects transplanted with such cells relative to unaltered wild-type cells.

[0316] The terms "treat" or "cure" or their equivalents refer to therapeutic intervention that alleviates (ameliorates) signs or symptoms.

[0317] The term "patient" or "host" or "mammalian host" or "subject" or its equivalent refers to a living vertebrate organism, a class that includes both humans and non-human mammals. In some embodiments, the subject is a human subject. The preferred treatment patient is a human. The target patient population may change over time with clinical use / experience in a manner independent of the combination product itself but rather related to the nature of the immunosuppressive regimen or the lack thereof. For example, the combination product can be used for a T1D population using a low immunogenic cell therapy in conjunction with an immunosuppressive drug (ISD) regimen to achieve surgical tolerance or low toxicity and side effects.

[0318] The term "blocking agent" herein refers to any agent that can bind to NK activating ligands on the surface of target cells, including but not limited to antibodies; or refers to an agent that prevents or inhibits the expression of NK activating ligands, including but not limited to proteins, enzymes or chemicals currently known or to be developed later.

[0319] The term "antibody" herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological blocking activity.

[0320] The term "antibody fragment" as used herein refers to a portion of an intact antibody, preferably comprising its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0321] 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, results in the prevention or reduction of the ability of NK cells to lyse the target cell.

[0322] As used herein, the term "syngeneic" or "syngeneic" refers to cells, tissues or organs that are genetically identical to or derived from a genetically identical source (such as identical twins) as the transplant recipient, particularly with respect to antigens or immune responses. Such cells, tissues or organs are referred to as syngeneic transplants. As used herein, the term "allogeneic" or "allogeneic" refers to cells, tissues or organs that are genetically non-identical to or derived from a source that is not genetically identical to the transplant recipient (e.g., an unrelated donor), particularly with respect to antigens or immune responses. Such cells, tissues or organs are referred to as allografts, allografts, allografts or xenografts.

[0323] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence necessary for the expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may be, for example, a promoter / regulatory sequence that expresses a gene product in a tissue-specific manner.

[0324] The term "effective amount" or "therapeutically effective amount" or its equivalent refers to the amount of an agent sufficient to achieve the desired effect in the subject or cell being treated. For example, this can be the amount of cells necessary to inhibit or measurably reduce blood glucose levels and ultimately achieve homeostatic glycemic control. It can also mean an effective amount of an agent to change the function or structure of a cell or subject. A therapeutically effective amount of an agent can be administered in a single dose or in several doses. However, the effective amount will depend on the specific agent applied, the subject being treated, the severity and type of illness, and the mode of administration.

[0325] The terms "reduce", "destroy", "reduced", "reduce" and "inhibit" are all used herein generally to mean reduction, specifically reduction of a statistically significant amount. However, for the avoidance of doubt, "reduced", "reduced", "reduced", "inhibited" include a reduction of at least 10% compared to a reference level, such as a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% reduction (i.e., no level compared to a reference sample), or any reduction between 10-100%, or at least about 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 reduction compared to a reference level, or any reduction between 2-fold and 10-fold or greater.

[0326] The terms "increased", "increase" or "enhance" or "activate" are all generally meant herein to increase by a statistically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhance" or "activate" mean an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase, or any increase between 10-100%, or at least about 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 compared to a reference level, or any increase between 2-fold and 10-fold or greater.

[0327] The term "statistically significant" or "significantly" refers to statistical significance and generally means two standard deviations (2SD) below the normal concentration or lower concentration of a reference. The term can also mean two standard deviations (2SD) higher than the normal concentration or higher concentration of a reference. The term refers to statistical evidence of the presence 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. The decision is usually made using a p-value.

[0328] As used herein, "reduced hypoglycemia" or its equivalent means a reduction in the number of hypoglycemic episodes without worsening glycemic control, which is defined as an increase in HbA1c of ≤ 0.2%.

[0329] 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, which is defined as an increase in HbA1c ≤ 0.2%.

[0330] As used herein, "tissue capsule" or its equivalent means a foreign body pocket formed around an implant or graft. The cell-containing combination product and / or device or perforated device is intended to remain within the capsule during implantation.

[0331] "Engraftment" or its equivalent refers to the differentiation of a progenitor or immature cell population into a mature cell type. For example, an implanted PDX1 positive pancreatic endoderm cell population matures into a pancreatic endocrine cell population.

[0332] "Graft" refers to a differentiated cell population encapsulated or delivered in a device herein. For example, cell populations include, but are not limited to, pancreatic endoderm, pancreatic progenitor cells, PDX-1 positive pancreatic endoderm, pancreatic endocrine precursors, pancreatic endocrine, single or multiple hormone endocrine, pre-β, β and / or insulin secreting grafts.

[0333] The term "substantially" or "essentially" or their equivalents means a majority or minimal or reduced amount of a component or cell present in any cell population or culture, such as an immature β cell culture is "substantially or substantially immature β cells that express INS, NKX6.1, and PDX1 and do not substantially or substantially express NGN3". Other examples include, but are not limited to, "substantially or substantially hES cells", "substantially or substantially definitive endoderm cells", "substantially or substantially foregut endoderm cells", "substantially or substantially PDX1-negative foregut endoderm cells", "substantially or substantially PDX1-positive pancreatic endoderm cells", "substantially or substantially pancreatic endocrine precursor cells", "substantially or substantially pancreatic endocrine cells", etc.

[0334] The term "substantially free," or its equivalent, with respect to cells in a cell culture or in a cell population, means that the cell culture or cell population does not contain 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 in the cell culture or cell population.

[0335] The term "nonwoven" or its equivalents includes, but is not limited to, bonded, formed or engineered fabrics made by processes other than weaving or knitting.

[0336] It should be understood that the invention disclosed herein is not limited in its application to the details set forth in the specification or as examples. The present invention encompasses other embodiments and can be practiced or executed in various ways. In addition, it should be understood that the words and terms used herein are for descriptive purposes and should not be considered as limiting.

[0337] Although certain compositions, methods, and assays of the present invention have been specifically described according to certain embodiments, the following examples are merely illustrative of the methods and compositions of the present invention and are not intended to limit the same.

[0338] The articles "a" and "an" as used herein in the specification and claims are to be understood to include plural referents unless expressly indicated to the contrary. Claims or specifications containing "or" between one or more members of the group are considered to be satisfied if one, more than one, or all of the group members are present, used, or otherwise associated with a given product or process, unless indicated to the contrary or otherwise obvious from the context. The invention includes embodiments in which exactly one member of the group is present, used, or otherwise associated with a given product or process. The invention also includes embodiments in which more than one or all of the group members are present, used, or otherwise associated with a given product or process.

[0339] In addition, it should be understood that the present invention encompasses all variations, combinations and permutations, wherein one or more limitations, elements, clauses, descriptive terms, etc. from one or more listed claims are introduced into another claim (or any other claim as related) that is dependent on the same basic claim, unless otherwise indicated or unless it is obvious to a person of ordinary skill in the art that contradictions or inconsistencies will occur. Where elements are presented in a list form (e.g., in a Markush group or similar format), it should be understood that each subgroup of the elements is also disclosed, and any one or more elements can be removed from the group. It should be understood that, in general, when the present invention or aspects of the present invention are referred to as including specific elements, features, etc., certain embodiments of the present invention or aspects of the present invention are composed of or substantially composed of such elements, features, etc. For simplicity, those embodiments are not specifically described in this article with too many words in any case. It should also be understood that any embodiment or aspects of the present invention can be explicitly excluded from the claims, regardless of whether specific exclusions are listed in the specification. The disclosure and other reference materials cited herein that describe the background of the present invention and provide additional details about its practice are incorporated herein by reference. The present disclosure is illustrated by the following non-limiting examples. Example

[0340] Example 1: Generation of B2M-deficient hES cells

[0341] B2M-deficient hES cells were generated using the CyT49 cell line, however any human pluripotent stem cell line can be used. Targeted disruption of the B2M gene generates cells that do not express any HLA-I class proteins on their cell surface. Using CRISPR / Cas9 technology and using the known techniques outlined in PCT Publication No. WO2016183041A (which is incorporated herein in its entirety), two alleles of the B2M locus in the CyT49 hESC line were destroyed. However, other nucleases including zinc finger nucleases (ZFNs) and transcription activator effector nucleases (TALENs) can also be used to edit genes, as well as traditional homologous recombination, etc. Examples of B2M public sequences are submitted as SEQ ID NOs: 1, 2, and 3. NEXTGEN TMCRISPR (Transposagen Inc., Lexington Kentucky) is used to edit the gene, which incorporates dual guide RNA and catalytically inactive Cas9 protein fused to FokI nuclease. Plasmids containing guide RNA and Cas9 are electroporated into CyT49 hESCs, and the cells are seeded on tissue culture plates. Twelve days after electroporation, cells that react negatively with B2M antibodies (BioLegend catalog number 316306) are sorted by fluorescence activated cell sorting (FACS). The sorted cells are spread at a clonal density. Select a single clone and spread it on about the 25th day. The clones are expanded and cryopreserved. Amplified clones that show normal karyotype by G banding and are negative for B2M protein expression and HLA-I class protein surface expression by flow cytometry and / or immunofluorescence are selected for further experiments.

[0342] B2M surface expression was assessed by flow cytometry in wild-type (WT) and knockout cells under normal and inflammatory conditions (following exposure to interferon (IFN)-γ). Figure 2A : Normal: untreated growth medium (line B); Inflammation: exposure to 100 ng / mL of IFN-γ for 18-24 hours (line A). Inflammatory responses occur in association with tissue trauma. This releases pro-inflammatory cytokines, some of which are IL-1-α, IL-1-β, TNF-α, IL-6, IL-8, and IFN-γ. Although WT and B2M- / - ESCs and PECs have been treated with IFN-γ, these observations can also be extended with other cytokines.

[0343] Figure 2A Shown is the expression of B2M in WT hES cells (B line) without IFN-γ, and the expression of B2M after WT hES cells are exposed to IFN-γ (A line). Compared with the background (shaded area), the fluorescence intensity of untreated WT hES cells (B line) shifts (increases) indicating that WT hES cells express B2M. After WT hES cells are exposed to IFN-γ, the further displacement (increase) of the fluorescence intensity beyond WT B2M expression (B line) indicates that B2M expression in WT hES cells after exposure to IFN-γ increases (A line). In this way, the expression of HLA-I class cell surface proteins can be upregulated during cell stress and inflammation (such as at least caused by IFN-γ treatment).

[0344] Figure 2BB2M expression in B2M knockout hES cells is shown, and knockout cells were generated using the CRISPR / Cas system. There is essentially no shift (increase) in fluorescence intensity compared to the background (shaded area) with or without exposure to IFN-γ, indicating that B2M knockout hES cells have reduced or eliminated B2M surface expression, and that B2M expression has not been induced by IFN-γ treatment. In such B2M knockouts, the expression of HLA-I class cell surface proteins is not upregulated upon cell stress and inflammation caused by IFN-γ treatment.

[0345] This example demonstrates that B2M knockout hES cells have reduced or eliminated B2M surface expression as visualized using a B2M antibody.

[0346] Example 2: Analysis of HLA class I cell surface protein expression in WT and B2M-deficient cells

[0347] Next, wild-type and B2M knockout hES cells were analyzed using Pan-HLA-ABC monoclonal antibodies (BD Pharmingen, catalog number 560169) to confirm that these knockout cells did not express HLA-I class proteins on the cell surface. Pan-HLA-ABC antibodies react with human major histocompatibility complex (MHC) class I proteins HLA-A, -B and -C. In normal and inflammatory states, after exposure to IFN-γ, the expression of Pan-HLA-ABC antibodies in wild-type and knockout cells was assessed by flow cytometry. Normal: no IFN-γ (B line); Inflammatory: exposure to 100ng / mL IFN-γ for 18-24 hours (A line).

[0348] Figure 3A Shown is Pan-HLA-ABC cell surface protein expression in WT hES cells (B line) and after treatment of WT hES cells with IFN-γ (A line). A shift (increase) in fluorescence intensity in untreated WT hES cells (B line) compared to background (shaded area) indicates that WT hES cells express Pan-HLA-ABC. A shift (increase) in fluorescence intensity beyond that expressed by WT hES after exposure to IFN-γ indicates increased expression of Pan-HLA-ABC in WT hES cells after exposure to IFN-γ.

[0349] Figure 3B Shown is the expression of Pan-HLA-ABC cell surface proteins in B2M knockout hES cells using the CRISPR / Cas system. There is no shift (increase) in fluorescence intensity compared to the background (shaded area) with or without exposure to IFN-γ, indicating that the knockout reduced or eliminated HLA-I class cell surface expression and that HLA-I class protein expression was not induced by IFN-γ treatment.

[0350] This example demonstrates that B2M knockout hES cells have reduced or eliminated HLA class I cell surface expression as shown using the Pan-HLA-ABC antibody.

[0351] Example 3: Differentiation of B2M-deficient cells into pancreatic lineage cells

[0352] B2M knockout hES cells were cultured, passaged and propagated under the same conditions as WT hES cells, as described in Schulz et al. A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells PLoS One 7: 51-17 (2012) and as described in U.S. Patent 8,895,300, both of which are incorporated herein by reference in their entirety. Specifically, Schulz et al. describe adherent hESC expansion and suspension-based differentiation.

[0353] Briefly, over the course of approximately 2 weeks (or 14 days), a four (4) stage procedure was used to separate WT hES cells and B2M - / -hES cells were differentiated in suspension aggregates to generate a population of pancreatic cell types, including pancreatic progenitor cells, endocrine progenitor cells, and hormone-expressing cells, collectively referred to as pancreatic endoderm cells (PECs). Human ES cells were isolated using accutase and single cells were aggregated in roller bottles. To initiate differentiation, the aggregates were pooled into one or more conical tubes and allowed to settle by gravity, then washed with stage 1 medium (RPMI + 0.2% vol / vol FBS, containing 1:5000 dilution of insulin-transferrin-selenium (ITS)) without growth factors. The aggregates were reprecipitated and then resuspended in day 1 medium (which contained RPMI + 0.2% vol / vol FBS, containing 1:5000 dilution of insulin-transferrin-selenium (ITS), activin A (100ng / mL) and wnt3a (50ng / mL)) and dispensed into roller bottles at a density of 2uL / mL. The roller bottles were placed on a FlexiRoll digital unit roller (Argos Technologies) at a speed of 31rpm. During the remainder of the differentiation process, the cultures were rotated at approximately 31 rpm and the media was changed daily to those described in Table 2 below, adapted from Schulz et al., (2012), supra. hES cell growth, passage, and proliferation were essentially as described in U.S. Pat. Nos. 7,964,402; 8,211,699; 8,334,138; 8,008,07; and 8,153,429. Table 2 below provides a standard manufacturing method for preparing pancreatic endoderm cells (PECs) derived from human embryonic stem cells.

[0354] Table 2: Standard manufacturing method for preparing hESC-derived pancreatic endoderm cells (PECs)

[0355]

[0356] 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 LifeTechnologies), 10 ng / mL heregulin-1β (Peprotech) and 10 ng / mL activin A (R&D Systems); SP: hESC SFM (Life Technologies); r0.2FBS: RPMI 1640 (Mediatech); 0.2% FBS (HyClone), 1x GlutaMAX-1 (Life Technologies), 1% v / v penicillin / streptomycin; ITS: 1:5000 or 1:1000 dilution of insulin-transferrin-selenium (Life Technologies); 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 0.5x 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).

[0357] As shown in Table 3, differentiated B2M- / - and WT PECs were analyzed using flow cytometry to determine the relative amounts of endocrine and pancreatic progenitor cells in the stage 4 population.

[0358] Table 3: Stage 4 pancreatic progenitor cell composition (percentage of total cells)

[0359]

[0360] 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 were largely similar to those observed in WT cells (upper row).

[0361] This example demonstrates that B2M - / - hES cells can differentiate along the pancreatic lineage like WT hES cells.

[0362] Example 4: Analysis of B2M expression in WT and B2M-deficient pancreatic endoderm cells

[0363] Next, wild-type and B2M knockout pancreatic endoderm cells (PECs) from Example 3 were analyzed using flow cytometry without and with IFN-γ treatment: without IFN-γ (line B); exposed to 100 ng / mL of IFN-γ for 18-24 hours (line A).

[0364] Figure 4A B2M expression in WT PEC cells without IFN-γ treatment (line B) and after treatment with IFN-γ (line A) is shown. 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. After exposing WT PEC to IFN-γ (line A), the shift (increase) in fluorescence intensity over WT expression indicates that B2M expression in WT PEC cells increases after exposure to IFN-γ. That is, exposure to IFN-γ increases B2M expression in WT PEC.

[0365] Figure 4B B2M expression in PEC cells derived from B2M knockout hES cells is shown. There was no change (increase) in fluorescence intensity compared to the background (shaded area) with or without exposure to IFN-γ, indicating that B2M knockout PECs reduced or abolished B2M cell surface expression, while B2M expression could not be induced in B2M knockout hES cell-derived PECs by IFN-γ treatment.

[0366] This example shows that hES cell-derived PECs in which expression of B2M is modulated / ablated have reduced or eliminated B2M surface expression.

[0367] Example 5: Analysis of HLA class I cell surface protein expression in WT and B2M knockout pancreatic endoderm cells

[0368] Wild-type and B2M knockout PECs were analyzed for HLA class I cell surface expression using Pan-HLA-ABC monoclonal antibody (BD Pharmingen, catalog number 560169) similar to Example 2. Expression was assessed in wild-type and knockout cells 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).

[0369] Figure 5AHLA-class I cell surface protein expression in WT PECs under untreated conditions (line B) and after treatment with IFN-γ (line A) is shown. The shift (increase) in fluorescence intensity of untreated WT PECs (line B) compared to background (shaded area) indicates that WT PECs express HLA-class I on the cell surface. After exposure to IFN-γ, the shift (increase) in fluorescence intensity over that of WT PECs indicates an increase in HLA class I cell surface expression in WT PEC cells after exposure to IFN-γ.

[0370] Figure 5B HLA class I cell surface protein expression in PECs derived from B2M knockout hES cells is shown. There was no change (increase) in fluorescence intensity compared to background (shaded area) in PEC cells with or without exposure to IFN-γ, indicating that the above knockout has reduced or eliminated HLA surface expression and that expression of HLA class I in B2M- / -PECs cannot be induced by IFN-γ treatment.

[0371] Thus, reduced or eliminated HLA class I cell surface expression was observed in those PEC cells in which expression of B2M was modulated / eliminated.

[0372] Example 6: Analysis of ICAM-1 cell surface protein expression in WT and B2M knockout HES cells

[0373] To further define the effects of IFN-γ treatment on target cells, ICAM-1 expression in WT and B2M knockout hES cells was evaluated by flow cytometry under the following two conditions: (1) untreated (B line) and (2) 100 ng / mL IFN-γ treated for 18-24 hours (A line). ICAM-1 is required for several immune functions including antigen presentation in target cells and is a known NK activating ligand. In vivo, it is possible to obtain immunological benefits by disrupting intercellular ICAM / LFA binding interactions using specific monoclonal antibodies ("mAbs"), i.e., anti-ICAM-1 or anti-LFA-1. See Isobe et al., Specific Acceptance of Cardiac Allograft After Treatment With Antibodies to ICAM-1andLFA-1, 255 SCIENCE 1125-1127 (February 1992). Applicants used ICAM-1 antibodies from Milteney Biotec Inc., catalog number 130-103-909.

[0374] Fig. 6AICAM-1 protein expression on the cell surface of untreated (line B) and IFN-γ treated WT hES cells (line A) is shown. The shift (increase) in fluorescence intensity in untreated WT hES cells (line B) compared to background (shaded area) indicates that WT hES cells express ICAM-1 protein on their cell surface. After WT hES cells were exposed to IFN-γ, the shift (increase) in fluorescence intensity over that of WT expression indicates that ICAM-1 expression is increased after exposure to IFN-γ.

[0375] Figure 6B Shown is the expression of ICAM-1 cell surface protein in B2M knockout hES cells untreated (line B) and after treatment with IFN-γ (line A). Figure 6B It was shown that ICAM-1 cell surface protein expression in B2M knockout hES cells was similar to that in WT hES cells.

[0376] This example demonstrates that treatment of WT and B2M knockout hES cells with IFN-γ increases cell surface protein expression of ICAM-1.

[0377] Example 7: Analysis of ICAM-1 cell surface protein expression in WT and B2M knockout pancreatic endoderm cells

[0378] Wild-type and B2M knockout PECs were analyzed using antibodies against known NK activating ligands, such as ICAM-1, similar to 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 18-24 hours.

[0379] Fig. 7A ICAM-1 cell surface protein expression in WT PEC (line B) and after treatment of WT PEC with IFN-γ (line A) is shown. The shift (increase) in fluorescence intensity in untreated WT PEC (line B) compared to background (shaded area) indicates that WT PEC expresses ICAM-1 protein on its cell surface. After exposure to IFN-γ, further shift (increase) in fluorescence intensity beyond WT PEC expression indicates increased ICAM-1 expression in WT PEC after exposure to IFN-γ. This demonstrates that NK activating ligands, especially ICAM-1, can be highly induced by IFN-γ stimulation.

[0380] Figure 7BICAM-1 cell surface protein expression in B2M knockout PECs is shown. ICAM-1 cell surface protein expression in B2M knockout PECs was similar to WT PECs with and without IFN-γ exposure, but with a small decrease in fluorescence. Thus, treatment of B2M knockout PECs with IFN-γ increased ICAM-1 cell surface protein expression.

[0381] Figure 8 RNA expression array data (Affymetrix) are shown, indicating that exposure to IFN-γ increases ICAM-1 expression in WThESC, B2M- / -hESC, WT PEC and B2M- / -PEC. at the mRNA level. Applicants found that cell surface protein expression of the NK activating ligand ICAM-1 in differentiated cell types (WT PEC and B2M- / -PEC) increased after exposure to IFN-γ. Therefore, ICAM-1 is highly induced by IFN-γ stimulation in PEC.

[0382] Example 8: Effects of IFN-treatment on additional NK activating ligands

[0383] To further characterize the effects of IFN-γ treatment on target cells, WT PECs were evaluated by flow cytometry and analyzed for cell surface protein expression of other known NK activating ligands under the following two conditions: (1) untreated (line B) and (2) treated with 100 ng / mL IFN-γ for 18-24 h.

[0384] Fig. 9 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 an antibody from BioLegend, catalog number 330909. A large shift (increase) in fluorescence intensity compared to background (line C) in untreated WThES cells (line B) indicates that most cells express CD58 protein on their surface. After exposure to IFN-γ, there is a small additional shift (increase) in fluorescence intensity.

[0385] Fig.10CD155 (also known as PVR, NECL-5, HVED) cell surface protein expression using an antibody from Milteneyi Biotech Inc., catalog number 130-105-905, in WT PECs untreated (line B) and after treatment with IFN-γ (line A) is shown. The shift (increase) in fluorescence intensity compared to background (line C) in untreated WT PEC cells (line B) indicates that WT PECs express CD155. After exposure to IFN-γ, there was no additional increase (increase) in fluorescence intensity over the untreated condition, indicating that there was no increase in CD155 expression in WT PECs after exposure to IFN-γ.

[0386] Fig.11 CEACAM1 (also known as CD66a, BGP and BGP1) cell surface protein expression using antibodies from Milteneyi Biotech Inc., catalog number 30-098-858, in WT PECs untreated (line B) and after treatment with IFN-γ (line A) is shown. The shift (increase) in fluorescence intensity compared to background (line C) in untreated WT hES cells (line B) indicates that WT PECs express CEACAM1 protein on the cell surface. After exposing WT PECs to IFN-γ, the shift (increase) in fluorescence intensity over untreated conditions indicates increased CEACAM1 protein expression on the cell surface in WT PECs after exposure to IFN-γ.

[0387] Fig.12 Shown is the expression of BAT3 (also known as BAG6) cell surface protein in WT PECs untreated (line B) and after treatment with IFN-γ (line A) using an antibody from Abcam Inc., catalog number ab210838. The shift (increase) in fluorescence intensity compared to background (line C) in untreated PECs (line B) indicates that the cells express CEACAM1. After exposure of WT PECs to IFN-γ, there was no additional shift (increase) in fluorescence intensity over untreated conditions, indicating that BAT3 expression in WT PECs was not increased after exposure to IFN-γ.

[0388] Fig.13CADM1 (also known as NECL2, TSLC1, IGSF4, RA175) cell surface protein expression using antibodies from MBLinternational Corporation, catalog number CM004-4, in untreated (line B) and after treatment with IFN-γ (line A) is shown. The shift (increase) in fluorescence intensity compared to background (line C) in the untreated condition (line B) indicates that WT PECs express CADM1 protein on the cell surface. After exposing PECs to IFN-γ, there is no additional shift (increase) in fluorescence intensity over the untreated condition indicating that CADM1 expression is not increased after exposure to IFN-γ.

[0389] Fig.14 CD112 (also known as Nectin-2, PVRR2, HVEB) expression using antibodies from Milteneyi Biotech Inc., catalog number 130-109-056, in untreated (line B) and WT PECs treated with IFN-γ (line A) is shown. The fluorescence intensity under untreated conditions (line B) is shifted (increased) compared to background (line C), indicating that WT PECs express CD112 protein on the cell surface. After exposing PECs to IFN-γ, there is no additional shift (increase) in fluorescence intensity over untreated conditions, indicating that CD112 expression is not increased after exposure to IFN-γ.

[0390] Example 9: MHC class I-deficient, NK cell-activating ligand-deficient cells prevent NK cell-mediated lysis

[0391] 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 cell lysis, ICAM-I expression was blocked on target cells using ICAM-1 blocking antibodies at concentrations of 5 and 10 ug / mL. Addition of blocking ICAM-1 antibodies to WT or B2M- / - ES cells or PECs following IFN-γ treatment resulted in reduced NK lysis of target cells ( Fig.15 ).

[0392] Staining of target cells with Calcein-AM

[0393] The calcein release assay is a non-radioactive alternative method for studying NK cell cytotoxicity. Target cells take up a fluorescent dye (Calcein AM) and convert it in the cytoplasm into an active fluorescent dye that is released from the cells only after lysis. Lysed cells release the fluorescent dye into the supernatant, which is then collected and the amount of fluorescence is quantified in a fluorimeter. The percentage of cell lysis is calculated based on the amount of fluorescence present in the supernatant after incubation in the presence or absence of NK cells (effectors), blocking antibodies, or both.

[0394] Target cells included WT ESCs, B2M - / - ESC, WT PEC or B2M - / - PEC. To prepare target cells, the target cell population was stained with 2 μg / ml Calcein AM staining medium (Enzo Biosciences 1 mg / mL stock solution (Cat. No. C3100MP)). The target cells were incubated at 37°C in an 8% CO2 incubator for 1 hour with intermittent mixing. The target cells were washed twice to remove any free Calcein AM and stained with 1 x 10 5 The cells / ml were resuspended in RPMI complete medium (RPMI, 10% heat-inactivated FBS and 1% antibiotics).

[0395] Co-culture of target, NK cells, and blocking antibodies

[0396] The target cells labeled with Calcein AM were then incubated with NK cells (effector cells) at an effector: target ratio (E:T ratio) of 10:1. Specifically, 100 μL of 1x 10 6 100 μL of calcein-stained ESCs or PECs (1x 10 5 Where indicated, a blocking antibody to the human ICAM-1 surface antigen (R&D Systems, Inc., Catalog No. BBA3) was added to the wells at concentrations of 5 and 10 ug / mL to determine whether NK-mediated cell lysis could be reduced.

[0397] The plate was incubated at 37° C. for 4 hours in an 8% CO2 incubator. After the incubation period, the plate was centrifuged at 200×g for 2 minutes. 100 μL of supernatant was carefully removed, transferred to a black 96-well plate, and fluorescence was measured using a Molecular Device plate reader (excitation filter: 485 nm / emission filter: 530 nm). Specific lysis was calculated by using the following formula: lysis %=100×[(average fluorescence with antibody-average autofluorescence) / (average maximum fluorescence-average autofluorescence)]. 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 in the absence of any antibody or effector cells.

[0398] result

[0399] like Figure 1 As shown, the goal is to move from situation C (NK cells attacking target cells) to situation A (no response or reduced response). Fig.15 This is shown in conditions 4 and 8. In conditions 4 and 8, target cells (B2M- / -hES cells or PECs) lack functional HLA-I class surface expression and are exposed to IFN-γ. As discussed above and seen in conditions 4 and 8, exposure to IFN-γ leads to an increase in NK activating ligands (ICAM-1) compared to HLA-I class knockouts that are not exposed to IFN-γ, followed by more cell lysis (compare condition 3 vs. 4 and the first vertical bar of condition 7 vs. 8). However, upon treatment with an ICAM-1 inhibitory antibody to block expression of NK activation signals on target cells, NK-mediated cell lysis decreased from 83% to 73% in B2M- / -hES cells and from 72% to 61% in B2M- / -PECs treated with IFN-γ. The percentage of NK-mediated cell lysis is not reduced to zero because the use of blocking antibodies may not completely block ICAM-1 cell surface protein expression, and as discussed above, target cells express more than one NK activating ligand. When ICAM-1 expression is further inhibited in target cells and other NK activating ligands are blocked, the percentage of NK-mediated cell lysis is expected to decrease to a greater extent. Therefore, ICAM-1 inhibitory antibodies can be combined with other NK activating ligand inhibitory antibodies, including those for any ligand listed in Table 1, preferably category 1 (known activating ligands) and 2 (potential activating ligand candidates identified from gene chip data, those upregulated in PEC and / or ESC after IFNγ). In one embodiment, the ICAM1 gene and other NK activating ligand genes are destroyed so as to completely block their activity.

[0400] Fig.15 The first bar of conditions 2 and 6 shows Figure 1 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 due to exposure to IFN-γ. This indicated that the expression of both HLA class I antigens and NK activating ligands was upregulated when the cells were incubated with ICAM-1 inhibitory antibodies. Figure 1 Scenario D was shifted to scenario B. Cell death was reduced from 79% to 60% in WT hES cells after incubation with ICAM1 inhibitory antibody, and from 53% to 27% in WT PECs exposed to IFN-γ. Indeed, when WT PECs were exposed to IFN-γ and incubated with ICAM-1 inhibitory antibody, the reduction in NK cell lysis was lower than that of untreated WT PECs: 27% for WT PECs with IFN-γ and ICAM-1 inhibitory antibody, compared to 37% for WT PECs with ICAM-1 inhibitory antibody and no IFN-γ.

[0401] From Figure 1 The situation B is Fig.15 The bars for ICAM1 antibody treatment in conditions 1, 2, 5, and 6 are exemplified. There, 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, the cell surface protein expression of ICAM-1 is not increased. As a result, the effect of the ICAM-1 inhibitory antibody is smaller. Therefore, cell lysis remains about the same: 58% to 57% in hES cells, and 33% to 37% in PEC cells.

[0402] From Figure 1 Case C and Fig.15 The first bars in conditions 3, 4, 7, and 8 were similar. Under these conditions, cells had no HLA class I cell surface expression due to B2M- / -. Therefore, when the NK activating ligands were not activated by IFN-γ, exposing the cells to ICAM-1 inhibitory antibodies had little effect: 71% to 70% in hES cells, and 54% to 57% in PECs.

[0403] As a general observation, compared with undifferentiated cell populations (WT hES and B2M - / - hES) compared to differentiated cell populations (WT PEC and B2M - / - When cells (WT or B2M- / - knockout, hES or PEC) were activated by IFN-γ, NK-mediated cell lysis increased to a greater extent.

[0404] Example 10: Generation of NK activating ligand-deficient B2M- / - knockout hES cells

[0405] Example 9 describes that inhibition or quenching of ICAM-1 expression protects IFN-γ-treated B2M- / -PECs from NK-mediated cell killing activity. Therefore, in order to protect B2M- / -PECs from NK cell-mediated killing after transplantation, destruction of NK cell activating ligand genes is desired. For example, based on the above example, ICAM-1, a known NK activating ligand gene, can be destroyed or "knocked out". Preferably, CRISPR / Cas9 or any other gene editing technology now known or to be known in the future can be used to destroy both alleles of the ICAM-1 locus in the B2M- / -CyT49 hESC line. See PCT Publication No. WO2016183041A, which is incorporated herein by reference in its entirety. Examples of public sequences of ICAM-1 are submitted as SEQ ID NOs.: 4, 5, and 6. For example, in one embodiment of the present invention, NEXTGEN TMCRISPR (Transposagen Inc., Lexington Kentucky), which incorporates dual guide RNAs and a catalytically inactive Cas9 protein fused to the FokI nuclease, is used to perform gene editing in cells.

[0406] Plasmids containing guide RNA and Cas9 can be electroporated into B2M- / -CyT49 hESCs and seeded onto tissue culture plates. After electroporation, cells can be sorted by flow cytometry for their negative reaction to ICAM-1 antibodies. Sorted cells can be plated at clonal density. Individual clones can be picked and re-plated. Clones can be expanded and cryopreserved. Amplified clones that show normal karyotype by G banding and are negative for ICAM-1 protein expression and ICAM-1 protein surface expression by flow cytometry and / or immunofluorescence are selected for further experiments.

[0407] As described above, ICAM1 and B2M-deficient cells fail to express at least one NK activating ligand and at least one or all MHC class I proteins on their cell surface and therefore should not bind to NK cell activating receptors and are protected from NK-mediated cell death.

[0408] Example 11: Generation of multiple NK activating ligand-deficient B2M- / - knockout hES cells

[0409] Examples 9 and 10 demonstrate that inhibition of functional cell surface expression of NK cell activating ligands (anti-NK activating ligands) and genetic disruption of NK cell activating ligands (e.g., ICAM1- / -) combined with disruption of MHC-I class cell surface expression (e.g., B2M- / -) can provide target cells with protection from NK-mediated cell death. Transplants of cells lacking more than one NK cell activating ligand can be generated and provide further protection from NK-mediated cell death.

[0410] The CD58 gene was selected as the NK activating ligand gene for knockout. 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 WO2016183041A. Examples of published sequences for CEACAM1 are submitted as SEQ ID NOs.: 7, 8, and 9. Similarly, the edited version can be NEXTGEN TM CRISPR (Transposagen Inc., Lexington Kentucky).

[0411] Plasmids containing guide RNA and Cas9 can be electroporated into B2M- / -, ICAM- / - knockout CyT49 hESCs and seeded onto tissue culture plates. After electroporation, cells can be sorted by flow cytometry for negative reactions to CD58 antibodies. Sorted cells can be plated at clonal density. Individual clones can be picked and re-plated. Clones can be expanded and cryopreserved. Amplified clones that show normal karyotype by G banding and are negative for CD58 protein expression and CD58 protein surface expression by flow cytometry and / or immunofluorescence are selected for further experiments.

[0412] As described above, cells with disrupted, deleted or modified ICAM1, CD58 and B2M cannot express ICAM1, CD58, nor MHC class I proteins on their cell surface and therefore should not bind NK cell activating receptors and are protected from NK-mediated cell death.

[0413] The CD155 (aka PVR) gene can also be selected as an NK activating ligand gene to be knocked out. CD155 can also be selected in addition to or instead of CD58. As above, the CD28 gene can be destroyed using CRISPR / Cas9 technology. The CD155 gene can also be selected as an NK activating ligand gene to be knocked out. CD155 can also be selected in addition to or instead of CD58. The CAECAM1 gene can also be selected as an NK activating ligand gene to be knocked out. CAECAM1 can also be selected in addition to or instead of CD58 and / or CD155.

[0414] Example 12: Inactivation of NK Activating Ligands ICAM1 and CD58

[0415] To completely eliminate NK activation, it may be necessary to eliminate / reduce multiple NK activating ligands by gene knockout in target cells (such as hES cell-derived cell therapy) or by using blocking antibodies or other strategies now known or developed in the future. To determine whether NK cytotoxicity can be reduced by inhibiting the action of NK activating ligands on target cells, ICAM1 and CD58 blocking antibodies that block ICAM1 and CD58 proteins on the surface of target cells can be used to block the expression of NK activating ligands in WT and B2M- / -hES cells and PECs. It is expected that cell lysis of target cells will be reduced (similar to Fig.15). Therefore, cell lysis by NK cells can be reduced by blocking NK activating ligands such as ICAM1 and CD58. Blocking ICAM1 and CD58 expression in B2M- / - cells using antibodies against NK activating ligands is a proof of concept for generating cells with three knockouts (HLA-I class gene knockout and NK activating ligand gene knockout). In this way, target cells (such as hES and / or pancreatic lineage cells) are removed from Figure 1 Situation C in is converted into A. Specifically, cells, tissues and organs of the present invention have suppressed or no HLA-I class cell surface protein expression (B2M- / -), and have suppressed or no NK activation ligand cell surface protein expression (such as ICAM1- / - and CD58- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or using antibodies to block the expression of proteins. Other strategies for interfering with cell surface protein expression include the use of antisense RNA, RNA baits, ribozymes, RNA aptamers, siRNA, shRNA / miRNA, trans-dominant negative proteins (TNPs), chimeric / fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFvs), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides and chemicals) and protease inhibitors. Multiple gene knockouts will effectively prevent cytotoxic T cell (CTL)-mediated and NK cell-mediated toxicity because almost no HLA class I and almost no NK activating ligand proteins are expressed on the cell surface for CTL or NK cell binding.

[0416] Example 13: Inactivation of NK Activating Ligands CADM1 and CD58

[0417] To completely eliminate NK activation, it may be necessary to eliminate / reduce multiple NK activating ligands by gene knockout in target cells (such as hES cell-derived cell therapy) or by using blocking antibodies or other strategies now 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, CADM1 and CD58 blocking antibodies that block CADM1 and CD58 proteins on the surface of target cells can be used to block the expression of NK activating ligands in WT and B2M- / - hES cells and PECs. It is expected that the cell lysis of target cells will be reduced (similar to Fig.15 ). Therefore, cell lysis by NK cells can be reduced by blocking NK activating ligands such as CADM1 and CD58. Blocking CADM1 and CD58 expression in B2M- / - cells using antibodies against NK activating ligands is a proof of concept for generating cells with three knockouts (HLA-I class gene knockout and NK activating ligand gene knockout). In this way, target cells (such as hES and / or pancreatic lineage cells) are removed from Figure 1Situation C in is converted into A. Specifically, cells, tissues and organs of the present invention have suppressed or no HLA-I class cell surface protein expression (B2M- / -), and have suppressed or no NK activation ligand cell surface protein expression (such as CADM1- / - and CD58- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or using antibody to block the expression of protein. Other strategies for interfering with cell surface protein expression include the use of antisense RNA, RNA bait, ribozyme, RNA aptamer, siRNA, shRNA / miRNA, trans-dominant negative protein (TNP), chimeric / fusion protein, nuclease, chemokine ligand, anti-infective cell protein, intracellular antibody (sFvs), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides and chemicals) and protease inhibitors. Multiple gene knockouts will effectively prevent cytotoxic T cell (CTL)-mediated and NK cell-mediated toxicity because almost no HLA class I and almost no NK activating ligand proteins are expressed on the cell surface for CTL or NK cell binding.

[0418] Example 14: Inactivation of NK Activating Ligands CD155 and CD58

[0419] To completely eliminate NK activation, it may be necessary to eliminate / reduce multiple NK activating ligands by gene knockout in target cells (such as hES cell-derived cell therapy) or by using blocking antibodies or other strategies now 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, CD155 and CD58 blocking antibodies that block CD155 and CD58 proteins on the surface of target cells can be used to block the expression of NK activating ligands in WT and B2M- / -hES cells and PECs. It is expected that the cell lysis of target cells will be reduced (similar to Fig.15 ). Therefore, cell lysis by NK cells can be reduced by blocking NK activating ligands such as CD155 and CD58. Blocking CD155 and CD58 expression in B2M- / - cells using antibodies against NK activating ligands is a proof of concept for generating cells with three knockouts (HLA-I class gene knockout and NK activating ligand gene knockout). In this way, target cells (such as hES and / or pancreatic lineage cells) are removed from Figure 1Situation C in is converted into A. Specifically, cells, tissues and organs of the present invention have suppressed or no HLA-I class cell surface protein expression (B2M- / -), and have suppressed or no NK activation ligand cell surface protein expression (such as CD155- / - and CD58- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or using antibody to block the expression of protein. Other strategies for interfering with cell surface protein expression include the use of antisense RNA, RNA bait, ribozyme, RNA aptamer, siRNA, shRNA / miRNA, trans-dominant negative protein (TNP), chimeric / fusion protein, nuclease, chemokine ligand, anti-infective cell protein, intracellular antibody (sFvs), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides and chemicals) and protease inhibitors. Multiple gene knockouts will effectively prevent cytotoxic T cell (CTL)-mediated and NK cell-mediated toxicity because almost no HLA class I and almost no NK activating ligand proteins are expressed on the cell surface for CTL or NK cell binding.

[0420] Example 15: Inactivation of NK activating ligands ICAM1, CD155 and CD58

[0421] To completely eliminate NK activation, it may be necessary to eliminate / reduce multiple NK activating ligands by gene knockout in target cells (such as hES cell-derived cell therapy) or by using blocking antibodies or other strategies now 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, ICAM1, CD155, and CD58 blocking antibodies that block ICAM1, CD155, and CD58 proteins on the surface of target cells can be used to block the expression of NK activating ligands in WT and B2M- / -hES cells and PECs. It is expected that the cell lysis of target cells will be reduced (similar to Fig.15 ). Therefore, cell lysis by NK cells can be reduced by blocking NK activating ligands such as ICAM1, CD58 and CD155. Blocking ICAM1, CD58 and CD155 expression in B2M- / - cells using antibodies against NK activating ligands is a proof of concept for generating cells with 4 knockouts (HLA-I class gene knockout and NK activating ligand gene knockout). In this way, target cells (such as hES and / or pancreatic lineage cells) are removed from Figure 1Situation C in is converted into A. Specifically, cells, tissues and organs of the present invention have suppressed or no HLA-I class cell surface protein expression (B2M- / -), and have suppressed or no NK activation ligand cell surface protein expression (such as ICAM1- / -, CD58- / - and CD155- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or using antibodies to block the expression of proteins. Other strategies for interfering with cell surface protein expression include the use of antisense RNA, RNA baits, ribozymes, RNA aptamers, siRNA, shRNA / miRNA, trans-dominant negative proteins (TNPs), chimeric / fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFvs), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides and chemicals) and protease inhibitors. Multiple gene knockouts will effectively prevent cytotoxic T cell (CTL)-mediated and NK cell-mediated toxicity because almost no HLA class I and almost no NK activating ligand proteins are expressed on the cell surface for CTL or NK cell binding.

[0422] Example 16: Inactivation of NK activating ligands ICAM1, CD155, CD58 and CADM1

[0423] To completely eliminate NK activation, it may be necessary to eliminate / reduce multiple NK activating ligands by gene knockout in target cells (such as hES cell-derived cell therapy) or by using blocking antibodies or other strategies now 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, ICAM1, CD155, CD58, and CADM1 blocking antibodies that block ICAM1, CD155, CD58, and CADM1 proteins on the surface of target cells can be used to block the expression of NK activating ligands in WT and B2M- / - hES cells and PECs. It is expected that the cell lysis of target cells will be reduced (similar to Fig.15 ). Therefore, cell lysis by NK cells can be reduced by blocking NK activating ligands such as ICAM1, CD58, CD155 and CADMI. Blocking ICAM1, CD58, CD155 and CADMI expression in B2M- / - cells using antibodies against NK activating ligands is a proof of concept for generating cells with 5 knockouts (HLA-I class I gene knockout and NK activating ligand gene knockout). In this way, target cells (such as hES and / or pancreatic lineage cells) are removed from Figure 1Situation C in is converted into A. Specifically, the cells, tissues and organs of the present invention have suppressed or no HLA-I class cell surface protein expression (B2M- / -), and have suppressed or no NK activation ligand cell surface protein expression (such as ICAM1- / -, CD58- / -, CD155- / - and CADM1- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or using antibodies to block the expression of proteins. Other strategies for interfering with cell surface protein expression include the use of antisense RNA, RNA baits, ribozymes, RNA aptamers, siRNA, shRNA / miRNA, trans-dominant negative proteins (TNPs), chimeric / fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFvs), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides and chemicals) and protease inhibitors. Multiple gene knockouts will effectively prevent cytotoxic T cell (CTL)-mediated and NK cell-mediated toxicity because almost no HLA class I and almost no NK activating ligand proteins are expressed on the cell surface for CTL or NK cell binding.

[0424] Example 17: Inactivation of NK activating ligands ICAM1, CD155 and CADM1

[0425] To completely eliminate NK activation, it may be necessary to eliminate / reduce multiple NK activating ligands by gene knockout in target cells (such as hES cell-derived cell therapy) or by using blocking antibodies or other strategies now 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, ICAM1, CD155, and CADM1 blocking antibodies that block ICAM1, CD155, and CADM1 proteins on the surface of target cells can be used to block the expression of NK activating ligands in WT and B2M- / - hES cells and PECs. Cell lysis of target cells can be expected to be reduced (similar to Fig.15 ). Therefore, cell lysis by NK cells can be reduced by blocking NK activating ligands such as ICAM1, CD155 and CADMI. Blocking ICAM1, CD155 and CADMI expression in B2M- / - cells using antibodies against NK activating ligands is a proof of concept for generating cells with 4 knockouts (HLA-I class I knockout and NK activating ligand knockout). In this way, target cells (such as hES and / or pancreatic lineage cells) are removed from Figure 1Situation C in is converted into A. Specifically, the cells, tissues and organs of the present invention have suppressed or no HLA-I class cell surface protein expression (B2M- / -), and have suppressed or no NK activation ligand cell surface protein expression (such as ICAM1- / -, CD155- / - and CADM1- / -). Inhibition of cell surface protein expression can be achieved by knocking out genes or using antibodies to block the expression of proteins. Other strategies for interfering with cell surface protein expression include the use of antisense RNA, RNA baits, ribozymes, RNA aptamers, siRNA, shRNA / miRNA, trans-dominant negative proteins (TNPs), chimeric / fusion proteins, nucleases, chemokine ligands, anti-infective cell proteins, intracellular antibodies (sFvs), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides and chemicals) and protease inhibitors. Multiple gene knockouts will effectively prevent cytotoxic T cell (CTL)-mediated and NK cell-mediated toxicity because almost no HLA class I and almost no NK activating ligand proteins are expressed on the cell surface for CTL or NK cell binding.

[0426] Blocking antibodies that can be used in the present invention are as follows: CADM1 antibody, clone 9D2, Creative Biolabs, catalog number BRD-0007MZ; CD155 antibody, clone SKII.4, Biolegend, catalog number 337602; and CD58 antibody, clone TS2 / 9, Biolegend, catalog number 330911.

Claims

1. A human ex vivo endocrine cell population, wherein the functions of at least one major histocompatibility complex (MHC) class I gene and at least one natural killer (NK) cell activating ligand are destroyed or inhibited in the human ex vivo endocrine cell population, resulting in reduced binding of the NK activating ligand to the NK activating receptor, wherein the MHC class I gene encodes beta-2 microglobulin (B2M) or human leukocyte antigen (HLA)-ABC cell surface protein, and wherein the NK cell activating ligand is intracellular adhesion molecule 1 (ICAM1) or one selected from the following combinations: a) CD58 and ICAM1; b) CD58, ICAM1 and CD155; c) CD58 and CADM1; d) CD58 and CD155; e) CD58, ICAM1, CD155 and CADM1; f) ICAM1, CADM1 and CD155; or g) ICAM1, CD58, CD155, CEACAM1, CADM1, MICA and MICB.

2. The human ex vivo endocrine cell population according to claim 1, wherein the NK cell activating ligand is: a) ICAM1 and CD58; b) ICAM1, CD58, CD155, CEACAM1, CADM1, MICA and MICB; c) ICAM-1, CD58 and CD155; d) ICAM1, CD58, CD155 and CADMI; e) CD58 and CADM1; or f) ICAM1, CADM1 and CD155.

3. The human ex vivo endocrine cell population of claim 1, wherein the human ex vivo endocrine cell population expresses a protein that, when expressed in the presence of a cell death inducer, is capable of killing cells in the cell population, wherein the protein is herpes simplex virus thymidine kinase, and the cell death inducer is ganciclovir. The human ex vivo endocrine cell population according to claim 1 , wherein the human ex vivo endocrine cell population is an immature endocrine cell population. The human ex vivo endocrine cell population according to claim 1 , further comprising an anti-NK cell activating ligand agent.

6. The human ex vivo endocrine cell population of claim 5, wherein the anti-NK cell activating ligand agent is an antibody or an antibody fragment.

7. The human exoendocrine cell population of claim 1, wherein the human exoendocrine cell population has been further altered to increase the expression of one or more tolerogenic factors.

8. The human ex vivo endocrine cell population of claim 7, wherein the tolerogenic 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.

9. The human ex vivo endocrine cell population of any one of claims 1-8, wherein the human ex vivo endocrine cell population is contained in a perforated cell delivery device.

10. Use of the human ex vivo endocrine cell population according to any one of claims 1 to 8 in the preparation of a medicament for treating type I diabetes.

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