Universal donor cells

Abstract

To provide genetically modified cells that are compatible with multiple subjects, e.g., universal donor cells, and to provide methods of generating the genetic modified cells.SOLUTION: The universal donor cells comprise at least one genetic modification within or near at least one gene encoding a survival factor, the genetic modification comprising the insertion of a polypeptide encoding a tolerogenic factor. The universal donor cell may further comprise at least one genetic modification within or near a gene encoding one or more MHC-I or MHC-II human leukocyte antigens or components or transcriptional regulators of the MHC-I or MHC-II complexes, the genetic modification comprising the insertion of a polypeptide encoding a second tolerogenic factor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 896,473, filed September 5, 2019, and U.S. Provisional Patent Application No. 62 / 979,771, filed February 21, 2020, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Incorporation by reference to sequence listing This application contains a Sequence Listing that has been submitted via EFS-Web in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy created on September 2, 2020, is entitled CT124-PCT-100867-666508-Sequence-Listing_ST25.txt and is approximately 53,000 bytes in size.

[0003] The present invention relates to the field of gene editing, and in some embodiments to genetic modification for the purpose of creating cells that are compatible with multiple subjects, e.g., universal donor cells. [Background technology]

[0004] Various approaches have been proposed to overcome allorejection of transplanted or engrafted cells, including HLA matching, blocking pathways that induce antibody-mediated T cell activation, the use of immunosuppressant cocktails, and autologous cell therapy. Another strategy to attenuate graft rejection involves minimizing allogeneic differences between the transplanted or engrafted cells and the recipient. Human leukocyte antigens (HLA), molecules expressed on the cell surface and encoded by genes located in the human major histocompatibility complex on chromosome 6, are the primary mediators of immune rejection. Mismatches of a single HLA gene between the donor and recipient can trigger a strong immune response (Fleischhauer K. et al., "Bone marrow-allograft rejection by T lymphocytes recognizing a single amino acid difference in HLA-B44," N Engl J Med., 1990, 323:1818-1822). HLA genes are classified into MHC class I (MHC-I) and MHC class II (MHC-II). MHC-I genes (HLA-A, HLA-B, and HLA-C) are expressed in almost all tissue types and present "non-self" antigen-processed peptides to CD8+ T cells, thereby promoting their activation into cytolytic CD8+ T cells. Transplanted or engrafted cells expressing "non-self" MHC-I molecules trigger a strong cellular immune response directed against these cells, ultimately leading to their elimination by activated cytolytic CD8+ T cells. MHC-I proteins essentially associate with beta-2-microglobulin (B2M) in the endoplasmic reticulum, which is essential for the formation of functional MHC-I molecules on the cell surface.

[0005] In contrast to the widespread expression of MHC-I genes, the expression of MHC-II genes is restricted to antigen-presenting cells such as dendritic cells, macrophages, and B cells. HLA antigen genes are the most polymorphic genes observed in the human genome (Rubinstein P., "HLA matching for bone marrow transplantation—how much is enough?" N Engl J Med., 2001, 345:1842-1844). The creation of "universal donor" cells compatible with any HLA genotype offers an alternative strategy that may address the immune rejection and economic costs associated with existing methodologies for immune evasion.

[0006] To generate such a universal donor cell line, one prior approach has been to functionally disrupt the expression of MHC-I and MHC-II class genes. This can be activated, for example, by disrupting both alleles of the gene encoding the MHC-I light chain, B2M. The resulting B2M-deficient cell line and its derivatives would be expected to exhibit a significant reduction in surface MHC-I and, therefore, reduced immunogenicity to allogeneic CD8+ T cells. Using a transcription activator-like effector nuclease (TALEN)-targeting approach, B2M-deficient hESC lines have been generated by deleting a few nucleotides in exon 2 of the B2M gene (Lu, P. et al., "Generating hypoimmunogenic human embryonic stem cells by the disruption of beta 2-microglobulin," Stem Cell Rev. 2013, 9:806-813). Although B2M-targeted hESC lines appeared to be surface HLA-I deficient, they were still found to contain mRNA specific for B2M and MHC-I. B2M and MHC-I mRNAs were expressed at levels equivalent to those of non-targeted hESCs (both constitutive and IFN-γ inducible). Thus, there is concern that these TALEN B2M-targeted hESC lines may express residual cell surface MHC-I that may be sufficient to cause immune rejection, as observed with B2M2 / 2 mouse cells, which also express B2M mRNA (Gross, R. and Rappuoli, R. "Pertussis toxin promoter sequences involved in modulation," Proc Natl Acad Sci, 1993, 90:3913-3917).Although TALEN B2M-targeted hESC lines were not tested for off-target cleavage, the occurrence of non-specific cleavage when using TALENs remains a serious problem that is thought to pose a major safety concern for their clinical use (Grau, J. et al. "TALEN offer: genome-wide TALEN off-target prediction", Bioinformatics, 2013, 29:2931-2932; Guilinger JP et al. "Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity", Nat Methods 2014, 11:429-435). Furthermore, in another report, IPS cells were generated that avoided allorecognition by knocking out the first B2M allele and knocking in the HLA-E gene with the second B2M allele, resulting in surface expression of HLA-E dimers or trimers in the absence of surface expression of HLA-A, HLA-B, or HLA-C (Gornalusse, GGet et al., "HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells", Nature Biotechnology, 2017, 35, 765-773). Summary of the Invention [Problem to be solved by the invention]

[0007] One potential limitation of the above strategies is that MHC class I-negative cells are susceptible to lysis by natural killer (NK) cells, because HLA molecules function as the primary ligand inhibitors for NK cells. Host NK cells have been shown to eliminate transplanted or engrafted B2M- / - donor cells, and a similar phenomenon occurs in vitro with MHC class I-negative human leukemia lines (Bix, M. et al., "Rejection of class I MHC-deficient hemopoietic cells by irradiated MHC-matched mice," Nature, 1991, 349, 329-331; Zarcone, D. et al., "Human leukemia-derived cell lines and clones as models for mechanistic analysis of natural killer cell-mediated cytotoxicity," Cancer Res., 1987, 47, 2674-2682). Therefore, there is a need to improve upon previous methods to generate universal donor cells that can evade immune responses and generate cells that can survive after engraftment. As described herein, cell survival after engraftment can be mediated by many other pathways that are independent of allogeneic rejection, such as hypoxia, reactive oxygen species, nutrient deprivation, and oxidative stress. Also, as described herein, genetic introduction of survival factors (genes and / or proteins) can promote cell survival after engraftment. As described herein, universal donor cell lines can combine properties that address both allogeneic rejection and survival after engraftment. [Means for solving the problem]

[0008] In some aspects, the disclosure encompasses methods for generating universal donor cells, the method comprising delivering to a cell (a) a site-specific nuclease that targets a site within or near a gene encoding a survival factor, and (b) a nucleic acid comprising a nucleotide sequence encoding a tolerogenic factor flanked by (i) a nucleotide sequence homologous to a region located to the left of the target site in (a) and (ii) a nucleotide sequence homologous to a region located to the right of the target site in (a), wherein the site-specific nuclease cleaves the target site in (a), and the nucleic acid in (b) is inserted at a site that partially overlaps, completely overlaps, or is contained within the site in (a), thereby generating a universal donor cell, the universal donor cell having increased cell survival compared to a cell in which the nucleic acid of (b) has not been inserted.

[0009] In some other embodiments, the survival factor is TXNIP, ZNF143, FOXO1, JNK, or MANF, and the immunogenic tolerance factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47. In particular embodiments, the survival factor is TXNIP, and the immunogenic tolerance factor is HLA-E. In embodiments in which the site-specific nuclease is a CRISPR system comprising a CRISPR nuclease and a guide RNA (gRNA), the CRISPR nuclease is a type II Cas9 nuclease or a type V Cfp1 nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal. In some embodiments, the gRNA targets a polynucleotide sequence selected from SEQ ID NOs: 15-24 or 45-54, and (i) consists essentially of the nucleotide sequence of SEQ ID NO: 25, and (ii) consists essentially of the nucleotide sequence of SEQ ID NO: 32.

[0010] In some embodiments, the method further comprises delivering to the cell (c) a nucleic acid comprising a site-specific nuclease that targets a site within or near a gene encoding one or more of the MHC-I or MHC-II human leukocyte antigens, or components or transcriptional regulators of the MHC-I or MHC-II complexes, and (d) a nucleotide sequence encoding a tolerogenic factor flanked by (iii) a nucleotide sequence homologous to a region located to the left of the target site in (c) and (iv) a nucleotide sequence homologous to a region located to the right of the target site in (c), wherein the tolerogenic factor in (d) is different from tolerogenic factor (b), the site-specific nuclease cleaves the target site in (c), and the nucleic acid in (d) is inserted at a site that partially overlaps, completely overlaps, or is contained within the site in (c), and the universal donor cell has increased immune evasion and / or cell survival compared to a cell in which the nucleic acid of (d) has not been inserted.

[0011] In some embodiments, the gene encoding one or more MHC-I or MHC-II human leukocyte antigens or components or transcriptional regulators of the MHC-I or MHC-II complex is an MHC-I gene selected from HLA-A, HLA-B, or HLA-C, an MHC-II gene selected from HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR, or a gene selected from B2M, NLRC5, CIITA, RFX5, RFXAP, or RFXANK, and the tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47. In specific embodiments, the gene encoding one or more MHC-I or MHC-II human leukocyte antigens or components or transcriptional regulators of the MHC-I or MHC-II complex is B2M, and the tolerogenic factor is PD-L1. In embodiments where the site-specific nuclease is a CRISPR system comprising a CRISPR nuclease and a gRNA, the CRISPR nuclease is a type II Cas9 nuclease or a type V Cfp1 nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal. In some embodiments, the gRNA targets a polynucleotide sequence selected from SEQ ID NOs: 1-3 or 35-44, and (iii) consists essentially of the nucleotide sequence of SEQ ID NO: 7, and (iv) consists essentially of the nucleotide sequence of SEQ ID NO: 13.

[0012] In some embodiments, the nucleotide sequences encoding the tolerogenic factors of (b) and (d) are operably linked to an exogenous promoter. The exogenous promoter may be selected from a constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoter. In some embodiments, the exogenous promoter is a CMV, EF1a, PGK, CAG, or UBC promoter. In a specific embodiment, the exogenous promoter is a CAG promoter.

[0013] The present disclosure also encompasses universal donor cells produced by the methods disclosed herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a pluripotent stem cell (PSC), embryonic stem cell (ESC), adult stem cell (ASC), induced pluripotent stem cell (iPSC), or hematopoietic stem or progenitor cell (HSPC) (also called hematopoietic stem cell (HSC)). In some embodiments, the cell is a differentiated cell. In some embodiments, the cell is a somatic cell.

[0014] Generally, the universal donor cells disclosed herein can differentiate into lineage-restricted progenitor cells or fully differentiated somatic cells. In some embodiments, the lineage-restricted progenitor cells are pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells, and the fully differentiated somatic cells are endocrine cells such as pancreatic beta cells, epithelial cells, endodermal cells, macrophages, hepatocytes, adipocytes, kidney cells, blood cells, or immune system cells. In some embodiments, the fully differentiated somatic cells are cardiomyocytes.

[0015] A further aspect of the present disclosure provides a method for treating a subject in need thereof, the method comprising obtaining or having obtained universal donor cells as disclosed herein following differentiation into lineage-restricted progenitor cells or fully differentiated somatic cells, and administering the lineage-restricted progenitor cells or fully differentiated somatic cells to the subject. Also provided is a method of obtaining cells for administration to a subject in need thereof, the method comprising obtaining or having universal donor cells as disclosed herein, and maintaining the universal donor cells for a time and under conditions sufficient for the cells to differentiate into lineage-restricted progenitor cells or fully differentiated somatic cells. In some embodiments, the subject is a human having, suspected of having, or at risk for a disease. In some embodiments, the disease is a genetically inherited disease.

[0016] Yet another embodiment of the present disclosure includes a gRNA that targets a polynucleotide sequence selected from SEQ ID NOs: 15-24 or 45-54.

[0017] While the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the drawings and detailed description set forth herein are not intended to limit the disclosure to the particular embodiments disclosed, but rather to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0018] Other features and advantages of the present disclosure will become apparent in the following detailed description of embodiments of the invention, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows TIDE analysis of B2M gRNA cleavage in CyT49 cells. B2M-1, B2M-2, and B2M-3 gRNAs were tested. [Figure 2A] Flow cytometry assessment of B2M expression in WT CyT49 cells (FIG. 2A) and B2M KO CyT49 cells (FIG. 2B) both with and without IFN-γ is shown. [Figure 2B] Flow cytometry assessment of B2M expression in WT CyT49 cells (FIG. 2A) and B2M KO CyT49 cells (FIG. 2B) both with and without IFN-γ is shown. [Figure 3] Figure 1 shows the plasmid map of the B2M-CAGGS-PD-L1 donor vector for HDR. [Figure 4] Flow cytometry analysis of pluripotency of B2M KO / PD-L1 KI CyT49 stem cells. Derived clones were >99% double positive for OCT4 and SOX2, two transcription factors essential for pluripotency. IgG was used as a negative control. [Figure 5A] Flow cytometry analysis of stem cell clones derived from WT CyT49 (Figure 5A) and B2M KO / PD-L1 KI (Figure 5B). WT cells upregulate B2M expression in response to IFNγ. B2M KO / PD-L1 KI clones express abundant PD-L1 and do not express B2M, regardless of whether or not they are treated with IFNγ. NT-1 = untreated. INTG-1 = cells treated with 50 ng / mL IFNγ for 48 hours. [Figure 5B] Flow cytometry analysis of stem cell clones derived from WT CyT49 (Figure 5A) and B2M KO / PD-L1 KI (Figure 5B). WT cells upregulate B2M expression in response to IFNγ. B2M KO / PD-L1 KI clones express abundant PD-L1 and do not express B2M, regardless of whether or not they are treated with IFNγ. NT-1 = untreated. INTG-1 = cells treated with 50 ng / mL IFNγ for 48 hours. [Figure 6] Flow cytometry for FOXA2 and SOX17 in stage 1 (definitive endoderm) cells differentiated from wild-type CyT49, PD-L1 KI / B2M KO, or B2M KO CyT49 cells. [Figure 7] Quantitative percentages of FOXA2 and SOX17 expression in stage 1 (definitive endoderm) cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells are shown. [Figure 8] Quantitative percentages of CHGA, PDX1, and NKX6.1 expression in stage 4 (PEC) cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells are shown. [Figure 9] Shown is a heterogeneous population of cells at stage 4 (PEC). [Figure 10] Figure 1 shows the expression of selected genes over a differentiation time course in cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells. [Figure 11A]Figure 11 shows the expression of selected genes over a differentiation time course in cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells. Figure 11A shows B2M expression in wild-type cells. [Figure 11B] Figure 11B shows the expression of selected genes over a differentiation time course in cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells. Figure 11B shows B2M expression in B2M KO cells. [Figure 11C] Figure 11C shows the expression of selected genes over a differentiation time course in cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells. Figure 11C shows B2M expression in PD-L1 KI / B2M KO cells. [Figure 11D] Figure 11D shows the expression of selected genes over a differentiation time course in cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells. Figure 11D shows PD-L1 expression in wild-type cells. [Figure 11E] Figure 11E shows the expression of selected genes over a differentiation time course in cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells. Figure 11F shows PD-L1 expression in B2M KO cells. [Figure 11F] Figure 11F shows the expression of selected genes over a differentiation time course in cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells. Figure 11F shows PD-L1 expression in PD-L1 KI / B2M KO cells. [Figure 12A] Figure 12 shows MHC class I and class II expression at the PEC stage in cells differentiated from wild-type, PD-L1 / B2M KO, or B2M KO cells. Figure 12A shows MHC class I expression in wild-type cells. [Figure 12B] Figure 12B shows MHC class I and class II expression at the PEC stage in cells differentiated from wild-type, PD-L1 / B2M KO, or B2M KO cells. Figure 12B shows MHC class I expression in B2M KO cells. [Figure 12C]Figure 12C shows MHC class I and class II expression at the PEC stage in cells differentiated from wild-type, PD-L1 / B2M KO, or B2M KO cells. Figure 12D shows MHC class I expression in PD-L1 KI / B2M KO cells. [Figure 12D] Figure 12D shows MHC class I and class II expression at the PEC stage in cells differentiated from wild-type, PD-L1 / B2M KO, or B2M KO cells. Figure 12D shows MHC class II PD-L1 expression in wild-type cells. [Figure 12E] Figure 12E shows MHC class I and class II expression at the PEC stage in cells differentiated from wild-type, PD-L1 / B2M KO, or B2M KO cells. Figure 12F shows MHC class II expression in B2M KO cells. [Figure 12F] Figure 12F shows MHC class I and class II expression at the PEC stage in cells differentiated from wild-type, PD-L1 / B2M KO, or B2M KO cells. Figure 12F shows MHC class II expression in PD-L1 KI / B2M KO cells. [Figure 13] Figure 1 shows TIDE analysis of TXNIP gRNA cleavage in TC1133 hiPSCs. Guide T5 appeared to be the most suitable for cleavage in exon 1. [Figure 14] Figure 1 shows the plasmid map of the TXNIP-CAGGS-HLA-E donor vector for HDR. [Figure 15] Flow cytometry analysis of pluripotency of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI CyT49 stem cells. Derived clones were >99% double positive for OCT4 and SOX2, two transcription factors essential for pluripotency. Clones also do not express B2M. Clones do not express MHC-I. [Figure 16] Flow cytometry analysis of pluripotency of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI CyT49 stem cells. Derived clones express PD-L1 and HLA-E after differentiation to stage 6 (immature cells). IgG was used as a negative control. [Figure 17] Quantitative percentages of CHGA, PDX1, and NKX6.1 expression in stage 4 (PEC) cells differentiated from wild-type, B2M KO, PD-L1 KI / B2M KO (V1A), or TXNIP KO / HLA-E KI (V1B) hESCs are shown. [Figures 18A-18B] Selected gene expression over a differentiation time course in TXNIP KO cells (FIG. 18A) or TXNIP KO / HLA-E KI(V1B) (FIG. 18B) cells is shown. [Figure 19A] Figure 19 shows flow cytometry analysis for T cell activation using a CFSE proliferation assay. Human primary CD3+ T cells were co-incubated with PECs derived from WT, B2M KO, B2M KO / PD-L1 KI, or B2M KO / PD-L1 KI+TXNIP KO / HLA-E KI CyT49 clones (Figure 19A). Figure 19B summarizes T cell activation in the various cells. One-way ANOVA (α=0.05 with Dunnett's multiple comparison test) with the "CFSE-T alone" set as a control. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. ns=non-significant. [Figure 19B] Figure 19 shows flow cytometry analysis for T cell activation using a CFSE proliferation assay. Human primary CD3+ T cells were co-incubated with PECs derived from WT, B2M KO, B2M KO / PD-L1 KI, or B2M KO / PD-L1 KI+TXNIP KO / HLA-E KI CyT49 clones (Figure 19A). Figure 19B summarizes T cell activation in the various cells. One-way ANOVA (α=0.05 with Dunnett's multiple comparison test) with the "CFSE-T alone" set as a control. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. ns=non-significant. [Figure 20] 1 shows selected gene expression over a differentiation time course of cells differentiated from TXNIP KO cells. [Figure 21]1 shows flow cytometry evaluation of PDX1 and NKX6.1 expression in PEC cells differentiated from TXNIP KO cells. [Figure 22] Morphology of various B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones ("S6-V1B-H9," "S6-V1B-3B11," "S6-V1B-1G7," and "S6-V1B-3C2") compared to wild-type ("WT") and uncut guide control ("NCG#1") cells after differentiation to stage 6 is shown. [Figures 23A-23B] Selected gene expression of clones following differentiation through stage 6 is shown. Figure 23A shows selected gene expression over the differentiation time course of cells differentiated from exemplary B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones. Figures 23B-23F show gene expression of INS (Figure 23B), NKX6.1 (Figure 23C), GCG (Figure 23D), SST (Figure 23E), and GCK (Figure 23F) in undifferentiated B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones ("ES-V1B-H9") and wild-type cells differentiated to stage 6 ("S6-Cyt49 WT"), uncleaved guide control ("S6-NCG#1") cells, and various B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones ("S6-V1B-H9," "S6-V1B-3B11," "S6-V1B-1G7," and "S6-V1B-3C2"), along with wild-type islets ("Islets") as a control. [Figures 23C-23D]Selected gene expression of clones following differentiation through stage 6 is shown. Figure 23A shows selected gene expression over the differentiation time course of cells differentiated from exemplary B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones. Figures 23B-23F show gene expression of INS (Figure 23B), NKX6.1 (Figure 23C), GCG (Figure 23D), SST (Figure 23E), and GCK (Figure 23F) in undifferentiated B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones ("ES-V1B-H9") and wild-type cells differentiated to stage 6 ("S6-Cyt49 WT"), uncleaved guide control ("S6-NCG#1") cells, and various B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones ("S6-V1B-H9," "S6-V1B-3B11," "S6-V1B-1G7," and "S6-V1B-3C2"), along with wild-type islets ("Islets") as a control. [Figures 23E-23F] Selected gene expression of clones following differentiation through stage 6 is shown. Figure 23A shows selected gene expression over the differentiation time course of cells differentiated from exemplary B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones. Figures 23B-23F show gene expression of INS (Figure 23B), NKX6.1 (Figure 23C), GCG (Figure 23D), SST (Figure 23E), and GCK (Figure 23F) in undifferentiated B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones ("ES-V1B-H9") and wild-type cells differentiated to stage 6 ("S6-Cyt49 WT"), uncleaved guide control ("S6-NCG#1") cells, and various B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones ("S6-V1B-H9," "S6-V1B-3B11," "S6-V1B-1G7," and "S6-V1B-3C2"), along with wild-type islets ("Islets") as a control. [Figures 24A-24B]Flow cytometry assessment of INS and GCG expression (Figure 24A) and INS and NKX6.1 expression (Figure 24B) in stage 6 cells differentiated from B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones. [Figures 25A-25B] Shown are the percentages of INS expression (Figure 25A) and NKX6.1 expression (Figure 25B) in stage 6 cells differentiated from wild-type cells ("S6-WT"), uncleaved guide control cells ("S6-NCG#1"), and two B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones ("S6-V1B003" and "V1B-H9"). [Figure 26A] Figure 1 shows flow cytometry assessment of PDX1 and NKX6.1 expression in stage 4 cells differentiated from clone 1 (B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI) with different seeding densities. [Figure 26B] Figure 1 shows flow cytometry assessment for PD-L1 and HLA-E expression in stage 4 cells differentiated from clone 1 (B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI) cells. [Figures 27A-27B] Figure 27 shows an analysis of the characterization of seeded clones differentiated to the PEC stage. Figure 27A shows morphology, and Figure 27B shows selected gene expression over the differentiation time course. [Figure 27C] Figure 27C shows the analysis of the characterization of seeded clones differentiated to the PEC stage. Figure 27C shows the percentage of CHGA- / NKX6.1+ / PDX1+ expressing cells in the differentiated population. [Figure 28] 1 shows selected gene expression over a differentiation time course of cells differentiated from a TXNIP KO / HLA-E KI clone. DETAILED DESCRIPTION OF THE INVENTION

[0020] I. Definition Deletion: As used herein, the term "deletion," which may be used interchangeably with the terms "gene deletion" or "knockout," generally refers to a genetic modification in which a site or region of genomic DNA is removed by any molecular biological method, such as those described herein, for example, by delivering an endonuclease and at least one gRNA to the site of genomic DNA. Any number of nucleotides can be deleted. In some embodiments, the deletion involves removal of at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 nucleotides. In some embodiments, the deletion involves removal of 10-50, 25-75, 50-100, 50-200, or more than 100 nucleotides. In some embodiments, the deletion involves removal of an entire target gene, such as the B2M gene. In some embodiments, the deletion involves removal of a portion of the target gene, such as all or part of the promoter and / or coding sequence of the B2M gene. In some embodiments, the deletion involves removal of a transcriptional regulator, e.g., the promoter region of the target gene. In some embodiments, the deletion involves removal of all or part of a coding region such that the product normally expressed by the coding region is no longer expressed, is expressed in a truncated form, or is expressed at a reduced level. In some embodiments, the deletion results in decreased expression of the gene relative to an unmodified cell.

[0021] Endonuclease: As used herein, the term "endonuclease" generally refers to an enzyme that cleaves phosphodiester bonds in a polynucleotide. In some embodiments, the endonuclease specifically cleaves phosphodiester bonds in a DNA polynucleotide. In some embodiments, the endonuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a homing endonuclease (HE), a meganuclease, a MegaTAL, or a CRISPR-associated endonuclease. In some embodiments, the endonuclease is an RNA-guided endonuclease. In certain aspects, the RNA-guided endonuclease is a CRISPR nuclease, such as a Type II CRISPR Cas9 endonuclease or a Type V CRISPR Cpfl endonuclease. In some embodiments, the endonuclease is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cm r3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease or a homolog thereof, a recombinant naturally occurring molecule thereof, a codon-optimized version thereof, or a modified version thereof, or a combination thereof. In some embodiments, the endonuclease may introduce one or more single-strand breaks (SSBs) and / or one or more double-strand breaks (DSBs).

[0022] Genetic modification: As used herein, the term "genetic modification" generally refers to a site of genomic DNA that has been genetically edited or manipulated using any molecular biological method, such as the methods described herein, for example, by delivering an endonuclease and at least one gRNA to the site of the genomic DNA. Examples of genetic modifications include insertions, deletions, duplications, inversions, and translocations, and combinations thereof. In some embodiments, the genetic modification is a deletion. In some embodiments, the genetic modification is an insertion. In other embodiments, the genetic modification is an insertion-deletion mutation (or indel) in which the reading frame of the target gene is shifted, resulting in a modified gene product or no gene product.

[0023] Guide RNA (gRNA): As used herein, the term "guide RNA" or "gRNA" generally refers to a short ribonucleic acid that can interact with, e.g., bind to, an endonuclease and bind or hybridize to a target genomic site or region. In some embodiments, the gRNA is a single-molecule guide RNA (sgRNA). In some embodiments, the gRNA may include a spacer extension region. In some embodiments, the gRNA may include a tracrRNA extension region. In some embodiments, the gRNA is single-stranded. In some embodiments, the gRNA comprises naturally occurring nucleotides. In some embodiments, the gRNA is a chemically modified gRNA. In some embodiments, the chemically modified gRNA is a gRNA that includes at least one nucleotide with a chemical modification, e.g., a 2'-O-methyl sugar modification. In some embodiments, the chemically modified gRNA comprises a modified nucleic acid backbone. In some embodiments, the chemically modified gRNA comprises 2'-O-methyl-phosphorothioate residues. In some embodiments, the gRNA may be pre-complexed with a DNA endonuclease.

[0024] Insertion: As used herein, the term "insertion," which may be used interchangeably with the terms "gene insertion" or "knock-in," generally refers to a genetic modification in which a polynucleotide is introduced or added to a site or region of genomic DNA by any molecular biological method, such as those described herein, such as delivering an endonuclease and at least one gRNA to the site of the genomic DNA. In some embodiments, the insertion can occur within or near a site of genomic DNA that was the site of a previous genetic modification, such as a deletion or an insertion-deletion mutation. In some embodiments, the insertion occurs at a site of genomic DNA that partially overlaps, completely overlaps, or is contained within the site of a previous genetic modification, such as a deletion or an insertion-deletion mutation. In some embodiments, the insertion occurs at a safe harbor locus. In some embodiments, the insertion comprises the introduction of a polynucleotide encoding a protein of interest. In some embodiments, the insertion comprises the introduction of a polynucleotide encoding a tolerogenic factor. In some embodiments, the insertion comprises the introduction of a polynucleotide encoding a survival factor. In some embodiments, the insertion comprises the introduction of an exogenous promoter, such as a constitutive promoter, such as a CAG promoter. In some embodiments, the insertion comprises the introduction of a polynucleotide encoding a non-coding gene. Generally, the inserted polynucleotide is flanked by sequences that share substantial sequence homology (e.g., homology arms) with the genomic DNA at or near the site of insertion.

[0025] Major Histocompatibility Complex Class I (MHC-I): As used herein, the term "major histocompatibility complex class I" or "MHC-I" generally refers to a class of biological molecules found on the cell surface of all nucleated cells in vertebrates, including mammals, e.g., humans; and functions to present non-self or foreign antigenic peptides, e.g., proteins, from within cells (i.e., the cytosol) to cytotoxic T cells, e.g., CD8+ T cells, to stimulate an immune response. In some embodiments, the MHC-I biological molecule is an MHC-I gene or MHC-I protein. Complex formation of the MHC-I protein with the beta-2 microglobulin (B2M) protein is required for cell surface expression of all MHC-I proteins. In some embodiments, reducing expression of MHC-I human leukocyte antigen (HLA) relative to unmodified cells comprises a reduction in expression of the MHC-I gene. In some embodiments, reducing expression of MHC-I human leukocyte antigen (HLA) relative to unmodified cells comprises a reduction in cell surface expression of the MHC-I protein. In some embodiments, the MHC-I biomolecule is HLA-A (NCBI Gene ID No.: 3105), HLA-B (NCBI Gene ID No.: 3106), HLA-C (NCBI Gene ID No.: 3107) or B2M (NCBI Gene ID No.: 567).

[0026] Major Histocompatibility Complex Class II (MHC-II): As used herein, the term "major histocompatibility complex class II" or "MHC-II" generally refers to a class of biological molecules typically found on the cell surface of antigen-presenting cells in vertebrates, including mammals, e.g., humans; and functions to present peptides, e.g., proteins, of non-self or foreign antigens from the outside (extracellular) of the cell to cytotoxic T cells, e.g., CD8+ T cells, to stimulate an immune response. In some embodiments, the antigen-presenting cell is a dendritic cell, macrophage, or B cell. In some embodiments, the MHC-II biological molecule is an MHC-II gene or MHC-II protein. In some embodiments, reducing expression of MHC-II human leukocyte antigen (HLA) relative to unmodified cells comprises reducing (or decreasing) expression of MHC-II genes. In some embodiments, reducing expression of MHC-II human leukocyte antigen (HLA) relative to unmodified cells comprises reducing (or decreasing) cell surface expression of MHC-II proteins. In some embodiments, the MHC-II biomolecule is HLA-DPA (NCBI Gene ID No.: 3113), HLA-DPB (NCBI Gene ID No.: 3115), HLA-DMA (NCBI Gene ID No.: 3108), HLA-DMB (NCBI Gene ID No.: 3109), HLA-DOA (NCBI Gene ID No.: 3111), HLA-DOB (NCBI Gene ID No.: 3112), HLA-DQA (NCBI Gene ID No.: 3117), HLA-DQB (NCBI Gene ID No.: 3119), HLA-DRA (NCBI Gene ID No.: 3122), or HLA-DRB (NCBI Gene ID No.: 3123).

[0027] Polynucleotide: As used herein, the term "polynucleotide," which may be used interchangeably with the term "nucleic acid," generally refers to a biomolecule comprising two or more nucleotides. In some embodiments, a polynucleotide comprises at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 250, at least 500, or any number of nucleotides. For example, a polynucleotide can comprise at least 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, at least about 1000 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, at least about 4500 nucleotides, or at least about 5000 nucleotides. A polynucleotide can be a DNA or RNA molecule or a hybrid DNA / RNA molecule. A polynucleotide can be single-stranded or double-stranded. In some embodiments, a polynucleotide is a site or region of genomic DNA. In some embodiments, the polynucleotide is an endogenous gene contained within the genome of an unmodified cell or a universal donor cell. In some embodiments, the polynucleotide is an exogenous polynucleotide that is not integrated into genomic DNA. In some embodiments, the polynucleotide is an exogenous polynucleotide that is integrated into genomic DNA. In some embodiments, the polynucleotide is a plasmid or an adeno-associated virus vector. In some embodiments, the polynucleotide is a circular or linear molecule.

[0028] Safe harbor locus: As used herein, the term "safe harbor locus" generally refers to any position, site, or region of genomic DNA that can accommodate a gene insertion at that position, site, or region without adverse effects on the cell. In some embodiments, a safe harbor locus is an intragenic or extragenic region. In some embodiments, a safe harbor locus is a region of genomic DNA that is typically transcriptionally silent. In some embodiments, a safe harbor locus is the AAVS1 (PPP1R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1, TF, or TTR locus. In some embodiments, a safe harbor locus is described in Sadelain, M. et al., "Safe harbors for the integration of new DNA in the human genome," Nature Reviews Cancer, 2012, Vol. 12, pages 51-58.

[0029] Safety switch: As used herein, the term "safety switch" generally refers to a biomolecule that causes a cell to undergo apoptosis. In some embodiments, the safety switch is a protein or a gene. In some embodiments, the safety switch is a suicide gene. In some embodiments, a safety switch, e.g., herpes simplex virus thymidine kinase (HSV-tk), causes a cell to undergo apoptosis by metabolizing a prodrug, e.g., ganciclovir. In some embodiments, the presence of an overexpressed safety switch causes a cell to undergo apoptosis. In some embodiments, the safety switch is a p53-based molecule, HSV-tk, or inducible caspase-9.

[0030] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate or rodent. In some embodiments, the subject is human. In some embodiments, the subject has, is suspected of having, or is at risk for a disease or disorder. In some embodiments, the subject has one or more symptoms of a disease or disorder.

[0031] Survival Factor: As used herein, the term "survival factor" generally refers to a protein (e.g., expressed by a polynucleotide as described herein) that, when increased or decreased in a cell, enables the cell, e.g., universal donor cell, to survive at a higher rate after transplantation or engraftment into a host subject relative to unmodified cells. In some embodiments, the survival factor is a human survival factor. In some embodiments, the survival factor is a member of a critical pathway involved in cell survival. In some embodiments, the critical pathway involved in cell survival has an effect on hypoxia, reactive oxygen species, nutrient deprivation, and / or oxidative stress. In some embodiments, genetic modification, e.g., deletion or insertion, of at least one survival factor enables the universal donor cell to survive for a longer period than unmodified cells after engraftment, e.g., at least 1.05-fold, at least 1.1-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold longer. In some embodiments, the survival factor is ZNF143 (NCBI Gene ID No.: 7702), TXNIP (NCBI Gene ID No.: 10628), FOXO1 (NCBI Gene ID No.: 2308), JNK (NCBI Gene ID No.: 5599), or MANF (NCBI Gene ID No.: 7873). In some embodiments, the survival factor is inserted into a cell, e.g., a universal donor cell. In some embodiments, the survival factor is deleted from a cell, e.g., a universal donor cell. In some embodiments, insertion of a polynucleotide encoding MANF enables the cell, e.g., universal donor cell, to survive at a higher rate after transplantation or engraftment into a host subject relative to an unmodified cell. In some embodiments, a deletion or insertion-deletion mutation in or near the ZFN143, TXNIP, FOXO1, or JNK gene enables the cell, e.g., universal donor cell, to survive at a higher rate after transplantation or engraftment into a host subject relative to an unmodified cell.

[0032] Tolerogenic Factor: As used herein, the term "tolerogenic factor" generally refers to a protein (e.g., expressed by a polynucleotide as described herein) that, when increased or decreased in a cell, enables the cell, e.g., universal donor cell, to inhibit or avoid immune rejection at a higher rate after transplantation or engraftment into a host subject relative to unmodified cells. In some embodiments, the tolerogenic factor is a human tolerogenic factor. In some embodiments, genetic modification of at least one tolerogenic factor (e.g., insertion or deletion of at least one tolerogenic factor) enables the cell, e.g., universal donor cell, to inhibit or avoid immune rejection at a rate at least 1.05-fold, at least 1.1-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold higher than unmodified cells after engraftment. In some embodiments, the tolerogenic factor is HLA-E (NCBI Gene ID No.: 3133), HLA-G (NCBI Gene ID No.: 3135), CTLA-4 (NCBI Gene ID No.: 1493), CD47 (NCBI Gene ID No.: 961), or PD-L1 (NCBI Gene ID No.: 29126). In some embodiments, the tolerogenic factor is inserted into cells, e.g., universal donor cells. In some embodiments, the tolerogenic factor is deleted from cells, e.g., universal donor cells. In some embodiments, insertion of a polynucleotide encoding HLA-E, HLA-G, CTLA-4, CD47, and / or PD-L1 enables the cells, e.g., universal donor cells, to inhibit or avoid immune rejection after transplantation or engraftment into a host subject.

[0033] MHC-I or MHC-II transcriptional regulator: As used herein, the term "MHC-I or MHC-II transcriptional regulator" generally refers to a biological molecule that regulates, e.g., increases or decreases, expression of MHC-I and / or MHC-II human leukocyte antigens. In some embodiments, the biological molecule is a polynucleotide, e.g., a gene or a protein. In some embodiments, the MHC-I or MHC-II transcriptional regulator increases or decreases cell surface expression of at least one MHC-I or MHC-II protein. In some embodiments, the MHC-I or MHC-II transcriptional regulator increases or decreases expression of at least one MHC-I or MHC-II gene. In some embodiments, the transcriptional regulator is CIITA (NCBI Gene ID No.: 4261) or NLRC5 (NCBI Gene ID No.: 84166). In some embodiments, deleting or reducing expression of CIITA or NLRC5 decreases expression of at least one MHC-I or MHC-II gene.

[0034] Universal donor cells: As used herein, the term "universal donor cells" generally refers to genetically modified cells that are less susceptible to allorejection during cell transplantation and / or demonstrate increased survival after transplantation relative to unmodified cells. In some embodiments, genetically modified cells as described herein are universal donor cells. In some embodiments, universal donor cells have increased immune evasion and / or cell survival compared to unmodified cells. In some embodiments, universal donor cells have increased cell survival compared to unmodified cells. In some embodiments, universal donor cells can be stem cells. In some embodiments, universal donor cells can be embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), or hematopoietic stem and progenitor cells (HSPCs) (also referred to as hematopoietic stem cells (HSCs)). In some embodiments, universal donor cells can be differentiated cells. In some embodiments, universal donor cells can be somatic cells (e.g., immune system cells). In some embodiments, universal donor cells are administered to a subject. In some embodiments, the universal donor cells are administered to a subject who has, is suspected of having, or is at risk for a disease. In some embodiments, the universal donor cells can differentiate into lineage-restricted progenitor cells or fully differentiated somatic cells. In some embodiments, the lineage-restricted progenitor cells are pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells. In some embodiments, the fully differentiated somatic cells are endocrine cells such as pancreatic beta cells, epithelial cells, endodermal cells, macrophages, hepatocytes, adipocytes, kidney cells, blood cells, or immune system cells. In some embodiments, the fully differentiated somatic cells are cardiomyocytes.

[0035] Unmodified cell: As used herein, the term "unmodified cell" refers to a cell that has not been subjected to genetic modification, including a polynucleotide or gene encoding an MHC-I, MHC-I, MHC-I or MHC-II transcriptional regulator, survival factor, and / or tolerogenic factor. In some embodiments, an unmodified cell can be a stem cell. In some embodiments, an unmodified cell can be an embryonic stem cell (ESC), adult stem cell (ASC), induced pluripotent stem cell (iPSC), or hematopoietic stem or progenitor cell (HSPC) (also referred to as hematopoietic stem cell (HSC)). In some embodiments, an unmodified cell can be a differentiated cell. In some embodiments, an unmodified cell can be selected from somatic cells (e.g., immune system cells, e.g., T cells, e.g., CD8+ T cells). When a universal donor cell is compared "against an unmodified cell," the universal donor cell and the unmodified cell are the same cell type or share a common parental cell line, e.g., a universal donor iPSC is compared to an unmodified iPSC.

[0036] Within or near a gene: As used herein, the term "within or near a gene" refers to a site or region of genomic DNA that is an intronic or extronic component of the gene or that is located adjacent to the gene. In some embodiments, a site of genomic DNA is within a gene if it comprises at least a portion of an intron or exon of the gene. In some embodiments, a site of genomic DNA located near a gene can be at the 5' or 3' end of the gene (e.g., the 5' or 3' end of the coding region of the gene). In some embodiments, a site of genomic DNA located near a gene can be a promoter or repressor region that regulates expression of the gene. In some embodiments, a site of genomic DNA located near a gene can be on the same chromosome as the gene. In some embodiments, a site or region of genomic DNA is near a gene if it is within 50 Kb, 40 Kb, 30 Kb, 20 Kb, 10 Kb, 5 Kb, 1 Kb, or closer to the 5' or 3' end of the gene (e.g., the 5' or 3' end of the coding region of the gene).

[0037] II. Genome editing methods Genome editing generally refers to a process of modifying the nucleotide sequence of a genome, preferably in a precise or predetermined manner. In some embodiments, genome editing methods as described herein, such as a CRISPR-endonuclease system, can be used to genetically modify cells as described herein, for example, to create universal donor cells. In some embodiments, genome editing methods as described herein, such as a CRISPR-endonuclease system, can be used to genetically modify cells as described herein, for example, to introduce into unmodified cells at least one genetic modification within or near at least one gene that reduces the expression of one or more MHC-I and / or MHC-II human leukocyte antigens or other components of the MHC-I or MHC-II complex; to introduce into unmodified cells at least one genetic modification that increases the expression of at least one polynucleotide encoding a tolerogenic factor; and / or to introduce into unmodified cells at least one genetic modification that increases or decreases the expression of at least one gene encoding a survival factor.

[0038] Examples of genome editing methods described herein include those that use site-specific nucleases to cleave deoxyribonucleic acid (DNA) at precise target locations in the genome, thereby creating single- or double-stranded DNA breaks at specific locations within the genome. Such breaks can be repaired, and are regularly repaired, by natural endogenous cellular processes such as homology-directed repair (HDR) and non-homologous end joining (NHEJ), as described in Cox et al., "Therapeutic genome editing: prospects and challenges," Nature Medicine, 2015, 21(2), 121-31. These two major DNA repair processes comprise a family of alternative pathways. NHEJ directly joins the DNA ends resulting from a double-stranded break and may involve the deletion or addition of nucleotide sequences, which can disrupt or enhance gene expression. HDR utilizes a homologous or donor sequence as a template for the insertion of a predetermined DNA sequence at the breakpoint. The homologous sequence may be present in the endogenous genome, such as a sister chromatid. Alternatively, the donor sequence may be an exogenous polynucleotide such as a plasmid, single-stranded oligonucleotide, double-stranded oligonucleotide, or virus that has regions of high homology with the nuclease cleavage locus (e.g., left and right homology arms) but may also contain additional sequences or sequence changes, including deletions, that can be incorporated into the cleaved target locus. A third repair mechanism may be microhomology-mediated end joining (MMEJ), also known as "alternative NHEJ," in that the genetic consequences are similar to NHEJ in that small deletions and insertions can occur at the cleavage site.MMEJ can utilize a few base pairs of homologous sequences flanking the DNA break site to promote a more favorable DNA end-joining repair outcome, and recent reports have further elucidated the molecular mechanisms of this process; see, e.g., Cho and Greenberg, Nature, 2015, 518, 174-76; Kent et al., Nature Structural and Molecular Biology, 2015, 22(3):230-7; Mateos-Gomez et al., Nature, 2015, 518, 254-57; Ceccaldi et al., Nature, 2015, 528, 258-62. In some cases, it may be possible to predict the repair outcome based on analysis of potential microhomologies at the site of the DNA break.

[0039] Each of these genome editing mechanisms can be used to create desired genetic modifications.The step in genome editing process can be to create one or two DNA breaks, the latter being double-strand breaks or two single-strand breaks at the target locus as the vicinity of the intended mutation site.This can be achieved through the use of endonuclease as described and shown herein.

[0040] CRISPR endonuclease system CRISPR-endonuclease system is a naturally occurring defense mechanism in prokaryotes that has been adapted as an RNA-guided DNA targeting platform for gene editing.CRISPR system includes type I, II, III, IV, V and VI systems.In some embodiments, CRISPR system is type II CRISPR / Cas9 system.In other embodiments, CRISPR system is type V CRISPR / Cprf system.CRISPR system relies on DNA nuclease, such as Cas9, and two non-coding RNAs, crisprRNA (crRNA) and transactivating RNA (tracrRNA), to target DNA cleavage.

[0041] The crRNA typically facilitates sequence recognition and specificity of the CRISPR-endonuclease complex through Watson-Crick base pairing with an approximately 20-nucleotide (nt) sequence in the target DNA. Varying the sequence of the 5' 20 nt in the crRNA allows for targeting of the CRISPR-endonuclease complex to a specific locus. The CRISPR-endonuclease complex will only bind to DNA sequences containing a sequence matching the first 20 nt of the single guide RNA (sgRNA) if the target sequence is followed by a specific short DNA motif (having the sequence NGG) called a protospacer adjacent motif (PAM).

[0042] TracrRNA hybridizes to the 3' end of crRNA to form an RNA duplex structure and is bound by the endonuclease to form a catalytically active CRISPR-endonuclease complex, which can then cleave the target DNA.

[0043] When the CRISPR-endonuclease complex binds to DNA at the target site, two independent nuclease domains within the endonuclease each cut one of the DNA strands three bases upstream of the PAM site, leaving a double-strand break (DSB) where both strands of DNA terminate in base pairs (blunt ends).

[0044] In some embodiments, the endonuclease is Cas9 (CRISPR-associated protein 9). In some embodiments, the Cas9 endonuclease is derived from Streptococcus pyogenes, although other Cas9 homologs, such as S. aureus Cas9, N. meningitidis Cas9, S. thermophilus CRISPR1 Cas9, S. thermophilus CRISPR3 Cas9, or T. denticola Cas9, can be used. In other cases, the CRISPR endonuclease is Cpf1, such as L. bacterium ND2006 Cpf1 or Acidaminococcus sp. BV3L6 Cpf1. In some embodiments, the endonuclease is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease. In some embodiments, wild-type variants may be used. In some embodiments, modified versions of the foregoing endonucleases (e.g., homologs thereof, recombinant naturally occurring molecules thereof, codon-optimized or modified versions thereof) may be used.

[0045] The CRISPR nuclease may be linked to at least one nuclear localization signal (NLS), which may be located at or within 50 amino acids of the amino terminus of the CRISPR nuclease, and / or which may be located at or within 50 amino acids of the carboxy terminus of the CRISPR nuclease.

[0046] Exemplary CRISPR / Cas polypeptides include the Cas9 polypeptide as published in Fonfara et al., "Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems," Nucleic Acids Research, 2014, 42:2577-2590. The nomenclature of CRISPR / Cas genes has undergone extensive rewriting since the discovery of Cas genes. Fonfara et al. also provide PAM sequences for Cas9 polypeptides from various species.

[0047] Zinc finger nuclease Zinc finger nucleases (ZFNs) are modular proteins composed of an engineered zinc finger DNA-binding domain linked to the catalytic domain of the type II endonuclease FokI. Because FokI functions only as a dimer, pairs of ZFNs must be designed to bind identical target "half-site" sequences on opposite DNA strands with precise spacing to allow the formation of catalytically active FokI dimers. Dimerization of the FokI domain, which has no sequence specificity by itself, generates a DNA double-strand break between the ZFN half-sites as the initiating step of genome editing.

[0048] The DNA-binding domain of each ZFN typically consists of three to six zinc fingers with abundant Cys2-His2 structures. Each finger primarily recognizes a triplet of nucleotides on one strand of the target DNA sequence, although cross-strand interactions with the fourth nucleotide may also be important. Amino acid modifications of the fingers at positions that make critical contacts with DNA alter the sequence specificity of a given finger. Thus, a four-finger zinc finger protein will selectively recognize a 12-bp target sequence, which is composed of triplet preferences conferred by each finger, although triplet preferences can be influenced to varying degrees by adjacent fingers. An important aspect of ZFNs is that they can be easily retargeted to almost any genomic address by simply modifying individual fingers. In most applications of ZFNs, proteins with four to six fingers are used, each recognizing 12 to 18 bp. Thus, a pair of ZFNs will typically recognize a combined target sequence of 24 to 36 bp, without the typical 5 to 7 bp spacer between half sites. Binding sites can be further separated by larger spacers, including 15-17 bp. Target sequences of this length are expected to be unique in the human genome, and repeated sequences or gene homologs are assumed to be excluded during the design process. Nevertheless, ZFN protein-DNA interactions are not absolute in their specificity, resulting in off-target binding and cleavage events, either as heterodimers between two ZFNs or as homodimers of one or the other ZFN. The latter possibility was effectively eliminated by engineering the dimerization interface of the FokI domain to create "plus" and "minus" variants, also known as obligate heterodimer variants, that can simply dimerize with each other but not with themselves. Enforcing obligate heterodimerization prevents homodimer formation. This significantly enhances the specificity of not only ZFNs but also any other nucleases employing these FokI variants.

[0049] Various ZFN-based systems have been described in the art, modifications of which are regularly reported, and numerous references describe the rules and parameters used to guide the design of ZFNs; see, e.g., Segal et al., Proc Natl Acad Sci, 1999 96(6):2758-63; Dreier B et al., J Mol Biol., 2000, 303(4):489-502; Liu Q et al., J Biol Chem., 2002, 277(6):3850-6; Dreier et al., J Biol Chem., 2005, 280(42):35588-97; and Dreier et al., J Biol Chem. 2001, 276(31):29466-78.

[0050] Transcription activator-like effector nucleases (TALENs) TALENs, like ZFNs, represent another form of modular nuclease in which an engineered DNA-binding domain is linked to a FokI nuclease domain, with the pair of TALENs acting in tandem to achieve targeted DNA cleavage. The primary difference between TALENs and ZFNs is the nature of the DNA-binding domain and the associated target DNA sequence recognition properties. TALEN DNA-binding domains are derived from TALE proteins, which were first described in the plant pathogenic microorganism Xanthomonas sp. TALEs consist of tandem arrays of 33-35 amino acid repeats, each of which typically recognizes a single base pair in a target DNA sequence up to 20 bp long, giving a total target sequence length of up to 40 bp. The nucleotide specificity of each repeat is determined by a repeat variable dinucleotide (RVD) comprising two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine, and thymine are primarily recognized by four RVDs: Asn-Asn, Asn-Ile, His-Asp, and Asn-Gly, respectively. This provides a much simpler recognition code than zinc fingers, thus giving TALENs an advantage over zinc fingers in nuclease design. Nevertheless, like ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs also benefit from the use of obligate heterodimeric variants of the FokI domain, which reduces off-target activity.

[0051] Additional variants of the FokI domain, which are catalytically inactive, have been created. When one member of either a TALEN or ZFN pair contains an inactive FokI domain, only single-stranded DNA nicking occurs at the target site, rather than a DSB. This result is comparable to the use of "nickase" mutants of CRISPR / Cas9 or CRISPR / Cpf1 (in which one of the Cas9 cleavage domains is inactivated). DNA nicks can be used to drive genome editing via HDR, but with lower efficiency than DSBs. A key advantage is that off-target nicks are repaired rapidly and accurately, unlike DSBs, which are prone to NHEJ-mediated misrepair.

[0052] Various TALEN-based systems have been described in the art, and modifications thereof are reported regularly; see, e.g., Boch, Science, 2009 326(5959):1509-12; Mak et al., Science, 2012, 335(6069):716-9; and Moscou et al., Science, 2009, 326(5959):1501. The use of TALENs based on the "Golden Gate" platform or cloning scheme has been described by several groups; see, for example, Cermak et al., Nucleic Acids Res., 2011, 39(12):e82; Li et al., Nucleic Acids Res., 2011, 39(14):6315-25; Weber et al., PLoS One., 2011, 6(2):e16765; Wang et al., J Genet Genomics, 2014, 41(6):339-47.; and Cermak T et al., Methods Mol Biol., 2015 1239:133-59.

[0053] Homing endonucleases Homing endonucleases (HEs) are sequence-specific endonucleases with long recognition sequences (14–44 base pairs) that cleave DNA with high specificity (often at unique sites in the genome). There are at least six known families of HEs, classified by their structure, including GIY-YIG, His-Cis box, HNH, PD-(D / E)xK, and VSR-like, derived from a wide range of hosts, including eukaryotes, protists, bacteria, archaea, cyanobacteria, and phages. Similar to ZFNs and TALENs, HEs can be used to induce DSBs at target loci as the first step in genome editing. Additionally, some natural and engineered HEs cleave only a single strand of DNA, thereby functioning as site-specific nickases. The broad target sequence of HEs and the specificity they offer make them attractive candidates for initiating site-specific DSBs.

[0054] Various HE-based systems have been described in the art, and modifications thereof are regularly reported; see, for example, the reviews by Steentoft et al., Glycobiology, 2014, 24(8):663-80; Belfort and Bonocora, Methods Mol Biol., 2014, 1123:1-26; and Hafez and Hausner, Genome, 2012, 55(8):553-69.

[0055] MegaTAL / Tev-mTALEN / MegaTev As further examples of hybrid nucleases, the MegaTAL and Tev-mTALEN platforms use a fusion of the DNA-binding domain of a TALE and a catalytically active HE, taking advantage of both the tunable DNA binding and specificity of the TALE and the cleavage sequence specificity of the HE; see, e.g., Boissel et al., Nucleic Acids Res., 2014, 42:2591-2601; Kleinstiver et al., G3, 2014, 4:1155-65; and Boissel and Scharenberg, Methods Mol. Biol., 2015, 1239:171-96.

[0056] In a further variant, the MegaTev structure is a fusion of the nuclease domain from Meganuclease (Mega) GIY-YIG homing endonuclease I-TevI ​​(Tev). The two active sites are located approximately 30 bp apart on the DNA substrate, generating two DSBs with incompatible cohesive ends; see, for example, Wolfs et al., Nucleic Acids Res., 2014, 42, 8816-29. It is expected that other combinations of existing nuclease-based methods will be developed and will be useful for achieving the targeted genome modification described herein.

[0057] dCas9-FokI or dCpf1-Fok1 and other nucleases Combining the structural and functional properties of the above-mentioned nuclease platforms provides an additional approach to genome editing that may overcome some of their inherent drawbacks. For example, CRISPR genome editing systems typically use a single Cas9 endonuclease to induce DSBs. Targeting specificity is promoted by a 20- or 24-nucleotide sequence in the guide RNA that undergoes Watson-Crick base pairing with the target DNA (plus, in the case of Cas9 derived from S. pyogenes, an additional two bases in the adjacent NAG or NGG PAM sequence). While such sequences are long enough to be unique in the human genome, the specificity of the RNA / DNA interaction is not absolute, and significant randomness may be tolerated, particularly in the 5' half of the target sequence, effectively reducing the number of bases that promote specificity. One solution to this problem is to completely inactivate the catalytic function of Cas9 or Cpf1 (retaining only the RNA-guided DNA-binding function) and instead fuse the FokI domain to the inactivated Cas9; see, e.g., Tsai et al., Nature Biotech, 2014, 32:569-76; and Guilinger et al., Nature Biotech., 2014, 32:577-82. Because FokI must dimerize to become catalytically active, two guide RNAs are required to tether two FokI fusions in close proximity to form dimers and cleave DNA. This essentially doubles the number of bases in the combined target site, thereby increasing the targeting stringency of CRISPR-based systems.

[0058] As a further example, fusion of a TALE DNA binding domain to a catalytically active HE such as I-TevI ​​takes advantage of both the tunable DNA binding and specificity of the TALE and the cleavage sequence specificity of I-TevI, with the expectation that off-target cleavage may be further reduced.

[0059] RNA-guided endonucleasesAs used herein, an RNA-guided endonuclease system can comprise an amino acid sequence having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid sequence identity to a wild-type exemplary endonuclease, such as Cas9 from S. pyogenes (US Patent Application Publication No. 2014 / 0068797 SEQ ID NO: 8 or Sapranauskas et al., Nucleic Acids Res, 39(21):9275-9282 (2011)). The endonuclease may comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity over 10 consecutive amino acids to a wild-type endonuclease (e.g., Cas9 from S. pyogenes, supra). The endonuclease may comprise up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity over 10 consecutive amino acids to a wild-type endonuclease (e.g., Cas9 from S. pyogenes, supra). The endonuclease may comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9 from S. pyogenes, supra) over 10 consecutive amino acids in the HNH nuclease domain of the endonuclease. The endonuclease may comprise up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9 from S. pyogenes, supra) over 10 consecutive amino acids in the HNH nuclease domain of the endonuclease.The endonuclease may comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in the RuvC nuclease domain of the endonuclease. The endonuclease may comprise up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in the RuvC nuclease domain of the endonuclease.

[0060] The endonuclease may include a modified form of a wild-type exemplary endonuclease. The modified form of a wild-type exemplary endonuclease may include a mutation that reduces the nucleic acid cleavage activity of the endonuclease. The modified form of a wild-type exemplary endonuclease may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of a wild-type exemplary endonuclease (e.g., Cas9 from S. pyogenes, supra). The modified form of the endonuclease may not have substantial nucleic acid cleavage activity. When an endonuclease is a modified form that does not have substantial nucleic acid cleavage activity, it is referred to herein as "without enzymatic activity."

[0061] Contemplated mutations may include substitutions, additions, deletions, or any combination thereof. The mutation converts the mutated amino acid to alanine. The mutation converts the mutated amino acid to another amino acid (e.g., glycine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, or arginine). The mutation converts the mutated amino acid to an unnatural amino acid (e.g., selenomethionine). The mutation converts the mutated amino acid to an amino acid mimic (e.g., a phosphomimic). The mutation may be a conservative mutation. For example, the mutation converts the mutated amino acid to an amino acid similar in size, shape, charge, polarity, conformation, and / or rotamer to the mutated amino acid (e.g., a cysteine / serine mutation, a lysine / asparagine mutation, a histidine / phenylalanine mutation). Mutations may cause reading frame shifts and / or the creation of premature stop codons. Mutations may cause changes to gene regulatory regions or loci that affect the expression of one or more genes.

[0062] guide RNA The present disclosure provides a guide RNA (gRNA) that can direct the activity of an associated endonuclease to a specific target site within a polynucleotide. The guide RNA can include at least one spacer sequence that hybridizes to a target nucleic acid sequence of interest and a CRISPR repeat sequence. In a type II CRISPR system, the gRNA also includes a second RNA called a tracrRNA sequence. In a type II CRISPR guide RNA (gRNA), the CRISPR repeat sequence and the tracrRNA sequence hybridize to each other to form a duplex. In a type V CRISPR system, the gRNA includes a crRNA that forms a duplex. In some embodiments, the gRNA can bind to an endonuclease, such that the gRNA and the endonuclease form a complex. The gRNA can provide target specificity to the complex due to its association with the endonuclease. Thus, the genome-targeting nucleic acid can direct the activity of an endonuclease.

[0063] Exemplary guide RNAs include a spacer sequence containing 15-200 nucleotides, and the gRNA targets a genomic location based on the GRCh38 human genome assembly. As will be understood by those skilled in the art, each gRNA can be designed to include a spacer sequence complementary to its genomic target site or region. See Jinek et al., Science, 2012, 337, 816-821 and Deltcheva et al., Nature, 2011, 471, 602-607.

[0064] The gRNA can be a bimolecular guide RNA. The gRNA can be a monomolecular guide RNA.

[0065] A bimolecular guide RNA may comprise two strands of RNA. The first strand comprises, from 5' to 3', an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence. The second strand may comprise a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0066] A single guide RNA (sgRNA) may comprise, from 5' to 3', an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension may comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single guide linker may link the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension may comprise one or more hairpins.

[0067] In some embodiments, the sgRNA comprises a spacer sequence of 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA comprises a spacer sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA comprises a spacer sequence longer than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA comprises a variable length spacer sequence having 17-30 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA comprises a spacer extension sequence having a length of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 nucleotides. In some embodiments, the sgRNA comprises a spacer extension sequence having a length of less than 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides.

[0068] In some embodiments, the sgRNA comprises a spacer extension sequence that includes another moiety (e.g., a stability control sequence, an endoribonuclease binding sequence, or a ribozyme). The moiety can reduce or increase the stability of the nucleic acid to which it targets. The moiety can be a transcription terminator segment (i.e., a transcription termination sequence). The moiety can function in eukaryotic cells. The moiety can function in prokaryotic cells. The moiety can function in both eukaryotic and prokaryotic cells. Non-limiting examples of suitable moieties include a 5' cap (e.g., a 7-methylguanylate cap (m7 G)), a riboswitch sequence (e.g., allowing for modulation of stability and / or modulation of accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplast, etc.), a modification or sequence that provides tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.), and / or a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including a transcriptional activator, a transcriptional repressor, a DNA methyltransferase, a DNA demethylase, a histone acetyltransferase, a histone deacetylase, etc.).

[0069] In some embodiments, the sgRNA comprises a spacer sequence that hybridizes to a sequence in the target polynucleotide. The spacer of the gRNA can interact with the target polynucleotide in a sequence-specific manner through hybridization (i.e., base pairing). The nucleotide sequence of the spacer can vary depending on the sequence of the target nucleic acid of interest.

[0070] In the CRISPR-endonuclease system, a spacer sequence can be designed to hybridize to the target polynucleotide located 5' of the PAM of the endonuclease used in the system. The spacer can perfectly match the target sequence or can have a mismatch. Each endonuclease, such as Cas9 nuclease, has a specific PAM sequence that it recognizes in the target DNA. For example, S. pyogenes Cas9 recognizes a PAM containing the sequence 5'-NRG-3' (where R contains either A or G, and N is any nucleotide, and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence).

[0071] The target polynucleotide sequence may comprise 20 nucleotides. The target polynucleotide may comprise fewer than 20 nucleotides. The target polynucleotide may comprise more than 20 nucleotides. The target polynucleotide may comprise at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. The target polynucleotide may comprise up to 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides. The target polynucleotide sequence may comprise 20 bases immediately 5' from the first nucleotide of the PAM.

[0072] The spacer sequence that hybridizes to the target polynucleotide may have a length of at least about 6 nucleotides (nt). The spacer sequence may be at least about 6 nt, at least about 10 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt, or at least about 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50 nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, from about 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about 45 nt, or from about The length can be 10 nt to about 40 nt, about 10 nt to about 35 nt, about 10 nt to about 30 nt, about 10 nt to about 25 nt, about 10 nt to about 20 nt, about 10 nt to about 19 nt, about 19 nt to about 25 nt, about 19 nt to about 30 nt, about 19 nt to about 35 nt, about 19 nt to about 40 nt, about 19 nt to about 45 nt, about 19 nt to about 50 nt, about 19 nt to about 60 nt, about 20 nt to about 25 nt, about 20 nt to about 30 nt, about 20 nt to about 35 nt, about 20 nt to about 40 nt, about 20 nt to about 45 nt, about 20 nt to about 50 nt, or about 20 nt to about 60 nt. In some examples, the spacer sequence can contain 20 nucleotides. In some examples, the spacer can contain 19 nucleotides. In some examples, the spacer can contain 18 nucleotides. In some examples, the spacer may comprise 22 nucleotides.

[0073] In some cases, the percent complementarity between the spacer sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some cases, the percent complementarity between the spacer sequence and the target nucleic acid is up to about 30%, up to about 40%, up to about 50%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, up to about 95%, up to about 97%, up to about 98%, up to about 99%, or 100%. In some instances, the percent complementarity between the spacer sequence and the target nucleic acid is 100% over the six contiguous 5'-most nucleotides of the target sequence on the complementary strand of the target nucleic acid. The percent complementarity between the spacer sequence and the target nucleic acid may be at least 60% over about 20 contiguous nucleotides. The lengths of the spacer sequence and the target nucleic acid may differ by 1 to 6 nucleotides, which may be considered one or more bulges.

[0074] The tracrRNA sequence may contain nucleotides that hybridize to the minimal CRISPR repeat sequence in a cell. The minimal tracrRNA sequence and the minimal CRISPR repeat sequence may form a duplex, i.e., a base-paired, double-stranded structure. Together, the minimal tracrRNA sequence and the minimal CRISPR repeat can bind to an RNA-guided endonuclease. At least a portion of the minimal tracrRNA sequence can hybridize to the minimal CRISPR repeat sequence. The minimal tracrRNA sequence may be at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the minimal CRISPR repeat sequence.

[0075] The minimal tracrRNA sequence can be about 7 nucleotides to about 100 nucleotides in length. For example, the minimal tracrRNA sequence can be about 7 nucleotides (nt) to about 50 nt, about 7 nt to about 40 nt, about 7 nt to about 30 nt, about 7 nt to about 25 nt, about 7 nt to about 20 nt, about 7 nt to about 15 nt, about 8 nt to about 40 nt, about 8 nt to about 30 nt, about 8 nt to about 25 nt, about 8 nt to about 20 nt, about 8 nt to about 15 nt, about 15 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt in length. The minimal tracrRNA sequence can be approximately 9 nucleotides in length. The minimal tracrRNA sequence can be approximately 12 nucleotides in length. The minimal tracrRNA can consist of tracrRNA nt 23-48 as described in Jinek et al., supra.

[0076] The minimal tracrRNA sequence can be at least about 60% identical to a reference minimal tracrRNA (e.g., a wild-type, S. pyogenes-derived tracrRNA) sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, the minimal tracrRNA sequence can be at least about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, about 99% identical, or 100% identical to the reference minimal tracrRNA sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0077] The duplex between the minimal CRISPR RNA and the minimal tracrRNA can comprise a double helix. The duplex between the minimal CRISPR RNA and the minimal tracrRNA can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. The duplex between the minimal CRISPR RNA and the minimal tracrRNA can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.

[0078] A duplex may contain mismatches (i.e., the two strands of the duplex are not 100% complementary). A duplex may contain at least about 1, 2, 3, 4, or 5 mismatches. A duplex may contain up to about 1, 2, 3, 4, or 5 mismatches. A duplex may contain no more than 2 mismatches.

[0079] In some embodiments, the tracrRNA can be a 3' tracrRNA. In some embodiments, the 3' tracrRNA sequence can include a sequence having at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).

[0080] In some embodiments, the gRNA may comprise a tracrRNA extension sequence. The tracrRNA extension sequence may be from about 1 nucleotide to about 400 nucleotides in length. The tracrRNA extension sequence may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or more than 200 nucleotides in length. The tracrRNA extension sequence may be from about 20 to about 5,000 or more nucleotides in length. The tracrRNA extension sequence may be less than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides in length. The tracrRNA extension sequence may comprise less than 10 nucleotides in length. The tracrRNA extension sequence may be 10 to 30 nucleotides in length. The tracrRNA extension sequence can be 30-70 nucleotides in length.

[0081] The tracrRNA extension sequence may contain functional moieties (e.g., stability control sequences, ribozymes, endoribonuclease binding sequences). Functional moieties may include transcription terminator segments (i.e., transcription termination sequences). Functional moieties may have a total length of about 10 nucleotides (nt) to about 100 nucleotides, about 10 nt to about 20 nt, about 20 nt to about 30 nt, about 30 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, about 90 nt to about 100 nt, about 15 nt to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt.

[0082] In some embodiments, the sgRNA can include a linker sequence having a length of about 3 nucleotides to about 100 nucleotides. For example, a simple four-nucleotide "tetraloop" (-GAAA-) was used in Jinek et al., supra (Jinek et al., Science, 2012, 337(6096):816-821). Exemplary linkers have lengths of about 3 nucleotides (nt) to about 90 nt, about 3 nt to about 80 nt, about 3 nt to about 70 nt, about 3 nt to about 60 nt, about 3 nt to about 50 nt, about 3 nt to about 40 nt, about 3 nt to about 30 nt, about 3 nt to about 20 nt, or about 3 nt to about 10 nt. For example, the linker may have a length of about 3 nt to about 5 nt, about 5 nt to about 10 nt, about 10 nt to about 15 nt, about 15 nt to about 20 nt, about 20 nt to about 25 nt, about 25 nt to about 30 nt, about 30 nt to about 35 nt, about 35 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt. The linker of the single-molecule guide nucleic acid may be 4 to 40 nucleotides. The linker can be at least about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides. The linker can be up to about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.

[0083] The linker can comprise any of a variety of sequences, but in some instances, the linker will not comprise a sequence with extensive regions of homology to other portions of the guide RNA, which may cause intramolecular binding that would interfere with other functional regions of the guide. In Jinek et al., supra, a simple four-nucleotide sequence -GAAA- was used (Jinek et al., Science, 2012, 337(6096):816-821), but numerous other sequences, including longer sequences, can be used as well.

[0084] The linker sequence may comprise a functional moiety. For example, the linker sequence may comprise one or more features including an aptamer, a ribozyme, a protein-interacting hairpin, a protein-binding site, a CRISPR array, an intron, or an exon. The linker sequence may comprise at least about 1, 2, 3, 4, or 5 or more functional moieties. In some examples, the linker sequence may comprise up to about 1, 2, 3, 4, or 5 or more functional moieties.

[0085] In some embodiments, the sgRNA does not contain any uracil, e.g., at the 3' end of the sgRNA sequence. In some embodiments, the sgRNA contains one or more uracils, e.g., at the 3' end of the sgRNA sequence. In some embodiments, the sgRNA contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 uracils (U) at the 3' end of the sgRNA sequence.

[0086] The sgRNA can be chemically modified. In some embodiments, the chemically modified gRNA is a gRNA that includes at least one nucleotide with a chemical modification, such as a 2'-O-methyl sugar modification. In some embodiments, the chemically modified gRNA includes a modified nucleic acid backbone. In some embodiments, the chemically modified gRNA includes 2'-O-methyl-phosphorothioate residues. In some embodiments, the chemical modification enhances stability, reduces the likelihood or extent of an innate immune response, and / or enhances other attributes, as described in the art.

[0087] In some embodiments, modified gRNAs may include modified backbones, such as phosphorothioates, phosphotriesters, morpholinos, methylphosphonates, short chain alkyl or cycloalkyl intersugar linkages, or short chain heteroatom or heterocyclic intersugar linkages.

[0088] Morpholino compounds are described in Braasch and David Corey, Biochemistry, 2002, 41(14):4503-4510; Genesis, 2001, Volume 30, Issue 3; Heasman, Dev. Biol., 2002, 243:209-214; Nasevicius et al., Nat. Genet., 2000, 26:216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97:9591-9596; and U.S. Patent No. 5,034,506, issued July 23, 1991.

[0089] Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 2000, 122:8595-8602.

[0090] In some embodiments, the modified gRNA may include one or more substituted sugar moieties at the 2' position, such as one of the following: OH, SH, SCH, F, OCN, OCH, OCHO(CH)CH, O(CH)NH, or O(CH)CH (n is 1 to about 10); C1-C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl, or aralkyl; Cl; Br; CN; CF; OCF; O- , S- or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving group; reporter group; interfering substance; 2'-O-(2-methoxyethyl); 2'-methoxy (2'-O-CH3); 2'-propoxy (2'-OCH2CH2CH3); and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the gRNA, particularly the 3' position of the sugar on the 3'-terminal nucleotide and the 5' position of the 5'-terminal nucleotide. In some instances, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units can be replaced with novel groups.

[0091] Guide RNAs may additionally or alternatively include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases that are found only rarely or occasionally in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also called 5-methyl-2'-deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl MHC and gentobiosyl HMC, as well as synthetic nucleobases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine or other heterosubstituted alkyl adenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6-diaminopurine. Kornberg, A., DNA Replication, W.H. Freeman & Co., San Francisco, pp. 75-77, 1980; Gebeyehu et al., Nucl. Acids Res. 1997, 15:4513. "Universal" bases known in the art, such as inosine, may also be included. 5-Me-C substitution has been shown to increase the stability of nucleic acid duplexes by 0.6 to 1.2°C (Sanghvi, Y.S., in Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and is an example of a base substitution.

[0092] Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-isopropyl ... Other synthetic and natural nucleobases may be included, such as auracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0093] Complex of genome-targeting nucleic acid and endonuclease The gRNA interacts with an endonuclease (e.g., an RNA-guided nuclease such as Cas9), thereby forming a complex. The gRNA guides the endonuclease to a target polynucleotide.

[0094] The endonuclease and gRNA can each be administered separately to a cell or a subject. In some embodiments, the endonuclease can be pre-complexed with one or more crRNAs combined with one or more guide RNAs or tracrRNAs. The pre-complexed material can then be administered to a cell or a subject. Such pre-complexed material is known as a ribonucleoprotein particle (RNP). The endonuclease in the RNP can be, for example, a Cas9 endonuclease or a Cpf1 endonuclease. The endonuclease can be flanked at the N-terminus, C-terminus, or both the N-terminus and C-terminus by one or more nuclear localization signals (NLSs). For example, the Cas9 endonuclease can be flanked by two NLSs, one NLS located at the N-terminus and a second NLS located at the C-terminus. The NLS can be any NLS known in the art, such as the SV40 NLS. The molar ratio of genome-targeting nucleic acid to endonuclease in the RNP can range from about 1:1 to about 10:1. For example, the molar ratio of sgRNA to Cas9 endonuclease in the RNP can be 3:1.

[0095] Nucleic acid coding system components The present disclosure provides nucleic acids comprising nucleotide sequences encoding the genome-targeting nucleic acids of the present disclosure, the endonucleases of the present disclosure, and / or any nucleic acid or proteinaceous molecule necessary to carry out aspects of the methods of the present disclosure. The encoding nucleic acid can be RNA, DNA, or a combination thereof.

[0096] Nucleic acids encoding genome-targeting nucleic acids of the present disclosure, endonucleases of the present disclosure, and / or any nucleic acid or proteinaceous molecules necessary to carry out aspects of the methods of the present disclosure can be comprised in a vector (e.g., a recombinant expression vector).

[0097] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector, in which additional nucleic acid segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0098] In some instances, vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or, more simply, "expression vectors," which serve the same function.

[0099] The term "operably linked" means that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence. The term "regulatory sequence" is intended to include, for example, promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology, 1990, 185, Academic Press, San Diego, CA. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be recognized by those skilled in the art that the design of an expression vector can depend on factors such as the choice of target cells and the level of expression desired.

[0100] Contemplated expression vectors include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors. Other vectors contemplated for eukaryotic target cells include, but are not limited to, the vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Other vectors may be used as long as they are compatible with the host cell.

[0101] In some instances, a vector may contain one or more transcriptional and / or translational control elements. Depending on the host / vector system utilized, any of several suitable transcriptional and translational control elements, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc., may be used in the expression vector. The vector may be a self-inactivating vector that inactivates either viral sequences or components of the CRISPR machinery or other elements.

[0102] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters that function in eukaryotic cells) include those derived from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, human elongation factor-1 alpha promoter (EF1 alpha), chicken beta-actin promoter (CBA), ubiquitin C promoter (UBC), a hybrid construct comprising a cytomegalovirus enhancer fused to the chicken beta-actin promoter (CAG), a hybrid construct comprising a cytomegalovirus enhancer fused to the promoter, first exon and first intron of the chicken beta-actin gene (CAG or CAGGS), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I promoter.

[0103] The promoter may be an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc.). The promoter may be a constitutive promoter (e.g., a CMV promoter, a UBC promoter, a CAG promoter). In some cases, the promoter may be a spatially constrained promoter and / or a temporally constrained promoter (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.).

[0104] Introduction of the complexes, polypeptides and nucleic acids of the present disclosure into cells can be carried out by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0105] III. Strategies to Evade the Immune Response and Increase Survival Described herein are strategies that allow genetically modified cells, i.e., universal donor cells, to increase their survival or viability after engraftment into a subject and / or evade an immune response. In some embodiments, these strategies allow universal donor cells to survive and / or evade an immune response at a higher success rate than unmodified cells. In some embodiments, the genetically modified cells comprise the introduction of at least one genetic modification within or near at least one gene encoding a survival factor, wherein the genetic modification comprises the insertion of a polypeptide encoding a tolerogenic factor. The universal donor cells may further comprise at least one genetic modification within or near a gene encoding one or more MHC-I or MHC-II human leukocyte antigens or components or transcriptional regulators of the MHC-I or MHC-II complexes, wherein the genetic modification comprises the insertion of a polypeptide encoding a second tolerogenic factor.

[0106] In some embodiments, the genetically modified cells comprise the introduction of at least one genetic modification within or near at least one gene that reduces expression of one or more MHC-I and MHC-II human leukocyte antigens relative to unmodified cells; at least one genetic modification that increases expression of at least one polynucleotide encoding a tolerogenic factor relative to unmodified cells; and at least one genetic modification that modifies expression of at least one gene encoding a survival factor relative to unmodified cells. In other embodiments, the genetically modified cells comprise at least one deletion or insertion-deletion mutation within or near at least one gene that modifies expression of one or more MHC-I and MHC-II human leukocyte antigens relative to unmodified cells; and at least one insertion of a polynucleotide encoding at least one tolerogenic factor at a site that partially overlaps, completely overlaps, or is contained within the site of a deletion of a gene that modifies expression of one or more MHC-I and MHC-II HLA. In yet other embodiments, the genetically modified cells comprise at least one genetic modification that modifies expression of at least one gene encoding a survival factor relative to unmodified cells.

[0107] Genes encoding the major histocompatibility complex (MHC) are located on human Chr. 6p21. The resulting proteins encoded by MHC genes are a series of surface proteins essential for donor compatibility during cell transplantation. MHC genes are classified into MHC class I (MHC-I) and MHC class II (MHC-II). MHC-I genes (HLA-A, HLA-B, and HLA-C) are expressed in almost all tissue types and present peptides processed from "non-self" antigens to CD8+ T cells, thereby promoting their activation as cytolytic CD8+ T cells. Transplanted or engrafted cells expressing "non-self" MHC-I molecules trigger a strong cellular immune response directed against these cells, ultimately leading to their elimination by activated cytolytic CD8+ T cells. MHC-I proteins essentially associate with beta-2-microglobulin (B2M) in the endoplasmic reticulum, which is essential for the formation of functional MHC-I molecules on the cell surface. In addition, there are three non-classical MHC-Ib molecules (HLA-E, HLA-F, and HLA-G) that have immunoregulatory functions. MHC-II biomolecules include HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR. Due to their key functions in the immune response, MHC-I and MHC-II biomolecules contribute to immune rejection after cell engraftment of non-host cells, for example, for regenerative medicine purposes.

[0108] MHC-I cell surface molecules are composed of an MHC-encoded heavy chain (HLA-A, HLA-B, or HLA-C) and the invariant subunit beta-2-microglobulin (B2M). Therefore, reducing the intracellular concentration of B2M provides an effective method for reducing cell surface expression of MHC-I cell surface molecules.

[0109] In some embodiments, the cells comprise a genetic modification of one or more MHC-I or MHC-II genes. In some embodiments, the cells comprise a genetic modification of one or more polynucleotide sequences that regulate expression of MHC-I and / or MHC-II. In some embodiments, the genetic modifications of the present disclosure are performed using any gene editing method, including, but not limited to, those described herein.

[0110] In some embodiments, reducing the expression of one or more MHC-I and MHC-II human leukocyte antigens relative to unmodified cells is achieved, for example, by targeting for gene deletion and / or insertion of at least one base pair directly in the MHC-I and / or MHC-II gene. In some embodiments, reducing the expression of one or more MHC-I and MHC-II human leukocyte antigens relative to unmodified cells is achieved, for example, by targeting for gene deletion the CIITA gene. In some embodiments, reducing the expression of one or more MHC-I and MHC-II human leukocyte antigens relative to unmodified cells is achieved, for example, by targeting for gene deletion at least one transcriptional regulator of MHC-I or MHC-II. In some embodiments, the MHC-I or MHC-II transcriptional regulator is the NLRC5 or CIITA gene. In some embodiments, the transcriptional regulator of MHC-I or MHC-II is an RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, IRF-1 and / or TAP1 gene.

[0111] In some embodiments, the genome of the cell has been modified to delete all or part of an HLA-A, HLA-B, and / or HLA-C gene. In some embodiments, the genome of the cell has been modified to delete all or part of a promoter region of an HLA-A, HLA-B, and / or HLA-C gene. In some embodiments, the genome of the cell has been modified to delete all or part of a gene encoding a transcriptional regulator of MHC-I or MHC-II. In some embodiments, the genome of the cell has been modified to delete all or part of a promoter region of a gene encoding a transcriptional regulator of MHC-I or MHC-II.

[0112] In some embodiments, the cell's genome is modified to reduce expression of beta-2-microglobulin (B2M). B2M is a non-polymorphic gene that encodes a shared protein subunit required for surface expression of all polymorphic MHC class I heavy chains. HLA-I proteins directly associate with B2M in the endoplasmic reticulum, which is essential for forming functional cell surface-expressed HLA-I molecules. In some embodiments, the gRNA targets a site in the B2M gene that contains the 5'-GCTACTCTCTCTTTCTGGCC-3' sequence (SEQ ID NO: 1). In some embodiments, the gRNA targets a site in the B2M gene that contains the 5'-GGCCGAGATGTCTCGCTCCG-3' sequence (SEQ ID NO: 2). In some embodiments, the gRNA targets a site in the B2M gene that contains the 5'-CGCGAGCACAGCTAAGGCCA-3' sequence (SEQ ID NO: 3). In alternative embodiments, the gRNA targets a site within the B2M gene comprising any of the following sequences: 5'-TATAAGTGGAGGCGTCGCGC-3' (SEQ ID NO: 35), 5'-GAGTAGCGCGAGCACAGCTA-3' (SEQ ID NO: 36), 5'-ACTGGACGCGTCGCGCTGGC-3' (SEQ ID NO: 37), 5'-AAGTGGAGGCGTCGCGCTGG-3' (SEQ ID NO: 38), 5-GGCCACGGAGCGAGACATCT-3' (SEQ ID NO: 39), 5'-GCCCGAATGCTGTCAGCTTC-3' (SEQ ID NO: 40), 5'-CTCGCGCTACTCTCTCTTTC-3' (SEQ ID NO: 41), 5'-TCCTGAAGCTGACAGCATTC-3' (SEQ ID NO: 42), 5'-TTCCTGAAGCTGACAGCATT-3' (SEQ ID NO: 43), or 5'-ACTCTCTCTTTCTGGCCTGG-3' (SEQ ID NO: 44). In some embodiments, the gRNA comprises the polynucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44. The gRNA / CRISPR nuclease complex targets and cleaves the target site in the B2M locus.Repair of the double-strand break by NHEJ can result in a deletion of at least one nucleotide and / or an insertion of at least one nucleotide, thereby disrupting or eliminating expression of B2M. Alternatively, the B2M locus may be targeted by at least two CRISPR systems, each containing a different gRNA, such that cuts at two sites in the B2M locus result in deletion of the sequence between the two cuts, thereby eliminating expression of B2M.

[0113] In some embodiments, the cell's genome is modified to reduce expression of thioredoxin interacting protein (TXNIP). In some embodiments, the gRNA targets a site in the TXNIP gene comprising the 5'-GAAGCGTGTCTTCATAGCGC-3' sequence (SEQ ID NO: 15). In some embodiments, the gRNA targets a site in the TXNIP gene comprising the 5'-TTACTCGTGTCAAAGCCGTT-3' sequence (SEQ ID NO: 16). In some embodiments, the gRNA targets a site in the TXNIP gene comprising the 5'-TGTCAAAGCCGTTAGGATCC-3' sequence (SEQ ID NO: 17). In some embodiments, the gRNA targets a site in the TXNIP gene comprising the 5'-GCCGTTAGGATCCTGGCTTG-3' sequence (SEQ ID NO: 18). In some embodiments, the gRNA targets a site in the TXNIP gene comprising the 5'-GCGGAGTGGCTAAAGTGCTT-3' sequence (SEQ ID NO: 19). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-TCCGCAAGCCAGGATCCTAA-3' sequence (SEQ ID NO: 20). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5-GTTCGGCTTTGAGCTTCCTC-3' sequence (SEQ ID NO: 21). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-GAGATGGTGATCATGAGACC-3' sequence (SEQ ID NO: 22). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-TTGTACTCATATTTGTTTCC-3' sequence (SEQ ID NO: 23). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-AACAAATATGAGTACAAGTT-3' sequence (SEQ ID NO: 24). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-GAAGCGTGTCTTCATAGCGCAGG-3' sequence (SEQ ID NO: 45). In some embodiments, the gRNA targets a site within the TXNIP gene that contains the sequence 5'-TTACTCGTGTCAAAGCCGTTAGG-3' (SEQ ID NO: 46).In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-TGTCAAAGCCGTTAGGATCCTGG-3' sequence (SEQ ID NO: 47). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-GCCGTTAGGATCCTGGCTTGCGG-3' sequence (SEQ ID NO: 48). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-GCGGAGTGGCTAAAGTGCTTTGG-3' sequence (SEQ ID NO: 49). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-TCCGCAAGCCAGGATCCTAACGG-3' sequence (SEQ ID NO: 50). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-GTTCGGCTTTGAGCTTCCTCAGG-3' sequence (SEQ ID NO: 51). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-GAGATGGTGATCATGAGACCTGG-3' sequence (SEQ ID NO: 52). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-TTGTACTCATATTTGTTTCCAGG-3' sequence (SEQ ID NO: 53). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the 5'-AACAAATATGAGTACAAGTTCGG-3' sequence (SEQ ID NO: 54). In some embodiments, the gRNA targets a site within the TXNIP gene comprising the polynucleotide sequence of any one of SEQ ID NOs: 15-24 or 45-54. In some embodiments, the gRNA targets the polynucleotide sequence of any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. The gRNA / CRISPR nuclease complex targets and cleaves the target site in the TXNIP gene locus. Repair of the double-strand break by NHEJ can result in a deletion of at least one nucleotide and / or an insertion of at least one nucleotide, thereby disrupting or eliminating the expression of TXNIP.Alternatively, insertion of a polynucleotide encoding a foreign gene into the TXNIP gene locus may disrupt or eliminate expression of TXNIP.

[0114] In some embodiments, the cell's genome is modified to reduce expression of class II transactivator (CIITA). CIITA is a member of the LR or nucleotide-binding domain (NBD) leucine-rich repeat (LRR) family of proteins that regulates MHC-II transcription by associating with the MHC enhanceosome. CIITA expression is induced in B cells and dendritic cells depending on developmental stage and is inducible by IFN-γ in most cell types.

[0115] In some embodiments, the cell genome is modified to reduce expression of NLR family CARD domain-containing 5 (NLRC5). NLRC5 is a critical regulator of MHC-I-mediated immune responses. Like CIITA, NLRC5 is highly inducible by IFN-γ and can translocate to the nucleus. NLRC5 activates the promoter of MHC-I genes, inducing the transcription of MHC-I and related genes involved in MHC-I antigen presentation.

[0116] In some embodiments, tolerogenic factors can be inserted or reinserted into genetically modified cells to create immune-privileged universal donor cells. In some embodiments, the universal donor cells disclosed herein are further modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, but are not limited to, one or more of HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, and IL-35. In some embodiments, genetic modification, e.g., insertion, of at least one polynucleotide encoding at least one tolerogenic factor enables the universal donor cells to inhibit or avoid immune rejection at least 1.05-fold, at least 1.1-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold higher than unmodified cells after engraftment. In some embodiments, insertion of polynucleotides encoding HLA-E, HLA-G, CTLA-4, CD47, and / or PD-L1 enables the universal donor cells to inhibit or avoid immune rejection after transplantation or engraftment into a host subject.

[0117] A polynucleotide encoding a tolerogenic factor generally comprises left and right homology arms flanking the sequence encoding the tolerogenic factor. The homology arms have substantial sequence homology with genomic DNA at or near the targeted insertion site. For example, the left homology arm can be a nucleotide sequence homologous to a region located to the left or upstream of the target site or cleavage site, and the right homology arm can be a nucleotide sequence homologous to a region located to the right or downstream of the target site or cleavage site. The proximal end of each homology arm can be homologous to a genomic DNA sequence adjacent to the cleavage site. Alternatively, the proximal end of each homology arm can be homologous to a genomic DNA sequence located up to about 10, 20, 30, 40, 50, 60, or 70 nucleic acid bases away from the cleavage site. Thus, a polynucleotide encoding a tolerogenic factor can be inserted into a targeted locus within about 10, 20, 30, 40, 50, 60, or 70 base pairs of the cleavage site, and additional genomic DNA adjacent to the cleavage site (and not sharing homology with the homology arms) can be deleted. Homology arms can range in length from about 50 nucleotides to several thousand nucleotides. In some embodiments, homology arms can range in length from about 500 nucleotides to about 1,000 nucleotides. Substantial sequence homology between the homology arms and the genomic DNA can be at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.

[0118] In some embodiments, homology arms are used with B2M guides (e.g., gRNAs comprising the nucleotide sequences of SEQ ID NOS: 1-3, 35-44). In some embodiments, homology arms are designed for use with any B2M guide that will remove the start site of the B2M gene. In some embodiments, the B2M homology arm can comprise or consist essentially of the polynucleotide sequence of SEQ ID NOS: 7 or 13, or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to that of SEQ ID NOS: 7 or 13. In some embodiments, the left B2M homology arm can comprise or consist essentially of SEQ ID NOS: 7 or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to that of SEQ ID NOS: 7. In some embodiments, the right B2M homology arm can comprise or consist essentially of SEQ ID NOS: 13 or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to that of SEQ ID NOS: 13.

[0119] In some embodiments, the homology arm is used with a TXNIP guide (e.g., a gRNA comprising the nucleotide sequence of SEQ ID NOs: 15-24). In some embodiments, the homology arm is designed for use with any TXNIP guide that targets exon 1 of TXNIP (e.g., a gRNA comprising the nucleotide sequence of SEQ ID NOs: 15-20). In some embodiments, the TXNIP homology arm can comprise or consist essentially of the polynucleotide sequence of SEQ ID NO: 25 or 32, or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to that of SEQ ID NO: 25 or 32. In some embodiments, the left TXNIP homology arm can comprise or consist essentially of SEQ ID NO: 25, or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to that of SEQ ID NO: 25. In some embodiments, the right TXNIP homology arm may comprise or essentially consist of SEQ ID NO: 32 or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to that of SEQ ID NO: 32.

[0120] At least one polynucleotide encoding at least one tolerogenic factor can be operably linked to an exogenous promoter. The exogenous promoter can be a constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoter. In some embodiments, the exogenous promoter is a CMV, EF1a, PGK, CAG, or UBC promoter.

[0121] In some embodiments, at least one polynucleotide encoding at least one tolerogenic factor is inserted into a safe harbor locus, e.g., the AAVS1 locus. In some embodiments, at least one polynucleotide encoding at least one tolerogenic factor is inserted into a site or region of genomic DNA that partially overlaps, completely overlaps, or is contained within (i.e., within or near) an MHC-I gene, an MHC-II gene, or a transcriptional regulator of MHC-I or MHC-II.

[0122] In some embodiments, the polynucleotide encoding PD-L1 is inserted at a site within or near the B2M locus. In some embodiments, the polynucleotide encoding PD-L1 is inserted at a site within or near the B2M locus that coincides with or follows a deletion of all or part of the B2M gene or promoter. The polynucleotide encoding PD-L1 is operably linked to an exogenous promoter. The exogenous promoter may be a CMV promoter. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 11.

[0123] In some embodiments, the polynucleotide encoding HLA-E is inserted at a site within or near the B2M locus. In some embodiments, the polynucleotide encoding HLA-E is inserted at a site within or near the B2M locus that coincides with or follows a deletion of all or a portion of the B2M gene or promoter. The polynucleotide encoding HLA-E is operably linked to an exogenous promoter. The exogenous promoter can be a CMV promoter. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 26, 27, 28, 29, 30, and / or 30. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 55.

[0124] In some embodiments, the polynucleotide encoding HLA-G is inserted at a site within or near the HLA-A, HLA-B, or HLA-C locus. In some embodiments, the polynucleotide encoding HLA-G is inserted at a site within or near the HLA-A, HLA-B, or HLA-C locus that coincides with or follows a deletion of the HLA-A, HLA-B, or HLA-C gene or promoter.

[0125] In some embodiments, the polynucleotide encoding CD47 is inserted at a site within or near the CIITA locus, hi some embodiments, the polynucleotide encoding CD47 is inserted at a site within or near the CIITA locus that coincides with or follows a deletion of the CIITA gene or promoter.

[0126] In some embodiments, the polynucleotide encoding HLA-G is inserted at a site within or near the HLA-A, HLA-B, or HLA-C locus that coincides with the insertion of the polynucleotide encoding CD47 at a site within or near the CIITA locus.

[0127] In some embodiments, at least one polynucleotide encoding at least one tolerogenic factor can be delivered to a cell as part of a vector. For example, the vector can be a plasmid vector. In various embodiments, the amount of plasmid vector delivered to a cell is from about 0.5 μg to about 10 μg (about 10 6 (per cell). In some embodiments, the amount of plasmid can range from about 1 μg to about 8 μg, about 2 μg to about 6 μg, or about 3 μg to about 5 μg. In certain embodiments, the amount of plasmid delivered to a cell can be about 4 μg.

[0128] In some embodiments, the cells comprise increased or decreased expression of one or more survival factors. In some embodiments, the cells comprise an insertion of one or more polynucleotide sequences encoding a survival factor. In some embodiments, the cells comprise a deletion of one of the additional survival factors. In some embodiments, the genetic modifications of the present disclosure are performed using any gene editing method, including but not limited to those described herein. In some embodiments, the cells comprise increased or decreased expression of at least one survival factor relative to unmodified cells. In some embodiments, the survival factor is a member or critical pathway involved in cell survival, such as hypoxia, reactive oxygen species, nutrient deprivation, and / or oxidative stress. In some embodiments, the genetic modification of at least one survival factor allows the universal donor cells to survive for a longer period after engraftment than unmodified cells, for example, at least 1.05-fold, at least 1.1-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold longer. In some embodiments, the survival factor is ZNF143, TXNIP, FOXO1, JNK, or MANF.

[0129] In some embodiments, the cells comprise an insertion of a polynucleotide encoding MANF, allowing the universal donor cells to survive at a higher rate after transplantation or engraftment into a host subject relative to unmodified cells. In some embodiments, the polynucleotide encoding MANF is inserted into a safe harbor locus. In some embodiments, the polynucleotide encoding MANF is inserted into a gene belonging to MHC-I, MHC-II, or a transcriptional regulator of MHC-I or MHC-II.

[0130] In some embodiments, the genome of the cell has been modified to delete all or part of the ZNF143, TXNIP, FOXO1 and / or JNK gene, hi some embodiments, the genome of the cell has been modified to delete all or part of the promoter region of the ZNF143, TXNIP, FOXO1 and / or JNK gene.

[0131] In some embodiments, two or more survival factors are genetically modified in the cells.

[0132] In certain embodiments, cells that have no MHC-II expression and moderate expression of MHC-I are genetically modified to have no surface expression of MHC-I or MHC-II. In another embodiment, cells that have no surface expression of MHC-I / II are further edited to have expression of PD-L1, e.g., an insertion of a polynucleotide encoding PD-L1. In yet another embodiment, cells that have no surface expression of MHC-I / II are further edited to have expression of PD-L1, e.g., an insertion of a polynucleotide encoding PD-L1, and are also genetically modified to increase or decrease the expression of at least one gene encoding a survival factor, relative to the unmodified cell.

[0133] In some embodiments, the cells further comprise, e.g., by genetic modification, increased or decreased expression of one or more additional genes not necessarily related to either immune evasion or cell survival after engraftment. In some embodiments, the cells further comprise increased expression of one or more safety switch proteins relative to unmodified cells. In some embodiments, the cells comprise increased expression of one or more additional genes encoding safety switch proteins. In some embodiments, the safety switch is also a suicide gene. In some embodiments, the safety switch is herpes simplex virus-1 thymidine kinase (HSV-tk) or inducible caspase-9. In some embodiments, a polynucleotide encoding at least one safety switch is inserted into the genome, e.g., at a safe harbor locus. In some other embodiments, the one or more additional genes to be genetically modified encode one or more of the following: a safety switch protein incorporated with the construct; a targeting modality; a receptor; a signaling molecule; a transcription factor; a pharmaceutically active protein or peptide; a potential drug target; and a protein that promotes engraftment, trafficking, migration, viability, self-renewal, persistence, and / or survival.

[0134] One aspect of the present invention provides a method for generating a genomically engineered universal donor cell (the universal donor cell containing at least one targeted genomic modification at one or more selected sites in the genome), the method comprising: genetically engineering a cell type as described herein by introducing into the cell one or more constructs that enable the targeted modification at the selected site; introducing into the cell one or more double-strand breaks at the selected site using one or more endonucleases capable of recognizing the selected site; and culturing the edited cell to enable endogenous DNA repair to generate a targeted insertion or deletion at the selected site, thereby obtaining a genomically engineered universal donor cell. The genomically engineered donor cell may undergo successive rounds of genomic modification such that multiple sites are targeted and modified. The genomically engineered cells are cultured, characterized, selected, and expanded using techniques well known in the art. The universal donor cell generated by this method will contain at least one functional targeted genomic modification, and the genomically engineered cells, if they are stem cells, can differentiate into progenitor cells or fully differentiated cells.

[0135] In some other embodiments, the genomically engineered universal donor cells comprise introduced or increased expression of at least one of HLA-E, HLA-G, CD47, or PD-L1. In some embodiments, the genomically engineered universal donor cells are HLA class I and / or class II deficient. In some embodiments, the genomically engineered universal donor cells comprise B2M null or deficient B2M. In some embodiments, the genomically engineered universal donor cells comprise an integrated or non-integrated exogenous polynucleotide encoding one or more of HLA-E, HLA-G, and PD-L1. In some embodiments, the introduced expression is increased expression from either a non-expressed or under-expressed gene contained in the cell. In some embodiments, the non-integrated exogenous polynucleotide is introduced using Sendai virus, AAV, an episome, or a plasmid. In some embodiments, the universal donor cells are B2M null with introduced expression of one or more of HLA-E, HLA-G, PD-L1, and increased or decreased expression of at least one safety switch protein. In another embodiment, the universal donor cells are HLA-A, HLA-B, and HLA-C null, with the introduction of expression of one or more of HLA-E, HLA-G, PD-L1, and at least one safety switch protein. In some embodiments, the universal donor cells are B2M null, with the introduction of expression of one or more of HLA-E, HLA-G, PD-L1, and increased or decreased expression of at least one survival factor, e.g., MANF. It is contemplated that the methods of making any of the genetically modified cells described herein are performed using at least any of the gene editing methods described herein.

[0136] IV.Cell type Cells as described herein, e.g., universal donor cells (and corresponding unmodified cells), can belong to any possible class of cell type. In some embodiments, cells, e.g., universal donor cells (and corresponding unmodified cells), can be mammalian cells. In some embodiments, cells, e.g., universal donor cells (and corresponding unmodified cells), can be human cells. In some embodiments, cells, e.g., universal donor cells (and corresponding unmodified cells), can be stem cells. In some embodiments, cells, e.g., universal donor cells (and corresponding unmodified cells), can be pluripotent stem cells (PSCs). In some embodiments, cells, e.g., universal donor cells (and corresponding unmodified cells), can be embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), or hematopoietic stem and progenitor cells (HSPCs) (also referred to as hematopoietic stem cells (HSCs)). In some embodiments, cells, e.g., universal donor cells (and corresponding unmodified cells), can be differentiated cells. In some embodiments, the cells, eg, universal donor cells (and the corresponding unmodified cells), can be somatic cells, eg, immune system cells, or contractile cells, eg, skeletal muscle cells.

[0137] The cells described herein, e.g., universal donor stem cells, can be differentiated into appropriate cell types for determining HLA expression and assessing the immunogenicity of the universal stem cell line. Generally, differentiation involves maintaining the cells of interest for a period of time and under conditions sufficient for the cells to differentiate into the differentiated cells of interest. For example, the universal stem cells disclosed herein can be differentiated into mesenchymal progenitor cells (MPCs), hypoimmunogenic cardiomyocytes, muscle progenitor cells, blast cells, endothelial cells (ECs), macrophages, hepatocytes, beta cells (e.g., pancreatic beta cells), pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, hematopoietic progenitor cells, or neural progenitor cells (NPCs). In some embodiments, the universal donor cells can be differentiated into definitive endoderm cells, primitive gut cells, posterior foregut cells, pancreatic endoderm cells (PECs), pancreatic endocrine cells, immature beta cells, or mature beta cells.

[0138] Stem cells have the ability to both proliferate and give rise to additional progenitor cells, which in turn have the capacity to generate a large number of mother cells that can give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and generate progeny that subsequently differentiate into one or more mature cell types, but also retain one or more cells with the developmental potential of their parent. The term "stem cell," then, refers to a cell that, under certain circumstances, has the ability or potential to differentiate into a more specific or differentiated phenotype, and that, under certain circumstances, retains the ability to proliferate without substantial differentiation. In one aspect, the term progenitor cell or stem cell refers to a general mother cell, whose progeny (progeny) often specialize in different directions by acquiring entirely individual characteristics, as seen in differentiation, e.g., the gradual diversification of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can be derived from pluripotent cells, which themselves are derived from pluripotent cells, etc. Each of these pluripotent cells can be considered a stem cell, but the range of cell types each can give rise to can vary greatly. Some differentiated cells also have the ability to give rise to cells with higher developmental potential. Such ability can be natural or can be artificially induced upon treatment with various factors. In many biological examples, stem cells can also be "pluripotent" because they can generate progeny of two or more different cell types, although this is not required for "stemness."

[0139] A "differentiated cell" is a cell that is further along a developmental pathway than the cell to which it is being compared. Thus, stem cells can differentiate into lineage-restricted precursor cells (such as myocyte progenitors), which can differentiate into other types of precursor cells (such as myocyte precursors) further along the pathway, and then into end-stage differentiated cells such as myocytes, which may or may not retain the ability to play characteristic roles in certain tissue types and further proliferate. In some embodiments, the differentiated cells can be pancreatic beta cells.

[0140] embryonic stem cells The cells described herein may be embryonic stem cells (ESCs). ESCs are derived from the blastocysts of mammalian embryos and can differentiate into any cell type and proliferate rapidly. ESCs also appear to have normal karyotypes, maintain high telomerase activity, and exhibit significant long-term proliferation potential, making these cells excellent candidates for use as universal donor cells.

[0141] adult stem cells The cells described herein may be adult stem cells (ASCs). ASCs are undifferentiated cells that can be found in mammals, e.g., humans. ASCs are defined by their ability to self-renew, e.g., be passaged through multiple rounds of cell replication while maintaining an undifferentiated state, and their ability to differentiate into multiple different cell types, e.g., glial cells. Adult stem cells are a broad class of stem cells that can include hematopoietic stem cells, mammary stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, and testicular cells.

[0142] induced pluripotent stem cells The cells described herein may be induced pluripotent stem cells (iPSCs). iPSCs can be generated directly from adult human cells by introducing genes encoding critical transcription factors involved in pluripotency, such as OCT4, SOX2, cMYC, and KLF4. iPSCs can be derived from the same subject to whom subsequent progenitor cells are administered. That is, somatic cells can be obtained from a subject, reprogrammed into induced pluripotent stem cells, and then redifferentiated into progenitor cells (e.g., autologous cells) that are subsequently administered to the subject. However, with autologous cells, there remains a risk of immune response and poor survival rate after engraftment.

[0143] Human hematopoietic stem and progenitor cells The cells described herein can be human hematopoietic stem and progenitor cells (hHSPCs). This stem cell lineage gives rise to all blood cell types, including erythroid (red blood cells or red blood cells (RBCs)), myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, megakaryocytes / platelets, and dendritic cells), and lymphoid (T cells, B cells, and NK cells). Blood cells are produced by the proliferation and differentiation of a very small number of multipotent hematopoietic stem cells (HSCs) that have the ability to replenish themselves through self-renewal. During differentiation, HSC progeny develop through various intermediate maturation stages, generating multipotent and lineage-committed progenitor cells before reaching maturity. The bone marrow (BM) is the primary site of hematopoietic development in humans, and under normal conditions, very few hematopoietic stem and progenitor cells (HSPCs) can be found in peripheral blood (PB). Treatment with cytokines, some myelosuppressive drugs used in cancer therapy, and compounds that disrupt interactions between hematopoietic and BM stromal cells can rapidly mobilize large numbers of stem and progenitor cells into the circulation.

[0144] Differentiation of cells into other cell types Another step of the disclosed method may include differentiating cells into differentiated cells. The differentiation step may be performed according to any method known in the art. For example, human iPSCs may be differentiated into definitive endoderm using various treatments, including activin and B27 supplements (Life Technologies). The definitive endoderm may be further differentiated into hepatocytes, using treatments including FGF4, HGF, BMP2, BMP4, oncostatin M, dexamethasone, etc. (Duan et al., Stem Cells, 2010;28:674-686; Ma et al., Stem Cells Translational Medicine, 2013;2:409-419). In another embodiment, the differentiation step may be performed according to Sawitza et al., Sci Rep. 2015;5:13320. The differentiated cells may be any somatic cells of a mammal, for example, a human. In some embodiments, the somatic cell can be an exocrine epithelial cell (e.g., a salivary gland mucous cell, a prostate cell), a hormone-secreting cell (e.g., anterior pituitary gland cell, intestinal cell, pancreatic islet), a keratinized epithelial cell (e.g., an epidermal keratinocyte), a moist stratified barrier epithelial cell, a sensory transduction cell (e.g., a photoreceptor), an autonomic neuron, a sensory and peripheral neuron supporting cell (e.g., a Schwann cell), a central nervous system neuron, a glial cell (e.g., an astrocyte, an oligodendrocyte), a lens cell, an adipocyte, a kidney cell, a barrier function cell (e.g., a duct cell), an extracellular matrix cell, a contractile cell (e.g., a skeletal muscle cell, a cardiac muscle cell, a smooth muscle cell), a blood cell (e.g., an erythrocyte), an immune system cell (e.g., a megakaryocyte, a microglial cell, a neutrophil, a mast cell, a T cell, a B cell, a natural killer cell), a germ cell (e.g., a sperm cell), a nurse cell, or a stromal cell.

[0145] Generally, populations of universal donor cells disclosed herein maintain expression of one or more inserted nucleotide sequences over time. For example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the universal donor cells express one or more tolerogenic factors. Furthermore, populations of lineage-restricted or fully differentiated cells derived from the universal donor cells disclosed herein maintain expression of one or more inserted nucleotide sequences over time. For example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the lineage-restricted or fully differentiated cells express one or more tolerogenic factors.

[0146] V. Formulation and Administration Formulation and Delivery for Gene Editing Guide RNAs, polynucleotides, such as polynucleotides encoding tolerogenic factors or polynucleotides encoding endonucleases and endonucleases as described herein, can be formulated and delivered to cells in any manner known in the art.

[0147] Guide RNAs and / or polynucleotides may be formulated with pharmaceutically acceptable excipients, such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending on the particular mode of administration and dosage form. Guide RNA and / or polynucleotide compositions may be formulated to achieve a physiologically compatible pH and a pH range of about 3 to about 11, or about 3 to about 7, depending on the formulation and route of administration. In some cases, the pH may be adjusted to a range of about pH 5.0 to about pH 8. In some cases, the composition may include a therapeutically effective amount of at least one compound as described herein along with one or more pharmaceutically acceptable excipients. Optionally, the composition may include a combination of compounds described herein, or may include a second active ingredient useful in the treatment or prevention of bacterial growth (e.g., but not limited to, an antibacterial or antimicrobial agent), or may include a combination of reagents of the present disclosure.

[0148] Suitable excipients include, for example, carrier molecules including large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients can include antioxidants (including, but not limited to, ascorbic acid), chelating agents (including, but not limited to, EDTA), carbohydrates (including, but not limited to, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (including, but not limited to, oils, water, saline, glycerol, and ethanol), wetting or emulsifying agents, pH buffering substances, and the like.

[0149] The guide RNA polynucleotide (RNA or DNA) and / or endonuclease polynucleotide (RNA or DNA) can be delivered by viral or non-viral delivery vehicles known in the art. Alternatively, the endonuclease polypeptide can be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles. In further alternative embodiments, the DNA endonuclease can be delivered as one or more polypeptides, alone or pre-complexed with one or more crRNAs in combination with one or more guide RNAs or tracrRNAs.

[0150] Polynucleotides can be delivered by non-viral vehicles, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras and RNA-fusion protein complexes.Some exemplary non-viral delivery vehicles are described in Peer and Lieberman, Gene Therapy, 2011, 18:1127-1133 (focusing on the non-viral delivery vehicles for siRNA, which are also useful for the delivery of other polynucleotides).

[0151] With respect to the polynucleotides of the present disclosure, formulations may be selected from any of those taught, for example, in International Application PCT / US2012 / 069610.

[0152] Polynucleotides such as guide RNAs, sgRNAs, and mRNAs encoding endonucleases can be delivered to cells or subjects by lipid nanoparticles (LNPs).

[0153] LNP refers to any particle with a diameter of less than 1000 nm, less than 500 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, less than 75 nm, less than 50 nm, or less than 25 nm. Alternatively, nanoparticles can range in size from 1 to 1000 nm, 1 to 500 nm, 1 to 250 nm, 25 to 200 nm, 25 to 100 nm, 35 to 75 nm, or 25 to 60 nm.

[0154] LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids, such as the membrane-fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include poor stability and rapid clearance, as well as low efficacy due to the generation of inflammatory or anti-inflammatory responses.

[0155] LNPs can also be composed of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0156] Any lipid or combination of lipids known in the art can be used to produce LNPs. Examples of lipids used to produce LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids include 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids include DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids include PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0157] Lipids can be combined in any number of molar ratios to produce LNPs. In addition, polynucleotides can be combined with lipids in a wide range of molar ratios to produce LNPs.

[0158] Recombinant adeno-associated virus (AAV) vectors can be used for delivery. Techniques for generating rAAV particles, in which a cell is provided with a packaged AAV genome containing the polynucleotide to be delivered, the rep and cap genes, and helper virus functions, are standard in the art. rAAV production typically requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from (i.e., not within) the rAAV genome, and the helper virus functions. The AAV rep and cap genes can be derived from any AAV serotype from which a recombinant virus can be derived, and can be derived from an AAV serotype different from the rAAV genome ITRs, including, but not limited to, the AAV serotypes described herein. The production of pseudotyped rAAV is disclosed, for example, in International Patent Application Publication No. WO 01 / 83692.

[0159] Formulation and Administration of Cells, E.g., Universal Donor Cells Genetically modified cells as described herein, e.g., universal donor cells, can be formulated and administered to a subject by any manner known in the art.

[0160] The terms "administer," "introduce," "implant," "engraft," and "transplant" are used interchangeably in reference to the placement of cells, e.g., progenitor cells, into a subject by a method or route that results in at least partial localization of the introduced cells at the desired site. Cells, e.g., progenitor cells or their differentiated progeny, can be administered by any suitable route that results in delivery to a desired location in a subject where at least a portion of the implanted cells or components of the cells remain viable. The survival period of the cells after administration to a subject can be as short as a few hours (e.g., 24 hours) to several days, years, or even the lifespan of the subject (i.e., long-term engraftment).

[0161] Genetically modified cells as described herein, e.g., universal donor cells, may be viable for a longer period of time after administration to a subject than that of unmodified cells.

[0162] In some embodiments, compositions comprising cells as described herein can be administered by a suitable route, which can include, for example, intravenous administration as a bolus over a period of time or by continuous infusion. In some embodiments, intravenous administration can be performed by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, or intrathecal routes. In some embodiments, the composition can be in solid, aqueous, or liquid form. In some embodiments, aqueous or liquid forms can be spray-dried or lyophilized. In some embodiments, spray-dried or lyophilized forms can be reconstituted with an aqueous or liquid solution.

[0163] The cell composition may also be emulsified or presented as a liposomal composition, provided that the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredients may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients, and in amounts suitable for use in the therapeutic methods described herein.

[0164] Additional agents included in the cell composition may contain pharmaceutically acceptable salts of the components. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0165] Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredient and water, or that contain buffers such as sodium phosphate, saline, or both at physiological pH values, such as phosphate-buffered saline. Additionally, aqueous carriers may contain two or more buffer salts, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions may also contain liquid phases in addition to and in addition to water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in the cell composition that will be effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0166] In some embodiments, the composition comprising the cells may be administered to a subject, e.g., a human subject, who has, is suspected of having, or is at risk for a disease. In some embodiments, the composition may be administered to a subject who does not have, is not suspected of having, or is not at risk for a disease. In some embodiments, the subject is a healthy human. In some embodiments, the subject is a subject, e.g., a human subject, who has, is suspected of having, or is at risk for a genetically inherited disease. In some embodiments, the subject is suffering from or at risk for developing symptoms of a disease. In some embodiments, the disease is diabetes, e.g., type I diabetes or type II diabetes.

[0167] VI. CERTAIN COMPOSITIONS AND METHODS OF THE DISCLOSURE Accordingly, the present disclosure relates, inter alia, to the following non-limiting compositions and methods:

[0168] In a first composition, Composition 1, the present disclosure provides a composition comprising universal donor cells comprising a nucleotide sequence encoding a first tolerogenic factor inserted within or near a gene encoding the survival factor, wherein the universal donor cells express the tolerogenic factor and have disrupted expression of the survival factor, and the universal donor cells have increased immune evasion and / or cell survival compared to control cells.

[0169] In another composition, Composition 2, the present disclosure provides a composition as provided in Composition 1, wherein the control cell is a wild-type cell or a cell that does not contain the inserted nucleotide sequence.

[0170] In another composition, Composition 3, the present disclosure provides a composition as provided in Composition 1 or 2, wherein the disrupted expression of the survival factor comprises reduced or eliminated expression.

[0171] In another composition, Composition 4, the present disclosure provides a composition as provided in any one of Compositions 1-3, wherein the first tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

[0172] In another composition, Composition 5, the present disclosure provides a composition as provided in any one of Compositions 1-4, wherein the survival factor is TXNIP, ZNF143, FOXO1, JNK, or MANF.

[0173] In another composition, composition 6, the present disclosure provides a composition as provided in any one of composition claims 1 to 5, wherein the first immune tolerogenic factor is HLA-E and the survival factor is TXNIP.

[0174] In another composition, composition 7, the present disclosure provides a composition as provided in composition 5 or 6, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide.

[0175] In another composition, composition 8, the present disclosure provides a composition as provided in composition 7, wherein the sequence encoding the HLA-E trimer consists essentially of SEQ ID NO:55.

[0176] In another composition, composition 9, the present disclosure provides a composition as provided in any one of compositions 1-8, wherein the nucleotide sequence encoding the first tolerogenic factor is operably linked to an exogenous promoter.

[0177] In another composition, composition 10, the present disclosure provides a composition as provided in composition 9, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0178] In another composition, composition 11, the present disclosure provides a composition as provided in any one of composition claims 1-10, further comprising a nucleotide sequence encoding a second tolerogenic factor inserted within or near a gene encoding MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of the MHC-I or MHC-II complex, wherein the universal donor cells express the tolerogenic factor and have disrupted expression of the MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of the MHC-I or MHC-II complex.

[0179] In another composition, composition 12, the present disclosure provides a composition as provided in composition 11, wherein the composition comprises reduced or eliminated expression of an MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of an MHC-I or MHC-II complex.

[0180] In another composition, composition 13, the present disclosure provides a composition as provided in composition 11 or 12, wherein the second tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

[0181] In another composition, composition 14, the present disclosure provides a composition as provided in any one of compositions 11-13, wherein the MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of the MHC-I or MHC-II complex is HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, B2M, NLRC5, CIITA, RFX5, RFXAP, or RFXANK.

[0182] In another composition, composition 15, the present disclosure provides a composition as provided in any one of compositions 11-14, wherein the second tolerogenic factor is PD-L1, and the MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of the MHC-I or MHC-II complex, is B2M.

[0183] In another composition, composition 16, the present disclosure provides a composition as provided in composition 15, wherein the nucleotide sequence encoding PD-L1 consists essentially of SEQ ID NO:11.

[0184] In another composition, composition 17, the present disclosure provides a composition as provided in any one of compositions 11-16, wherein the nucleotide sequence encoding a second tolerogenic factor is operably linked to an exogenous promoter.

[0185] In another composition, composition 18, the present disclosure provides a composition as provided in composition 17, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0186] In another composition, composition 19, the present disclosure provides a composition as provided in any one of compositions 11-18, wherein the first tolerogenic factor is HLA-E, the survival factor is TXNIP, the second tolerogenic factor is PD-L1, and the MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of the MHC-I or MHC-II complex is B2M.

[0187] In another composition, composition 20, the present disclosure provides a composition as provided in any one of compositions 1-19, wherein the cells are stem cells.

[0188] In another composition, composition 21, the present disclosure provides a composition as provided in composition 20, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0189] In another composition, composition 22, the present disclosure provides a composition as provided in any one of compositions 1-19, wherein the cell is a differentiated cell or a somatic cell.

[0190] In another composition, composition 23, the present disclosure provides a composition as provided in any one of compositions 1-19, wherein the cells are capable of differentiating into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0191] In another composition, composition 24, the present disclosure provides a composition as provided in composition 23, wherein the lineage-restricted progenitor cells are pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells.

[0192] In another composition, composition 25, the present disclosure provides a composition as provided in composition 23, wherein the fully differentiated somatic cell is a pancreatic beta cell, an epithelial cell, an endodermal cell, a macrophage, a hepatocyte, an adipocyte, a kidney cell, a blood cell, a cardiac muscle cell, or an immune system cell.

[0193] In another composition, composition 26, the present disclosure provides a composition as provided in any one of compositions 1-25, wherein the composition comprises a plurality of universal donor cells.

[0194] In another composition, composition 27, the present disclosure provides a composition as provided in composition 26, wherein the composition is comprised of a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from a plurality of universal donor cells.

[0195] In another composition, composition 28, the present disclosure provides a composition as provided in composition 27, wherein the lineage-restricted progenitor cell is a pancreatic endodermal progenitor cell, a pancreatic endocrine progenitor cell, a mesenchymal progenitor cell, a muscle progenitor cell, a blast cell, a hematopoietic progenitor cell, or a neural progenitor cell, and the fully differentiated somatic cell is a pancreatic beta cell, an epithelial cell, an endodermal cell, a macrophage, a hepatocyte, an adipocyte, a kidney cell, a blood cell, a cardiomyocyte, or a cell of the immune system.

[0196] In another composition, composition 29, the present disclosure provides a composition as provided in composition 6 or 19, wherein the composition comprises a plurality of universal donor cells.

[0197] In another composition, composition 30, the present disclosure provides a composition as provided in composition 29, wherein the composition is comprised of a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from a plurality of universal donor cells.

[0198] In another composition, composition 31, the present disclosure provides a composition as provided in composition 30, wherein the lineage-restricted progenitor cell is a definitive endoderm cell, a primitive gut cell, a posterior foregut cell, a pancreatic endoderm progenitor cell, a pancreatic endocrine progenitor cell, an immature beta cell, or a mature beta cell, and the fully differentiated somatic cell is a pancreatic beta cell.

[0199] In another composition, composition 32, the present disclosure provides a composition as provided in composition 26 or 29, wherein at least about 50%, at least about 70%, or at least about 90% of the cells express the first tolerogenic factor, the second tolerogenic factor, or the first and second tolerogenic factors.

[0200] In another composition, composition 33, the present disclosure provides a composition as provided in any one of compositions 27, 28, 30, or 31, wherein at least about 50%, at least about 70%, or at least about 90% of the cells express the first tolerogenic factor, the second tolerogenic factor, or the first and second tolerogenic factors.

[0201] In another composition, composition 34, the present disclosure provides a composition comprising a plurality of cells of composition 26 or a population of cells of composition 27 or 28.

[0202] In another composition, composition 35, the present disclosure provides a composition as provided in composition 34 for use in treating a subject in need thereof.

[0203] In another composition, composition 36, the present disclosure provides a composition as provided in composition 35, wherein the subject has, is suspected of having, or is at risk of having a disease.

[0204] In another composition, composition 37, the present disclosure provides a composition as provided in composition 36, wherein the disease is a genetically inherited disease.

[0205] In another composition, composition 38, the present disclosure provides a composition comprising a plurality of cells of composition 29 or a population of cells of composition 30 or 31.

[0206] In another composition, composition 39, the present disclosure provides a composition as provided in composition 38 for treating diabetes in a subject in need thereof.

[0207] In another composition, composition 40, the present disclosure provides a composition as provided in composition 39, wherein the subject has type I diabetes or type II diabetes.

[0208] In another composition, composition 41, the present disclosure provides a composition as provided in any one of compositions 35-40, wherein the subject is a human.

[0209] In a first method, Method 1, the present disclosure provides a method of obtaining cells for administration to a subject in need thereof, the method comprising: (a) obtaining or having obtained a plurality of universal donor cells of any one of Compositions 26, 29, or 32; and (b) maintaining the plurality of universal donor cells for a time and under conditions sufficient for the cells to differentiate into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0210] In another method, Method 2, the disclosure provides a method for performing treatment in a subject in need thereof, the method comprising: (a) obtaining or having obtained a plurality of universal donor cells of any one of Compositions 26, 29, or 32 after differentiation into lineage-restricted progenitor cells or fully differentiated somatic cells; and (b) administering the lineage-restricted progenitor cells or fully differentiated somatic cells to the subject.

[0211] In another method, Method 3, the present disclosure provides a method as provided in Method 2, wherein administering comprises implanting into the subject a device comprising lineage-restricted progenitor cells or fully differentiated somatic cells.

[0212] In another method, Method 4, the disclosure provides a method as provided in any one of Methods 1-3, wherein the lineage-restricted progenitor cell is a pancreatic endodermal progenitor cell, a pancreatic endocrine progenitor cell, a mesenchymal progenitor cell, a muscle progenitor cell, a blast cell, a hematopoietic progenitor cell, or a neural progenitor cell, and the fully differentiated somatic cell is a pancreatic beta cell, an epithelial cell, an endodermal cell, a macrophage, a hepatocyte, an adipocyte, a kidney cell, a blood cell, a cardiomyocyte, or a cell of the immune system.

[0213] In another method, Method 5, the disclosure provides a method as provided in any one of Methods 1-4, wherein the subject has, is suspected of having, or is at risk of having the disease.

[0214] In another method, method 6, the disclosure provides a method as provided in method 5, wherein the disease is a genetically inherited disease.

[0215] In another method, Method 7, the disclosure provides a method as provided in any one of Methods 1-6, wherein the subject is a human.

[0216] In another method, Method 8, the present disclosure provides a method for treating diabetes in a subject in need thereof, comprising: (a) obtaining or having obtained a plurality of universal donor cells of Compositions 29 or 32 following differentiation into pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, mature beta cells, or pancreatic beta cells; and (b) administering the pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, mature beta cells, or pancreatic beta cells to the subject.

[0217] In another method, Method 9, the present disclosure provides a method as provided in Method 8, wherein administering comprises implanting into the subject a device comprising pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, mature beta cells, or pancreatic beta cells.

[0218] In another method, method 10, the disclosure provides a method as provided in method 8 or 9, wherein the subject has type I diabetes or type II diabetes.

[0219] In another method, Method 11, the disclosure provides a method as provided in any one of Methods 8-10, wherein the subject is a human.

[0220] In another composition, composition 41, the present disclosure provides a composition comprising universal donor cells comprising a nucleotide sequence encoding an HLA class I histocompatibility antigen, alpha chain E (HLA-E), inserted within or near a gene encoding thioredoxin interacting protein (TXNIP), wherein the universal donor cells express HLA-E and have disrupted expression of TXNIP, and the universal donor cells have increased immune evasion and / or cell survival compared to a control.

[0221] In another composition, composition 42, the present disclosure provides a composition as provided in composition 41, wherein the control cell is a wild-type cell or a cell that does not contain the inserted nucleotide sequence.

[0222] In another composition, composition 43, the present disclosure provides a composition as provided in composition 41, wherein the disrupted expression of TXNIP comprises reduced or eliminated expression.

[0223] In another composition, composition 44, the present disclosure provides a composition as provided in composition 41, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide.

[0224] In another composition, composition 45, the present disclosure provides a composition as provided in composition 44, wherein the sequence encoding the HLA-E trimer consists essentially of SEQ ID NO:55.

[0225] In another composition, composition 46, the present disclosure provides a composition as provided in composition 41, wherein the nucleotide sequence encoding HLA-E is operably linked to an exogenous promoter.

[0226] In another composition, composition 47, the present disclosure provides a composition as provided in composition 41, wherein the exogenous promoter is a CAG promoter.

[0227] In another composition, composition 48, the present disclosure provides a composition as provided in composition 41, wherein the cells are stem cells.

[0228] In another composition, composition 49, the present disclosure provides a composition as provided in composition 48, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0229] In another composition, composition 50, the present disclosure provides a composition as provided in composition 41, wherein the cell is a differentiated cell or a somatic cell.

[0230] In another composition, composition 51, the present disclosure provides a composition as provided in composition 41, wherein the cells are capable of differentiating into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0231] In another composition, composition 52, the present disclosure provides a composition as provided in composition 51, wherein the lineage-restricted progenitor cells are definitive endoderm cells, primitive gut cells, posterior foregut cells, pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, immature beta cells or mature beta cells, and the fully differentiated somatic cells are pancreatic beta cells.

[0232] In another composition, composition 53, the present disclosure provides a composition comprising a plurality of universal donor cells as provided in composition 41.

[0233] In another composition, composition 54, the present disclosure provides a composition as provided in composition 53, wherein at least about 50% of the cells express HLA-E.

[0234] In another composition, composition 55, the present disclosure provides a composition as provided in composition 53, wherein at least about 70% of the cells express HLA-E.

[0235] In another composition, composition 56, the present disclosure provides a composition as provided in composition 53, wherein at least about 90% of the cells express HLA-E.

[0236] In another composition, composition 57, the present disclosure provides a composition comprising a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from a plurality of universal donor cells of composition 53.

[0237] In another composition, composition 58, the present disclosure provides a composition as provided in composition 57, wherein the lineage-restricted progenitor cell is a definitive endoderm cell, a primitive gut cell, a posterior foregut cell, a pancreatic endoderm progenitor cell, a pancreatic endocrine progenitor cell, an immature beta cell, or a mature beta cell, and the fully differentiated somatic cell is a pancreatic beta cell.

[0238] In another composition, composition 59, the present disclosure provides a composition as provided in composition 58, wherein at least about 50% of the cells express HLA-E.

[0239] In another composition, composition 60, the present disclosure provides a composition as provided in composition 59, wherein at least about 70% of the cells express HLA-E.

[0240] In another composition, composition 61, the present disclosure provides a composition as provided in composition 59, wherein at least about 90% of the cells express HLA-E.

[0241] In another composition, composition 62, the present disclosure provides a composition comprising genetically modified cells having introduced or increased expression of HLA class I histocompatibility antigen, alpha chain E (HLA-E) and disrupted expression of thioredoxin interacting protein (TXNIP), wherein the genetically modified cells have increased immune evasion and / or cell survival compared to unmodified cells.

[0242] In another composition, composition 63, the present disclosure provides a composition as provided in composition 62, comprising a nucleotide sequence encoding HLA-E inserted within or near the gene encoding TXNIP, thereby disrupting the TXNIP gene.

[0243] In another composition, composition 64, the present disclosure provides a composition as provided in composition 62, wherein the disrupted expression of TXNIP comprises reduced or eliminated expression.

[0244] In another method, Method 12, the present disclosure provides a method for treating diabetes in a subject in need thereof, comprising (a) obtaining or having obtained a plurality of universal donor cells of Composition 53 following differentiation into pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, mature beta cells, or pancreatic beta cells; and (b) administering the pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, mature beta cells, or pancreatic beta cells to the subject.

[0245] In another method, Method 13, the present disclosure provides a method as provided in Method 12, wherein administering comprises implanting into the subject a device comprising pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, mature beta cells, or pancreatic beta cells.

[0246] In another method, method 14, the present disclosure provides a method as provided in method 12, wherein the subject has type I diabetes or type II diabetes.

[0247] In another method, method 15, the disclosure provides a method as provided in method 12, wherein the subject is a human.

[0248] In another composition, composition 65, the present disclosure provides a composition comprising universal donor cells comprising (a) a nucleotide sequence encoding programmed death-ligand 1 (PD-L1) inserted within or near a gene encoding beta-2 microglobulin (B2M), and (b) a nucleotide sequence encoding HLA class I histocompatibility antigen, alpha chain E (HLA-E) inserted within or near a gene encoding thioredoxin interacting protein (TXNIP), wherein the universal donor cells express PD-L1 and HLA-E and have disrupted expression of B2M and TXNIP, and the universal donor cells have increased immune evasion and / or cell survival compared to a control.

[0249] In another composition, composition 66, the present disclosure provides a composition as provided in composition 65, wherein the control cell is a wild-type cell or a cell that does not contain the inserted nucleotide sequence.

[0250] In another composition, composition 67, the present disclosure provides a composition as provided in composition 65, wherein the disrupted expression of B2M comprises reduced or eliminated expression of B2M, and the disrupted expression of TXNIP comprises reduced or eliminated expression of TXNIP.

[0251] In another composition, composition 68, the present disclosure provides a composition as provided in composition 65, wherein the nucleotide sequence encoding PD-L1 consists essentially of SEQ ID NO:11.

[0252] In another composition, composition 69, the present disclosure provides a composition as provided in composition 65, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide.

[0253] In another composition, composition 70, the present disclosure provides a composition as provided in composition 69, wherein the sequence encoding the HLA-E trimer consists essentially of SEQ ID NO:55.

[0254] In another composition, composition 71, the present disclosure provides a composition as provided in composition 65, wherein a nucleotide sequence encoding PD-L1 is operably linked to an exogenous promoter, and a nucleotide sequence encoding HLA-E is operably linked to an exogenous promoter.

[0255] In another composition, composition 72, the present disclosure provides a composition as provided in composition 71, wherein the exogenous promoter is a CAG promoter.

[0256] In another composition, composition 73, the present disclosure provides a composition as provided in composition 65, wherein the cells are stem cells.

[0257] In another composition, composition 74, the present disclosure provides a composition as provided in composition 73, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0258] In another composition, composition 75, the present disclosure provides a composition as provided in composition 65, wherein the cell is a differentiated cell or a somatic cell.

[0259] In another composition, composition 76, the present disclosure provides a composition as provided in composition 65, in which the cells are capable of differentiating into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0260] In another composition, composition 77, the present disclosure provides a composition as provided in composition 76, wherein the lineage-restricted progenitor cell is a definitive endoderm cell, a primitive gut cell, a posterior foregut cell, a pancreatic endoderm progenitor cell, a pancreatic endocrine progenitor cell, an immature beta cell, or a mature beta cell, and the fully differentiated somatic cell is a pancreatic beta cell.

[0261] In another composition, composition 78, the present disclosure provides a composition comprising a plurality of universal donor cells as provided in composition 65.

[0262] In another composition, composition 79, the present disclosure provides a composition as provided in composition 78, wherein at least about 50% of the cells express PD-L1 and / or at least about 50% of the cells express HLA-E.

[0263] In another composition, composition 80, the present disclosure provides a composition as provided in composition 78, wherein at least about 70% of the cells express PD-L1 and / or at least about 70% of the cells express HLA-E.

[0264] In another composition, composition 81, the present disclosure provides a composition as provided in composition 78, wherein at least about 90% of the cells express PD-L1 and / or at least about 90% of the cells express HLA-E.

[0265] In another composition, composition 82, the present disclosure provides a composition comprising a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from a plurality of universal donor cells as provided in composition 78.

[0266] In another composition, composition 83, the present disclosure provides a composition as provided in composition 82, wherein the lineage-restricted progenitor cell is a definitive endoderm cell, a primitive gut cell, a posterior foregut cell, a pancreatic endoderm progenitor cell, a pancreatic endocrine progenitor cell, an immature beta cell, or a mature beta cell, and the fully differentiated somatic cell is a pancreatic beta cell.

[0267] In another composition, composition 84, the present disclosure provides a composition as provided in composition 83, wherein at least about 50% of the cells express PD-L1 and / or at least about 50% of the cells express HLA-E.

[0268] In another composition, composition 85, the present disclosure provides a composition as provided in composition 83, wherein at least about 70% of the cells express PD-L1 and / or at least about 70% of the cells express HLA-E.

[0269] In another composition, composition 86, the present disclosure provides a composition as provided in composition 83, wherein at least about 90% of the cells express PD-L1 and / or at least about 90% of the cells express HLA-E.

[0270] In another composition, composition 87, the present disclosure provides a composition comprising genetically modified cells having introduced or increased expression of PD-L1 and HLA-E and disrupted expression of B2M and TXNIP, wherein the genetically modified cells have increased immune evasion and / or cell survival compared to unmodified cells.

[0271] In another composition, composition 88, the present disclosure provides a composition as provided in composition 87, comprising a nucleotide sequence encoding PD-L1 inserted within or near the gene encoding B2M, thereby disrupting the B2M gene, and comprising a nucleotide sequence encoding HLA-E inserted within or near the gene encoding TXNIP, thereby disrupting the TXNIP gene.

[0272] In another composition, composition 89, the present disclosure provides a composition as provided in composition 87, wherein the disrupted expression of B2M and TXNIP comprises reduced or eliminated expression of B2M and TXNIP.

[0273] In another method, Method 16, the present disclosure provides a method for treating diabetes in a subject in need thereof, comprising: (a) obtaining or having obtained a plurality of universal donor cells of Composition 78 following differentiation into pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, mature beta cells, or pancreatic beta cells; and (b) administering the pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, mature beta cells, or pancreatic beta cells to the subject.

[0274] In another method, Method 17, the present disclosure provides a method as provided in Method 16, wherein administering comprises implanting into the subject a device comprising pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, mature beta cells, or pancreatic beta cells.

[0275] In another method, method 18, the present disclosure provides a method as provided in method 16, wherein the subject has type I diabetes or type II diabetes.

[0276] In another method, method 19, the disclosure provides a method as provided in method 16, wherein the subject is a human.

[0277] In another method, Method 20, the present disclosure provides a method for generating a universal donor cell, comprising delivering to a cell (a) a first site-specific nuclease that targets a site within or near a gene encoding a survival factor; and (b) a first nucleic acid comprising: (i) a nucleotide sequence homologous to a region located to the left of the target site in (a) and (ii) a nucleotide sequence encoding a first tolerogenic factor, flanked by nucleotide sequences homologous to a region located to the right of the target site in (a), wherein the first site-specific nuclease cleaves the target site in (a), and the first nucleic acid in (b) is inserted at a site that partially overlaps, completely overlaps, or is contained within the site in (a), thereby generating a universal donor cell, and the universal donor cell has increased cell survival compared to a cell in which the nucleic acid of (b) has not been inserted.

[0278] In another method, method 21, the disclosure provides a method as provided in method 20, wherein the survival factor is TXNIP, ZNF143, FOXO1, JNK, or MANF.

[0279] In another method, method 22, the present disclosure provides a method as provided in methods 20 or 21, wherein the first tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

[0280] In another method, Method 23, the present disclosure provides a method as provided in any one of Methods 20-22, wherein the survival factor is TXNIP.

[0281] In another method, method 24, the present disclosure provides a method as provided in method 23, wherein the first tolerogenic factor is HLA-E.

[0282] In another method, Method 25, the present disclosure provides a method as provided in any one of Methods 20-24, wherein the first site-specific nuclease is a CRISPR system comprising a CRISPR nuclease and a guide RNA (gRNA).

[0283] In another method, Method 26, the present disclosure provides a method as provided in any one of Methods 20-25, wherein the CRISPR nuclease is a Type II Cas9 nuclease or a Type V Cfp1 nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal.

[0284] In another method, Method 27, the present disclosure provides a method as provided in any one of Methods 20-26, wherein the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NOs: 15-24.

[0285] In another method, Method 28, the present disclosure provides the method as provided in any one of Methods 25-27, wherein the nucleotide sequence of (b)(i) consists essentially of SEQ ID NO:25 and the nucleotide sequence of (b)(ii) consists essentially of SEQ ID NO:32.

[0286] In another method, Method 29, the disclosure provides a method for producing a nuclease comprising: (c) a second site-specific nuclease that targets a site within or near a gene encoding one or more of an MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of an MHC-I or MHC-II complex; and (d) a second site-specific nuclease that includes a nucleotide sequence encoding a second tolerogenic factor, flanked by (iii) a nucleotide sequence homologous to a region located to the left of the target site in (c) and (iv) a nucleotide sequence homologous to a region located to the right of the target site in (c). 29. A method as provided in any one of methods 20-28, further comprising delivering a nucleic acid to the cell, wherein the second tolerogenic factor of (d) is different from the first tolerogenic factor (b), the second site-specific nuclease cleaves the target site of (c), and the second nucleic acid of (d) is inserted at a site that partially overlaps, completely overlaps, or is contained within the site of (c), and the universal donor cell has increased immune evasion and / or cell survival compared to a cell in which the second nucleic acid of (d) has not been inserted.

[0287] In another method, method 30, the present disclosure provides a method as provided in method 29, wherein the MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of the MHC-I or MHC-II complex is HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, B2M, NLRC5, CIITA, RFX5, RFXAP, or RFXANK.

[0288] In another method, method 31, the present disclosure provides a method as provided in method 29 or 30, wherein the second tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

[0289] In another method, method 32, the present disclosure provides a method as provided in any one of methods 29-31, wherein the MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of an MHC-I or MHC-II complex is B2M.

[0290] In another method, method 33, the present disclosure provides a method as provided in method 32, wherein the second tolerogenic factor is PD-L1.

[0291] In another method, method 34, the present disclosure provides a method as provided in any one of methods 29-33, wherein the second site-specific nuclease is a CRISPR system comprising a CRISPR nuclease and a gRNA.

[0292] In another method, method 35, the present disclosure provides a method as provided in method 34, wherein the CRISPR nuclease is a Type II Cas9 nuclease or a Type V Cfp1 nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal.

[0293] In another method, method 36, the present disclosure provides a method as provided in method 34 or 35, wherein the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NOs: 1-3 or 35-44.

[0294] In another method, Method 37, the present disclosure provides the method as provided in any one of Methods 34-36, wherein the nucleotide sequence of (d)(iii) consists essentially of SEQ ID NO:7 and the nucleotide sequence of (d)(iv) consists essentially of SEQ ID NO:13.

[0295] In another method, method 38, the present disclosure provides a method as provided in any one of methods 25-28 or 34-37, wherein the CRISPR nuclease and gRNA are present in a molar ratio of 1:3.

[0296] In another method, Method 39, the present disclosure provides a method as provided in any one of Methods 20-38, wherein a nucleotide sequence encoding a first tolerogenic factor is operably linked to an exogenous promoter, and a nucleotide sequence encoding a second tolerogenic factor is operably linked to an exogenous promoter.

[0297] In another method, method 40, the disclosure provides a method as provided in method 39, wherein the exogenous promoter is a constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoter, optionally, the exogenous promoter is a CMV, EFIα, PGK, CAG, or UBC promoter.

[0298] In another method, method 41, the disclosure provides a method for generating a universal donor cell, comprising: (a) a first site-specific nuclease that targets a site within or near a gene encoding a survival factor; (b) a first nucleic acid comprising a nucleotide sequence encoding a first tolerogenic factor, flanked by (i) a nucleotide sequence homologous to a region located to the left of the target site in (a) and (ii) a nucleotide sequence homologous to a region located to the right of the target site in (a), wherein the first site-specific nuclease is a first nucleic acid that serves as a template for cleaving the target site of a) and, through a process of homologous recombination, inserting a nucleotide sequence encoding a first tolerogenic factor into a site that partially overlaps, completely overlaps, or is contained within the site of (a), thereby disrupting the gene of (a); (c) a first nucleic acid that is used as a template for cleaving the target site of a) and inserting a nucleotide sequence encoding a first tolerogenic factor into a site that partially overlaps, completely overlaps, or is contained within the site of (a), thereby disrupting the gene of (a); a second site-specific nuclease that targets a site in the vicinity of (c); and (d) a second nucleic acid comprising a nucleotide sequence encoding a second tolerogenic factor flanked by (iii) a nucleotide sequence homologous to a region located to the left of the target site in (c) and (iv) a nucleotide sequence homologous to a region located to the right of the target site in (c), wherein the tolerogenic factor in (d) is different from the tolerogenic factor (b), and the second site-specific nuclease cleaves the target site in (c) and, through the process of homologous recombination, and (d) a second nucleic acid inserting a nucleotide sequence encoding a second tolerogenic factor into a site that partially overlaps, completely overlaps, or is contained within the site in (c), thereby serving as a template for disrupting the gene in (c), delivering the second nucleic acid to the cell, thereby creating a universal donor cell, wherein the universal donor cell has increased cell survival compared to a cell not having the first nucleic acid in (b) and the second nucleic acid in (d) inserted therein.

[0299] In another method, method 42, the present disclosure provides a method as provided in method 41, wherein the survival factor is TXNIP, the first tolerogenic factor is HLA-E, the MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of the MHC-I or MHC-II complex is B2M, and the second tolerogenic factor is PD-L1.

[0300] In another method, method 43, the present disclosure provides a method as provided in any one of methods 20-42, wherein the cell is a mammalian cell, and optionally, the cell is a human cell.

[0301] In another method, method 44, the present disclosure provides a method as provided in any one of methods 20-43, wherein the cell is a stem cell.

[0302] In another method, method 45, the present disclosure provides a method as provided in any one of methods 20 to 43, wherein the cell is a pluripotent stem cell, an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.

[0303] In another method, method 46, the present disclosure provides a method as provided in any one of methods 20-43, wherein the cell is a differentiated cell or a somatic cell.

[0304] In another method, method 47, the present disclosure provides a method as provided in any one of methods 20-43, wherein the universal donor cells are capable of differentiating into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0305] In another method, method 48, the present disclosure provides a method as provided in method 47, wherein the lineage-restricted progenitor cell is a pancreatic endodermal progenitor cell, a pancreatic endocrine progenitor cell, a mesenchymal progenitor cell, a muscle progenitor cell, a blast cell, a hematopoietic progenitor cell, or a neural progenitor cell.

[0306] In another method, method 49, the present disclosure provides a method as provided in method 47, wherein the fully differentiated somatic cell is an endocrine cell such as a pancreatic beta cell, an epithelial cell, an endodermal cell, a macrophage, a hepatocyte, an adipocyte, a kidney cell, a blood cell, or an immune system cell.

[0307] In another method, method 49A, the present disclosure provides a method as provided in method 47, wherein the fully differentiated somatic cell is a cardiomyocyte or an immune system cell.

[0308] In another composition, composition 90, the present disclosure provides a composition comprising a plurality of universal donor cells produced by any one of methods 20-49.

[0309] In another composition, composition 91, the present disclosure provides a composition as provided in composition 90, maintained for a time and under conditions sufficient for the cells to undergo differentiation.

[0310] In another composition, composition 92, the present disclosure provides a composition as provided in composition 90 or 91 for use in treating a subject in need thereof.

[0311] In another composition, composition 93, the present disclosure provides a composition as provided by composition 92, in which the subject has, is suspected of having, or is at risk of having a disease.

[0312] In another method, method 50, the present disclosure provides a method comprising administering a plurality of universal donor cells of composition 90 or 91 to a subject.

[0313] In another method, method 51, the present disclosure provides a method for performing treatment in a subject in need thereof, the method comprising: (a) obtaining or having obtained a plurality of universal donor cells of composition 90 after differentiation into lineage-restricted progenitor cells or fully differentiated somatic cells; and (b) administering the lineage-restricted progenitor cells or fully differentiated somatic cells to the subject.

[0314] In another method, method 52, the present disclosure provides a method of obtaining cells for administration to a subject in need thereof, the method comprising: (a) obtaining or having obtained a universal donor cell of claim 31; and (b) maintaining the universal donor cell for a time and under conditions sufficient for the cell to differentiate into a lineage-restricted progenitor cell or a fully differentiated somatic cell.

[0315] In another method, method 53, the present disclosure provides a method as provided by method 51 or 52, wherein the lineage-restricted progenitor cell is a pancreatic endodermal progenitor cell, a pancreatic endocrine progenitor cell, a mesenchymal progenitor cell, a muscle progenitor cell, a blast cell, a hematopoietic progenitor cell, or a neural progenitor cell.

[0316] In another method, method 54, the present disclosure provides a method as provided by method 51 or 52, wherein the fully differentiated somatic cell is an endocrine cell such as a pancreatic beta cell, an epithelial cell, an endodermal cell, a macrophage, a hepatocyte, an adipocyte, a kidney cell, a blood cell, or an immune system cell.

[0317] In another method, method 54A, the present disclosure provides a method as provided in method 51 or 52, wherein the fully differentiated somatic cells are cardiomyocytes.

[0318] In another method, method 55, the disclosure provides a method as provided by methods 50-54, wherein the subject is a human having, suspected of having, or at risk of having a disease.

[0319] In another method, method 56, the disclosure provides a method as provided by method 55, wherein the disease is a genetically inherited disease.

[0320] In another composition, composition 93, the present disclosure provides a guide RNA comprising a spacer sequence corresponding to a target sequence consisting of SEQ ID NOs: 15-24.

[0321] In another method, Method 57, the present disclosure provides an in vitro method for generating a universal donor cell, comprising delivering to a stem cell a vector ((i) flanked by (ii) and (iii)) comprising: (a) an RNA-guided nuclease; (b) a guide RNA (gRNA) targeting a target site in a thioredoxin-interacting protein (TXNIP) gene locus; and (c) a nucleic acid, wherein the vector comprises: (i) a nucleotide sequence encoding an immune tolerogenic factor; (ii) a nucleotide sequence consisting essentially of SEQ ID NO: 25 and having sequence homology with a genomic region located to the left and within 50 nucleic acid bases of the target site; and (iii) a nucleic acid comprising: a nucleotide sequence consisting essentially of SEQ ID NO: 32 and having sequence homology with a genomic region located to the right and within 50 nucleic acid bases of the target site; wherein the TXNIP gene locus is cleaved at the target site and the nucleic acid is inserted into the TXNIP gene locus, thereby disrupting the TXNIP gene and generating a universal donor cell, wherein the universal donor cell has increased immune evasion and / or cell survival compared to a control cell.

[0322] In another method, method 57A, the disclosure provides a method as provided by method 57, wherein the nucleic acid is inserted into the TXNIP gene locus within 50 base pairs of the target site.

[0323] In another method, method 58, the disclosure provides a composition as provided by method 57, wherein the control cell is a wild-type cell or a cell that does not contain the inserted nucleic acid.

[0324] In another method, method 59, the disclosure provides a method as provided by method 57, wherein the disrupted TXNIP gene has reduced or eliminated expression of TXNIP.

[0325] In another method, method 60, the present disclosure provides a method as provided by method 57, wherein the gRNA comprises a spacer sequence corresponding to a sequence consisting of SEQ ID NOs: 15-24.

[0326] In another method, method 61, the present disclosure provides a method as provided by method 57, wherein the gRNA comprises a spacer sequence corresponding to the sequence consisting of SEQ ID NO:20.

[0327] In another method, method 62, the present disclosure provides a method as provided by method 57, wherein the vector is a plasmid vector.

[0328] In another method, method 63, the present disclosure provides a method as provided by method 57, wherein the tolerogenic factor is HLA class I histocompatibility antigen, alpha chain E (HLA-E).

[0329] In another method, method 64, the present disclosure provides a method as provided by method 63, wherein the nucleotide sequence encoding HLA-E includes a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide.

[0330] In another method, method 65, the disclosure provides a method as provided by method 63, wherein the sequence encoding the HLA-E trimer consists essentially of SEQ ID NO:55.

[0331] In another method, method 66, the present disclosure provides a method as provided by method 65, wherein the sequence encoding the HLA-E trimer is operably linked to an exogenous promoter.

[0332] In another method, method 67, the disclosure provides a method as provided by method 66, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0333] In another method, method 68, the disclosure provides a method as provided by method 57, wherein the RNA-guided nuclease is a Cas9 nuclease.

[0334] In another method, method 69, the disclosure provides a method as provided by method 68, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0335] In another method, method 70, the present disclosure provides a method as provided by method 69, wherein the Cas9 nuclease and gRNA are present in a molar ratio of 1:3.

[0336] In another method, method 71, the present disclosure provides a composition as provided by method 57, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0337] In another method, method 72, the present disclosure provides a method as provided by method 57, wherein the stem cells are human hepatocytes.

[0338] In another method, Method 73, the present disclosure provides an in vitro method for generating a universal donor cell, comprising delivering to a stem cell a vector comprising: (a) an RNA-guided nuclease; (b) a guide RNA (gRNA) that targets a target site in a thioredoxin-interacting protein (TXNIP) gene locus; and (c) a nucleic acid, the vector comprising: (i) a nucleotide sequence encoding an immune tolerogenic factor; (ii) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site and within 50 nucleic acid bases; and (iii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site and within 50 nucleic acid bases ((i) is adjacent to (ii) and (iii), and the vector comprises a nucleotide sequence consisting of SEQ ID NO: 34 or 56, wherein the TXNIP gene locus is cleaved at the target site and the nucleic acid is inserted into the TXNIP gene locus, thereby disrupting the TXNIP gene), to generate a universal donor cell, wherein the universal donor cell has increased immune evasion and / or cell survival compared to a control cell.

[0339] In another method, method 73A, the disclosure provides a method as provided by method 73, wherein the nucleic acid is inserted into the TXNIP gene locus within 50 base pairs of the target site.

[0340] In another method, method 74, the present disclosure provides a composition as provided by method 73, wherein the control cell is a wild-type cell or a cell that does not contain the inserted nucleic acid.

[0341] In another method, method 75, the disclosure provides a method as provided by method 73, wherein the disrupted TXNIP gene has reduced or eliminated expression of TXNIP.

[0342] In another method, method 76, the present disclosure provides a method as provided by method 73, wherein the gRNA comprises a spacer sequence corresponding to a sequence consisting of SEQ ID NOs: 15-24.

[0343] In another method, method 77, the present disclosure provides a method as provided by method 73, wherein the gRNA comprises a spacer sequence corresponding to the sequence consisting of SEQ ID NO:20.

[0344] In another method, method 78, the present disclosure provides a method as provided by method 73, wherein the vector is a plasmid vector.

[0345] In another method, method 79, the present disclosure provides a method as provided by method 73, wherein the tolerogenic factor is HLA class I histocompatibility antigen, alpha chain E (HLA-E).

[0346] In another method, method 80, the disclosure provides a method as provided by method 73, wherein the RNA-guided nuclease is a Cas9 nuclease.

[0347] In another method, method 81, the disclosure provides a method as provided by method 80, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0348] In another method, method 82, the present disclosure provides a method as provided by method 80, wherein the Cas9 nuclease and gRNA are present in a molar ratio of 1:3.

[0349] In another method, method 83, the present disclosure provides a method as provided by method 73, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0350] In another method, method 84, the present disclosure provides a method as provided by method 73, wherein the stem cells are human hepatocytes.

[0351] In another method, Method 85, the disclosure provides an in vitro method for generating a universal donor cell, comprising: (a) a first ribonucleoprotein (RNP) complex comprising an RNA-guided nuclease and a guide RNA (gRNA) that targets a target site in a beta-2 microglobulin (B2M) gene locus; (b) a nucleic acid that comprises: (i) a nucleotide sequence encoding a first tolerogenic factor; and (ii) a genomic region that consists essentially of SEQ ID NO:7 and is located to the left of and within 50 nucleobases of the target site in the B2M gene locus. and (iii) a first vector comprising a nucleic acid consisting essentially of SEQ ID NO: 13 and comprising a nucleotide sequence having sequence homology to a genomic region located to the right of and within 50 nucleic acid bases of a target site in the B2M gene locus ((i) is flanked by (ii) and (iii); the B2M gene locus is cleaved at the target site and a nucleic acid comprising a nucleotide sequence encoding a first tolerogenic factor is inserted into the B2M gene locus, thereby disrupting the B2M gene); (c) a thioredoxin-interacting tag a second RNP complex comprising an RNA-guided nuclease and a gRNA that targets a target site in the TXNIP gene locus; and (d) a nucleic acid comprising: (i) a nucleotide sequence encoding a second tolerogenic factor; (ii) a nucleotide sequence consisting essentially of SEQ ID NO:25 and having sequence homology to a genomic region located to the left and within 50 nucleobases of the target site in the TXNIP gene locus; and (iii) a nucleotide sequence consisting essentially of SEQ ID NO:32 and located to the right and within 50 nucleobases of the target site in the TXNIP gene locus. The method includes delivering a second vector ((i) flanked by (ii) and (iii)); the TXNIP gene locus is cleaved at the target site, and a nucleic acid comprising a nucleotide sequence encoding a second immune tolerogenic factor is inserted into the TXNIP gene locus, thereby disrupting the TXNIP gene) to the stem cell to create a universal donor cell, wherein the universal donor cell has increased immune evasion and / or cell survival compared to the control cell.

[0352] In another method, method 85A, the present disclosure provides a method as provided by method 85, wherein the nucleic acid in (b) is inserted into a B2M gene locus within 50 base pairs of the target site, and / or the nucleic acid in (d) is inserted into a TXNIP gene locus within 50 base pairs of the target site.

[0353] In another method, method 86, the disclosure provides a method as provided by method 85, wherein the control cell is a wild-type cell or a cell that does not contain the inserted nucleic acid.

[0354] In another method, method 87, the present disclosure provides a method as provided by method 85, wherein the disrupted B2M gene has reduced or eliminated expression of B2M and the disrupted TXNIP gene has reduced or eliminated expression of TXNIP.

[0355] In another method, method 88, the present disclosure provides a method as provided by method 85, wherein the gRNA of the first RNP complex comprises a spacer sequence corresponding to a sequence consisting of SEQ ID NOs: 1-3 or 35-44, and the gRNA of the second RNP complex comprises a spacer sequence corresponding to a sequence consisting of SEQ ID NOs: 15-24.

[0356] In another method, method 89, the present disclosure provides a method as provided by method 85, wherein the gRNA of the first RNP complex comprises a spacer sequence corresponding to the sequence consisting of SEQ ID NO:2, and the gRNA of the second RNP complex comprises a spacer sequence corresponding to the sequence consisting of SEQ ID NO:20.

[0357] In another method, method 90, the present disclosure provides a method as provided by method 85, wherein the first vector is a plasmid vector and the second vector is a plasmid vector.

[0358] In another method, method 91, the present disclosure provides a method as provided by method 85, wherein the first tolerogenic factor is programmed death-ligand 1 (PD-L1) and the second tolerogenic factor is HLA class I histocompatibility antigen, alpha chain E (HLA-E).

[0359] In another method, method 92, the disclosure provides a method as provided by method 91, wherein the nucleotide sequence encoding PD-L1 consists essentially of SEQ ID NO:11.

[0360] In another method, method 93, the present disclosure provides a method as provided by method 91, wherein the nucleotide sequence encoding HLA-E includes a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide, and the sequence encoding the HLA-E trimer consists essentially of SEQ ID NO:55.

[0361] In another method, method 94, the present disclosure provides a method as provided by method 85, wherein a nucleotide sequence encoding a first tolerogenic factor is operably linked to an exogenous promoter, and a nucleotide sequence encoding a second tolerogenic factor is operably linked to an exogenous promoter.

[0362] In another method, method 95, the disclosure provides a method as provided by method 94, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0363] In another method, method 96, the present disclosure provides a method as provided by method 85, wherein the first RNP complex and the second RNP complex each comprise a molar ratio of RNA-guided nuclease to gRNA of 1:3.

[0364] In another method, method 97, the disclosure provides a method as provided by method 85, wherein the RNA-guided nuclease of each of the first RNP complex and the second RNP complex is a Cas9 nuclease.

[0365] In another method, method 98, the disclosure provides a method as provided by method 97, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0366] In another method, method 99, the present disclosure provides a composition as provided by method 85, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0367] In another method, method 100, the present disclosure provides a method as provided by method 85, wherein the stem cells are human hepatocytes.

[0368] In another method, method 101, the disclosure provides an in vitro method for generating a universal donor cell, comprising: (a) a first ribonucleoprotein (RNP) complex comprising an RNA-guided nuclease and a guide RNA (gRNA) that targets a target site in a beta-2 microglobulin (B2M) gene locus; (b) a nucleic acid, the nucleic acid comprising: (i) a nucleotide sequence encoding a first tolerogenic factor; and (ii) nucleotides having sequence homology to a genomic region located to the left of and within 50 nucleobases of the target site in the B2M gene locus. and (iii) a first vector comprising a nucleic acid comprising a nucleotide sequence having sequence homology to a genomic region located to the right of and within 50 nucleobases of a target site in the B2M gene locus ((i) is adjacent to (ii) and (iii), and the first vector comprises a nucleotide sequence consisting of SEQ ID NO: 33; the B2M gene locus is cleaved at the target site, and a nucleic acid comprising a nucleotide sequence encoding a first tolerogenic factor is inserted into the B2M gene locus, thereby disrupting the B2M gene); (c) a thioredoxin-interacting protein a second RNP complex comprising an RNA-guided nuclease and a gRNA that targets a target site in the TXNIP gene locus; and (d) a nucleic acid comprising: (i) a nucleotide sequence encoding a second tolerogenic factor; (ii) a nucleotide sequence having sequence homology to a genomic region located to the left of and within 50 nucleobases of the target site in the TXNIP gene locus; and (iii) a nucleotide sequence having sequence homology to a genomic region located to the right of and within 50 nucleobases of the target site in the TXNIP gene locus. The present invention provides a method for producing a universal donor cell, comprising delivering a vector ((i) flanking (ii) and (iii), and a second vector comprising a nucleotide sequence consisting of SEQ ID NO: 34 or 56, wherein the TXNIP gene locus is cleaved at a target site, and a nucleic acid comprising a nucleotide sequence encoding a second immune tolerogenic factor is inserted into the TXNIP gene locus, thereby disrupting the TXNIP gene) to a stem cell, wherein the universal donor cell has increased immune evasion and / or cell survival compared to a control cell.

[0369] In another method, method 101A, the present disclosure provides a method as provided by method 101, wherein the nucleic acid in (b) is inserted into a B2M gene locus within 50 base pairs of the target site, and / or the nucleic acid in (d) is inserted into a TXNIP gene locus within 50 base pairs of the target site.

[0370] In another method, method 102, the present disclosure provides a composition as provided by method 101, wherein the control cell is a wild-type cell or a cell that does not contain the inserted nucleic acid.

[0371] In another method, method 103, the present disclosure provides a method as provided by method 10, wherein the disrupted B2M gene has reduced or eliminated expression of B2M and the disrupted TXNIP gene has reduced or eliminated expression of TXNIP.

[0372] In another method, method 104, the present disclosure provides a method as provided by method 101, wherein the gRNA of the first RNP complex comprises a spacer sequence corresponding to a sequence consisting of SEQ ID NOs: 1-3 or 35-44, and the gRNA of the second RNP complex comprises a spacer sequence corresponding to a sequence consisting of SEQ ID NOs: 15-24.

[0373] In another method, method 105, the present disclosure provides a method as provided by method 101, wherein the gRNA of the first RNP complex comprises a spacer sequence corresponding to the sequence consisting of SEQ ID NO:2, and the gRNA of the second RNP complex comprises a spacer sequence corresponding to the sequence consisting of SEQ ID NO:20.

[0374] In another method, method 106, the present disclosure provides a method as provided by method 101, wherein the first vector is a plasmid vector and the second vector is a plasmid vector.

[0375] In another method, method 107, the present disclosure provides a method as provided by method 101, wherein the first tolerogenic factor is programmed death-ligand 1 (PD-L1) and the second tolerogenic factor is HLA class I histocompatibility antigen, alpha chain E (HLA-E).

[0376] In another method, method 108, the present disclosure provides a method as provided by method 101, wherein the first RNP complex and the second RNP complex each comprise a molar ratio of RNA-guided nuclease to gRNA of 1:3.

[0377] In another method, method 109, the disclosure provides a method as provided by method 101, wherein the RNA-guided nuclease of each of the first RNP complex and the second RNP complex is a Cas9 nuclease.

[0378] In another method, method 110, the disclosure provides a method as provided by method 109, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0379] In another method, method 111, the present disclosure provides a composition as provided by method 101, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0380] In another method, method 112, the present disclosure provides a method as provided by method 101, wherein the stem cells are human hepatocytes.

[0381] In a first process, Process 1, the disclosure provides a process for generating universal donor cells, the process comprising: (a) modifying stem cells by inserting a nucleotide sequence encoding programmed death-ligand 1 (PD-L1) into or near a gene encoding beta-2 microglobulin (B2M), thereby generating PD-L1 positive cells; (b) enriching for the PD-L1 positive cells; (c) modifying the PD-L1 positive cells from (b) by inserting a nucleotide sequence encoding HLA class I histocompatibility antigen, alpha chain E (HLA-E) into or near a gene encoding thioredoxin interacting protein (TXNIP), thereby generating PD-L1, HLA-E double positive cells; (d) enriching for the PD-L1, HLA-E double positive cells; (e) performing single cell sorting to select for the PD-L1, HLA-E double positive cells; (f) characterizing the cells from (e) as universal donor cells; and (g) freezing the universal donor cells for long-term storage.

[0382] In another process, Process 2, the present disclosure provides a process as provided in Process 1, wherein the modification in (a) comprises delivering to the stem cell a first vector comprising: (1) a first ribonucleoprotein (RNP) complex comprising an RNA-guided nuclease and a guide RNA (gRNA) that targets a target site in the B2M gene locus; and (2) a nucleic acid comprising: (i) a nucleotide sequence homologous to a region located to the left of the target site in the B2M gene locus, (ii) a nucleotide sequence encoding PD-L1, and (iii) a nucleotide sequence homologous to a region located to the right of the target site in the B2M gene locus; wherein the B2M gene locus is cleaved at the target site, and the nucleic acid comprising the nucleotide sequence encoding PD-L1 is inserted into the B2M gene locus, thereby disrupting the B2M gene.

[0383] In another process, Process 2A, the disclosure provides a method as provided by Process 2, wherein the nucleic acid is inserted into the B2M gene locus within 50 base pairs of the target site.

[0384] In another process, Process 3, the disclosure provides a process as provided in Process 2, wherein the RNA-guided nuclease of the first RNP complex is a Cas9 nuclease, and the gRNA of the first RNP complex comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NO:2.

[0385] In another process, Process 4, the disclosure provides a process as provided in Process 3, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0386] In another process, Process 5, the disclosure provides a process as provided in Process 2, wherein the first RNP comprises a molar ratio of gRNA:RNA-guided nuclease of 3:1.

[0387] In another process, Process 6, the present disclosure provides a process as provided in Process 2, wherein the nucleotide sequence of (a)(2)(i) consists essentially of SEQ ID NO:7, and the nucleotide sequence of (a)(2)(iii) consists essentially of SEQ ID NO:13.

[0388] In another process, Process 7, the present disclosure provides a process as provided in Process 2, wherein the nucleotide sequence encoding PD-L1 consists essentially of SEQ ID NO:11.

[0389] In another process, Process 8, the present disclosure provides a process as provided in Process 2, wherein the nucleotide sequence encoding PD-L1 is operably linked to a CAG promoter.

[0390] In another process, Process 9, the present disclosure provides a process as provided in Process 2, wherein the first vector is a plasmid vector and comprises a nucleotide sequence consisting of SEQ ID NO:33.

[0391] In another process, Process 10, the present disclosure provides a process as provided in Process 2, wherein the delivery of (a)(1) and (a)(2) comprises electroporation.

[0392] In another process, Process 11, the present disclosure provides the process as provided in Process 1, wherein the enrichment of PD-L1 positive cells in (b) comprises magnetic assisted cell sorting (MACS), single cell cloning, expanding the PD-L1 positive cells, or a combination thereof.

[0393] In another process, Process 12, the present disclosure provides a process as provided in Process 1, wherein the modification in (c) includes delivering to the PD-L1-positive cells a second vector comprising: (1) a second RNP complex comprising an RNA-guided nuclease and a gRNA that targets a target site in the TXNIP gene locus; and (2) a nucleic acid comprising: (i) a nucleotide sequence homologous to a region located to the left of the target site in the TXNIP gene locus, (ii) a nucleotide sequence encoding HLA-E, and (iii) a nucleotide sequence homologous to a region located to the right of the target site in the TXNIP gene locus; wherein the TXNIP gene locus is cleaved at the target site, and the nucleic acid comprising the nucleotide sequence encoding HLA-E is inserted into the TXNIP gene locus, thereby disrupting the TXNIP gene.

[0394] In another process, Process 12A, the disclosure provides a method as provided by Process 12, wherein a nucleic acid is inserted into the TXNIP gene locus within 50 base pairs of the target site.

[0395] In another process, Process 13, the present disclosure provides a process as provided in Process 12, wherein the RNA-guided nuclease of the second RNP complex is a Cas9 nuclease, and the gRNA of the second RNP complex comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NO:20.

[0396] In another process, Process 14, the disclosure provides a process as provided in Process 13, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0397] In another process, Process 15, the present disclosure provides a process as provided in Process 12, wherein the second RNP comprises a molar ratio of gRNA:RNA-guided nuclease of 3:1.

[0398] In another process, Process 16, the present disclosure provides the process as provided in Process 12, wherein the nucleotide sequence of (c)(2)(i) consists essentially of SEQ ID NO:25, and the nucleotide sequence of (c)(2)(iii) consists essentially of SEQ ID NO:32.

[0399] In another process, Process 17, the present disclosure provides a process as provided in Process 12, wherein the nucleotide sequence encoding HLA-E includes a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide.

[0400] In another process, Process 18, the present disclosure provides the process as provided in Process 17, wherein the sequence encoding the HLA-E trimer consists essentially of SEQ ID NO:55.

[0401] In another process, Process 19, the present disclosure provides a process as provided in Process 12, wherein the nucleotide sequence encoding HLA-E is operably linked to a CAG promoter.

[0402] In another process, Process 20, the present disclosure provides the process as provided in Process 12, wherein the second vector is a plasmid vector and comprises a nucleotide sequence consisting of SEQ ID NO: 34 or 56.

[0403] In another process, Process 21, the present disclosure provides a process as provided in Process 12, wherein the delivery of (c)(1) and (c)(2) comprises electroporation.

[0404] In another process, Process 22, the present disclosure provides the process as provided in Process Claim 1, wherein the enrichment of PD-L1, HLA-E double positive cells in (d) comprises magnetic assisted cell sorting, single cell cloning, expanding the PD-L1, HLA-E double positive cells, or a combination thereof.

[0405] In another process, Process 23, the present disclosure provides the process as provided in Process 1, wherein the single cell sorting in (e) comprises fluorescence activated cell sorting (FACS), single cell cloning, expanding the single cell sorted cells, or a combination thereof.

[0406] In another process, Process 24, the present disclosure provides a process as provided in Process 1, wherein the characterization in (f) includes DNA analysis for zygosity status and / or indel profile.

[0407] In another process, Process 25, the present disclosure provides the process as provided in Process 1, wherein the characterization in (f) includes analyzing cells for morphology, viability, karyotype analysis, endotoxin levels, mycoplasma levels, on / off-target analysis, random vector insertions, residual Cas9, residual vector, pluripotent state, differentiation potential, or a combination thereof.

[0408] In another process, Process 26, the present disclosure provides a process as provided in Process 1, further comprising freezing prior to characterization in (f).

[0409] In another process, Process 27, the present disclosure provides a process as provided in Process 1, further comprising: (a) expanding the generated PD-L1 positive cells; (c) expanding the generated PD-L1, HLA-E double positive cells; (e) expanding the selected PD-L1, HLA-E double positive cells, or a combination thereof.

[0410] In another process, Process 28, the disclosure provides a process for generating universal donor cells, comprising: (a) modifying stem cells by inserting a nucleotide sequence encoding a first tolerogenic factor within or near a gene encoding an MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of MHC-I or MHC-II, thereby generating first tolerogenic factor-positive cells; (b) enriching for the first tolerogenic factor-positive cells; and (c) inserting a second tolerogenic factor within or near a gene encoding a survival factor. (d) modifying the first tolerogenic factor-positive cells from (b) by inserting a nucleotide sequence encoding the first tolerogenic factor-positive cell, thereby generating first tolerogenic factor-positive / second tolerogenic factor-positive cells; (e) performing single cell sorting to select the first tolerogenic factor-positive / second tolerogenic factor-positive cells; (f) characterizing the cells from (e) as universal donor cells; and (g) freezing the universal donor cells for long-term storage.

[0411] In another process, Process 29, the present disclosure provides the process as provided in Process 28, wherein enriching the first tolerogenic factor-positive cells in (b) comprises magnetic assisted cell sorting (MACS), single cell cloning, expanding the first tolerogenic factor-positive cells, or a combination thereof.

[0412] In another process, Process 30, the present disclosure provides a process as provided in Process 28 or 29, wherein the enrichment of first tolerogenic factor-positive / second tolerogenic factor-positive cells in (d) comprises magnetic-assisted cell sorting, single-cell cloning, expanding the first tolerogenic factor-positive / second tolerogenic factor-positive cells, or a combination thereof.

[0413] In another process, Process 31, the present disclosure provides a process as provided in any one of Processes 28-30, further comprising: (a) expanding the generated first tolerogenic factor-positive cells; (c) expanding the generated first tolerogenic factor-positive / second tolerogenic factor-positive cells; (e) expanding the selected first tolerogenic factor-positive / second tolerogenic factor-positive cells, or a combination thereof.

[0414] In another process, Process 32, the present disclosure provides a method for modifying a gene encoding a first tolerogenic factor, the method comprising the steps of: (1) a first RNA-guided nuclease and a first guide RNA (gRNA) that target a target site in the gene locus of an MHC-I or MHC-II human leukocyte antigen, or a component of an MHC-I or MHC-II complex, or a transcriptional regulator; and (2) a first nucleic acid, the first nucleic acid comprising: (i) a nucleotide sequence homologous to a region located to the left of the target site in the gene locus of an MHC-I or MHC-II human leukocyte antigen, or a component of an MHC-I or MHC-II complex, or a transcriptional regulator; (ii) a nucleotide sequence encoding a first tolerogenic factor; and (iii) a nucleotide sequence encoding a first tolerogenic factor. 32. A process as provided in any one of Processes 28 to 31, comprising delivering to a stem cell a first vector comprising a first nucleic acid comprising a nucleotide sequence homologous to a region located to the right of the target site in the target site, wherein the gene locus of MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of the MHC-I or MHC-II complex, is cleaved at the target site, and a first nucleic acid comprising a nucleotide sequence encoding a first tolerogenic factor is inserted into the gene locus of MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of the MHC-I or MHC-II complex, thereby disrupting the gene for MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of the MHC-I or MHC-II complex.

[0415] In another process, Process 32A, the present disclosure provides a method as provided by Process 32, wherein the nucleic acid is inserted into a gene locus of an MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of an MHC-I or MHC-II complex, within 50 base pairs of a target site.

[0416] In another process, Process 33, the present disclosure provides a process as provided in Process 32, wherein the first RNA-guided nuclease and the first gRNA form a first ribonucleoprotein (RNP) complex.

[0417] In another process, Process 34, the present disclosure provides a method for preparing a nucleic acid sequence encoding a nucleic acid sequence encoding a MHC-I or MHC-II human leukocyte antigen, or a component of an MHC-I or MHC-II complex or a transcriptional regulator, wherein the modification in (a) comprises: (1) a first ribonucleoprotein (RNP) complex comprising a first RNA-guided nuclease and a first guide RNA (gRNA) that targets a target site in the gene locus of an MHC-I or MHC-II human leukocyte antigen, or a component of an MHC-I or MHC-II complex or a transcriptional regulator; and (2) a first nucleic acid comprising: (i) a nucleotide sequence homologous to a region located to the left of the target site in the gene locus of an MHC-I or MHC-II human leukocyte antigen, or a component of an MHC-I or MHC-II complex or a transcriptional regulator; (ii) a nucleotide sequence encoding a first tolerogenic factor; and (iii) a nucleotide sequence encoding a MHC-I or MHC-II human leukocyte antigen, or a component of an MHC-I or MHC-II complex or a transcriptional regulator. 34. A process as provided in any one of processes 28 to 33, comprising delivering to a stem cell a first vector comprising a first nucleic acid comprising a nucleotide sequence homologous to a region located to the right of a target site in a gene locus of a transcriptional regulator, wherein the gene locus of the MHC-I or MHC-II human leukocyte antigen, or a component of the MHC-I or MHC-II complex, or the transcriptional regulator is cleaved at the target site, and a first nucleic acid comprising a nucleotide sequence encoding a first tolerogenic factor is inserted into the gene locus of the MHC-I or MHC-II human leukocyte antigen, or a component of the MHC-I or MHC-II complex, or the transcriptional regulator, thereby disrupting the gene for the MHC-I or MHC-II human leukocyte antigen, or a component of the MHC-I or MHC-II complex, or the transcriptional regulator.

[0418] In another process, Process 34A, the present disclosure provides a method as provided by Process 34, wherein the nucleic acid is inserted into the locus of an MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of an MHC-I or MHC-II complex, within 50 base pairs of the target site.

[0419] In another process, Process 35, the present disclosure provides the process as provided in any one of Processes 28 to 34, wherein the gene for the MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of the MHC-I or MHC-II complex, is HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, B2M, NLRC5, CIITA, RFX5, RFXAP, or RFXANK.

[0420] In another process, Process 36, the present disclosure provides the process as provided in any one of Processes 28 to 35, wherein the gene for the MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of the MHC-I or MHC-II complex, is B2M.

[0421] In another process, Process 37, the present disclosure provides the process as provided in Process 36, wherein the nucleotide sequence of (a)(2)(i) consists essentially of SEQ ID NO:7, and the nucleotide sequence of (a)(2)(iii) consists essentially of SEQ ID NO:13.

[0422] In another process, Process 38, the present disclosure provides a process as provided in Process 36 or 37, wherein the first gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NO:2.

[0423] In another process, Process 39, the disclosure provides a process as provided in any one of Processes 32-38, wherein the first RNA-guided nuclease is a Cas9 nuclease.

[0424] In another process, process 40, the present disclosure provides a process as provided in process 39, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0425] In another process, Process 41, the present disclosure provides a process as provided in any one of Processes 32-40, wherein the first RNP comprises a molar ratio of first gRNA:first RNA-guided nuclease of 3:1.

[0426] In another process, Process 42, the present disclosure provides the process as provided in any one of Processes 28 to 41, wherein the first tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

[0427] In another process, Process 43, the present disclosure provides a process as provided in any one of Processes 28 to 42, wherein the nucleotide sequence encoding the first tolerogenic factor is operably linked to an exogenous promoter.

[0428] In another process, Process 44, the present disclosure provides a process as provided in Process 43, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0429] In another process, Process 45, the present disclosure provides a process as provided in any one of Processes 28-44, wherein the first tolerogenic factor is PD-L1.

[0430] In another process, Process 46, the present disclosure provides the process as provided in Process 45, wherein the nucleotide sequence encoding PD-L1 consists essentially of SEQ ID NO:11.

[0431] In another process, Process 47, the present disclosure provides a process as provided in Process 46, wherein the nucleotide sequence encoding PD-L1 is operably linked to a CAG promoter.

[0432] In another process, Process 48, the present disclosure provides the process as provided in any one of Processes 45-47, wherein the first vector comprises a nucleotide sequence consisting of SEQ ID NO:33.

[0433] In another process, Process 49, the present disclosure provides a process as provided in any one of Processes 28 to 48, wherein the modification in (c) comprises delivering to the stem cell a second vector comprising (1) a second RNA-guided nuclease and a second guide RNA (gRNA) that target a target site in the survival factor gene locus, and (2) a second nucleic acid, the second nucleic acid comprising (i) a nucleotide sequence homologous to a region located to the left of the target site in the survival factor gene locus, (ii) a nucleotide sequence encoding a second tolerogenic factor, and (iii) a nucleotide sequence homologous to a region located to the right of the target site in the survival factor gene locus, wherein the survival factor gene locus is cleaved at the target site, and the second nucleic acid comprising the nucleotide sequence encoding the second tolerogenic factor is inserted into the survival factor gene locus, thereby disrupting the survival factor gene.

[0434] In another process, process 49A, the disclosure provides a method as provided by process 49, wherein the nucleic acid is inserted into a survival factor gene locus within 50 base pairs of the target site.

[0435] In another process, process 50, the present disclosure provides a process as provided in process 49, wherein a second RNA-guided nuclease and a second gRNA form a second ribonucleoprotein (RNP) complex.

[0436] In another process, Process 51, the present disclosure provides a process as provided in any one of Processes 28 to 48, wherein the modification in (c) comprises delivering to the first tolerogenic factor-positive cell a second vector comprising: (1) a second ribonucleoprotein (RNP) complex comprising a second RNA-guided nuclease and a second guide RNA (gRNA) that targets a target site in the survival factor gene locus; and (2) a second nucleic acid, the second nucleic acid comprising (i) a nucleotide sequence homologous to a region located to the left of the target site in the survival factor gene locus, (ii) a nucleotide sequence encoding a second tolerogenic factor, and (iii) a nucleotide sequence homologous to a region located to the right of the target site in the second survival factor gene locus, wherein the survival factor gene locus is cleaved at the target site, and the second nucleic acid comprising the nucleotide sequence encoding the second tolerogenic factor is inserted into the survival factor gene locus, thereby disrupting the survival factor gene.

[0437] In another process, process 51A, the present disclosure provides a method as provided by process 51, wherein a nucleic acid is inserted into a survival factor gene locus within 50 base pairs of the target site.

[0438] In another process, Process 52, the present disclosure provides a process as provided in any one of Processes 28-51, wherein the survival gene is TXNIP, ZNF143, FOXO1, JNK, or MANF.

[0439] In another process, process 53, the present disclosure provides a process as provided in process 52, wherein the survival gene is TXNIP.

[0440] In another process, Process 54, the present disclosure provides a process as provided in Process 53, wherein the second gRNA includes a spacer sequence corresponding to a target sequence consisting of SEQ ID NO:20.

[0441] In another process, Process 55, the present disclosure provides the process as provided in Process 52 or 53, wherein the nucleotide sequence of (c)(2)(i) consists essentially of SEQ ID NO:25 and the nucleotide sequence of (c)(2)(iii) consists essentially of SEQ ID NO:32.

[0442] In another process, Process 56, the present disclosure provides a process as provided in any one of Processes 49-55, wherein the second RNA-guided nuclease is a Cas9 nuclease.

[0443] In another process, Process 57, the present disclosure provides a process as provided in Process 56, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0444] In another process, Process 58, the present disclosure provides the process as provided in any one of Processes 49-57, wherein the second RNP comprises a molar ratio of second gRNA:second RNA-guided nuclease of 3:1.

[0445] In another process, Process 59, the present disclosure provides the process as provided in any one of Processes 49-58, wherein the second tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

[0446] In another process, Process 60, the present disclosure provides a process as provided in any one of Processes 28 to 59, wherein the nucleotide sequence encoding the second tolerogenic factor is operably linked to an exogenous promoter.

[0447] In another process, Process 61, the present disclosure provides a process as provided in Process 60, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0448] In another process, Process 62, the present disclosure provides a process as provided in any one of Processes 28-61, wherein the second tolerogenic factor is HLA-E.

[0449] In another process, Process 63, the present disclosure provides a process as provided in Process 62, wherein the nucleotide sequence encoding HLA-E includes a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide.

[0450] In another process, Process 64, the present disclosure provides the process as provided in Process 63, wherein the sequence encoding the HLA-E trimer consists essentially of SEQ ID NO:55.

[0451] In another process, Process 65, the present disclosure provides a process as provided in Process 63 or 64, wherein the nucleotide sequence encoding HLA-E is operably linked to a CAG promoter.

[0452] In another process, Process 66, the present disclosure provides the process as provided in any one of Processes 62 to 65, wherein the second vector comprises a nucleotide sequence consisting of SEQ ID NO: 34 or 56.

[0453] In another process, Process 67, the present disclosure provides the process as provided in any one of Processes 28 to 66, wherein the single cell sorting in (e) comprises fluorescence activated cell sorting (FACS), single cell cloning, expanding the single cell sorted cells, or a combination thereof.

[0454] In another process, Process 68, the present disclosure provides a process as provided in any one of Processes 28-67, wherein the characterization in (f) includes DNA analysis for zygosity status and / or indel profile.

[0455] In another process, Process 69, the present disclosure provides the process as provided in any one of Processes 28 to 68, wherein the characterization in (f) includes cell analysis for morphology, viability, karyotype analysis, endotoxin levels, mycoplasma levels, on / off-target analysis, random vector insertions, residual Cas9, residual vector, pluripotent state, differentiation potential, or a combination thereof.

[0456] In another process, Process 70, the present disclosure provides a process as provided in any one of Processes 28-69, further comprising freezing prior to characterization in (f). [Example]

[0457] The examples below describe the generation and characterization of certain universal donor cells according to the present disclosure.

[0458] Example 1: Cell maintenance and proliferation Maintenance of hESC / hiPSC. Cells of the human embryonic stem cell line CyT49 (proprietary hES cell line, ViaCyte, Inc., San Diego, CA) were maintained, cultured, passaged, expanded, and plated as described in Schulz et al. (2012) PLoS ONE 7(5):e37004. CyT49 cells were dissociated using ACCUTASE® (Stemcell Technologies 07920 or equivalent).

[0459] Human induced pluripotent stem cells (hiPSCs), such as the TC1133 cell line (Lonza), were maintained in StemFlex Complete (Life Technologies, A3349401) on tissue culture plates coated with BIOLAMININ 521 CTG (BioLamina Cat#CT521). Plates were pre-coated with a 1:10 or 1:20 dilution of BIOLAMININ, calcium, and magnesium (Life Technologies, 14040133) in DPBS for 2 hours at 37°C. Cells were fed daily with StemFlex medium. For cell passage, the same cell density as for CyT49 was used. To seed cells as single cells, cells were seeded on BIOLAMININ-coated plates in StemFlex with 1% RevitaCell™ supplement (100x) (Thermofisher Cat#A2644501).

[0460] Single-cell cloning of hPSCs. For single-cell cloning, hPSCs (hESCs or hiPSCs) were fed with StemFlex Complete with Revitacell (1x Revitacell final concentration) 3–4 hours before dissociation with ACCUTASE®. After dissociation, cells were sorted as single cells per well of a biolaminin-coated 96-well tissue culture plate. Single cells were sorted into wells using a WOLF FACS sorter (Nanocellect). Plates were pre-filled with 100–200 μL of StemFlex Complete with Revitacell. Three days after cell seeding, cells were fed with fresh StemFlex and continued to be fed with 100–200 μL of medium every other day. After 10 days of expansion, cells were fed with StemFlex daily until days 12–14. At this point, the plates were dissociated with ACCUTASE®, and the collected cell suspension was split 1:2: one half was placed in a new 96-well plate for maintenance, and the other half was placed in QuickExtract™ DNA Extraction Solution (Lucigen). After DNA extraction, PCR was performed to assess the presence or absence of the desired gene editing at the targeted DNA locus. Sanger sequencing was used to verify the desired editing.

[0461] Expansion of single-cell-derived hPSC clones. For CyT49 (ViaCyte), successfully targeted clones were passaged onto 24-well plates containing pure 10% XF KSR A10H10 medium but coated with biolaminin. After the 24-well stage, CyT49 clones were passaged as described in Schulz et al. (2012) PLoS ONE 7(5):e37004.

[0462] For hiPSCs (TC1133), cells were maintained in StemFlex Complete throughout the periodic maintenance process on BIOLAMININ-coated plates with Revitacell during the cloning and passaging steps.

[0463] Example 2: Generation of B2M knockout (KO) human pluripotent stem cells (hPSCs) Guide RNA (gRNA) selection for B2M in hPSCs. Three B2M-targeting gRNAs were designed to target exon 1 of the B2M coding sequence. These gRNAs had low off-target scores predicted based on sequence homology prediction using gRNA design software. The target sequences of the gRNAs are shown in Table 1. The gRNAs contain RNA sequences that correspond to the target DNA sequence.

[0464] [Table 1]

[0465] To evaluate their cleavage efficiency in hPSCs, CyT49 cells (a proprietary hES cell line from ViaCyte) were electroporated with a ribonucleoprotein (RNP) mixture of Cas9 protein (Biomay) and guide RNA (Synthego) (see Table 3 for gRNA sequences) at a molar ratio of 3:1 (gRNA:Cas9) with absolute values ​​of 125 pmol Cas9 and 375 pmol gRNA using a Neon electroporator (Neon transfection system ThermoFisher Cat#MPK5000). To form the RNP complex, gRNA and Cas9 were combined in one vessel with R-buffer (Neon transfection system 100 μL kit ThermoFisher Cat#MPK10096) to a total volume of 25 μL and incubated at room temperature for 15 min. Cells were dissociated using ACCUTASE®, then resuspended in DMEM / F12 medium (Gibco, cat#11320033), counted using an NC-200 (Chemometec), and centrifuged. A total of 1 × 10 6Cells were resuspended with the RNP complex, and R-buffer was added to a total volume of 125 μL. The mixture was then electroporated with two 30-second pulses at 1100 V. After electroporation, the cells were pipetted into an Eppendorf tube filled with StemFlex medium containing RevitaCell. The cell suspension was then plated onto tissue culture dishes pre-coated with BIOLAMININ 521 CTG at a 1:20 dilution. The cells were cultured for 48 hours in a normoxic incubator (37°C, 8% CO2). After 48 hours, genomic DNA was collected from the cells using QuickExtract (Lucigen, Middleton, WI; Cat# QE09050).

[0466] PCR for the target B2M sequence was performed, and the resulting amplified DNA was evaluated for cleavage efficiency by TIDE analysis. PCR for the relevant region was performed using Platinum Taq Supermix (Invitrogen, cat#125320176 and cat#11495017). The sequences of the PCR primers are shown in Table 2; cycling conditions are provided in Table 3.

[0467] [Table 2]

[0468] [Table 3]

[0469] The resulting amplified products were subjected to PCR purification and Sanger sequencing. The Sanger sequencing results, along with the guide sequences, were entered into Tsunami software. The percentage and identity of indels were calculated by the software. Specific gRNAs were then selected based on their indel frequency in hPSCs. Figure 1 shows the cleavage efficiency of B2M-1, B2M-2, and B2M-3 gRNAs.

[0470] The off-target potential of the selected gRNAs was assessed in stem cell-derived DNA using hybrid capture analysis of predicted sequence similarity sites. The B2M-2 and B2M-3 guides showed no detectable off-target effects. The B2M-2 gRNA was selected for further cloning due to its high on-target activity and undetectable off-target activity.

[0471] B2M KO hPSC clone generation and characterization. CyT49 hESCs (ViaCyte) were electroporated using B2M-2 gRNA and single-cell sorted into BIOLAMININ 521 CTG-coated 96-well plates containing StemFlex and Revitacell using a WOLF FACS-sorter (Nanocellect) three days after electroporation. Seeded single cells were grown in a normoxic incubator (37°C, 8% CO2) with medium changes every other day until colonies were large enough to be replated as single cells. Upon confluence, samples were split for maintenance and genomic DNA extraction.

[0472] The B2M KO status of the clones was confirmed via PCR and Sanger sequencing. The resulting DNA sequences of the targeted B2M region were aligned in Snapgene software to determine indel identity and zygosity. Clones with the desired edits were expanded and further verified via flow cytometry evaluation for B2M expression (see Table 4 for a list of antibodies utilized). Clones were evaluated with and without interferon-gamma treatment (25 ng / mL, R&D Systems, 285-IF). Figure 2A shows B2M expression in wild-type cells, and Figure 2B shows B2M expression in B2M KO cells. The karyotype status of the clones was assessed via Cell Line Genetics Services (Madison, WI), and a normal karyotype was reported.

[0473] [Table 4]

[0474] Clones were confirmed to retain pluripotency by intracellular flow cytometry for the pluripotency markers OCT4 and SOX2, and were differentiated into pancreatic endocrine progenitor cells using a previously established method (Schulz et al. (2012) PLoS ONE 7(5):e37004).

[0475] Example 3: Generation of B2M KO / PD-L1 knock-in (KI) human pluripotent stem cells (hPSCs) Design of the B2M-KO / PD-L1 KI Strategy. A plasmid for inserting PD-L1 (CD274) into the B2M locus was designed so that the start codon of B2M would be removed after homology-directed repair (HDR), allowing PD-L1 to be inserted and eliminating the chance of partial B2M expression. Figure 3 shows a schematic diagram of the plasmid, and Table 5 identifies the elements and locations therein. The donor plasmid contained a CAGGS promoter-driven cDNA of PD-L1 flanked by 800 base pair homology arms with sequence identical to the B2M locus around exon 1. The complete sequence of the plasmid comprises SEQ ID NO:33.

[0476] [Table 5]

[0477] B2M-2 gRNA was used to facilitate insertion of the PD-L1 transgene at the targeted B2M locus. The PD-L1 donor plasmid was co-transfected with an RNP complex composed of the B2M-targeting gRNA and Cas9 protein. Four micrograms of plasmid DNA was delivered with the RNP per million CyT49 cells (ViaCyte). Electroporation was performed as described in Example 2. Seven days after electroporation, cells were sorted for PD-L1 surface expression using a WOLF FACS-sorter (Nanocellect) onto BIOLAMININ 521 CTG-coated 96-well plates containing StemFlex with Revitacell. For FACS-sorting, unedited cells served as a negative control. PD-L1-positive cells were selected for sorting and single-cell cloning.

[0478] An anti-PD-L1 fluorescent antibody was used to detect PD-L1 surface expression (see Table 4). Seeded single cells were grown in a normoxic incubator (37°C, 8% CO2) with medium changes every other day until colonies were large enough to be replated as single cells. Upon confluence, samples were split for maintenance and genomic DNA extraction.

[0479] Correctly targeted clones were identified via PCR using primers amplifying a region from outside the homology arms of the plasmid to the PD-L1 cDNA insert, allowing for amplification of only the KI-integrated DNA for the PD-L1 knock-in (KI) insert. The on-target insert was tested for zygosity by PCR to assess whether the KI occurred in a heterozygous or homozygous fashion. If a heterozygous clone was identified, the KI-negative allele was sent for Sanger sequencing to verify that it contained a B2M-disrupting indel in the non-KI allele. Correct KI clones with complete B2M disruption (either due to the KI insert or indel formation) were expanded in an incremental tissue culture format until they reached a population size of 30 million cells. Approximately 10 clones were expanded in this format and confirmed to be pluripotent by testing for OCT4 and SOX2 via intracellular flow cytometry (Figure 4). Clones that passed the above tests were then further tested for karyotype analysis (Cell Line Genetics) as described below. Furthermore, clones were tested for their ability to differentiate into pancreatic endoderm progenitor cells (PECs) via an established protocol (Schulz et al. (2012) PLoS ONE 7(5):e37004) as described below. Loss of B2M was further confirmed by the lack of B2M expression in the presence or absence of interferon-gamma treatment (25 ng / mL, R&D Systems, 285-IF) via flow cytometry. Figures 5A and 5B show PD-L1 expression in wild-type and B2M KO / PD-L1 KI cells, respectively.

[0480] Example 4: Karyotype analysis of edited clones G-banded karyotyping of edited embryonic stem (ES) cells. One million edited ES cells were passaged into T-25 culture flasks containing culture medium (DMEM / F12 + 10% Xeno-free KSR with 10 ng / mL activin and 10 ng / mL heregulin). After overnight culture, three T25 culture flasks were sent to a cytogenetics laboratory (Cell Line Genetics, Inc.) for karyotyping; FISH analysis for chromosomes 1, 12, 17, and 20; and array comparative genomic hybridization (aCGH) analysis using a standard 8x60K array. G-banding results for selected cells electroporated with the non-cutting guide ("NCG"), B2M KO clone, and B2M KO / PD-L1 KI clone ("V1-A") are shown in Table 6.

[0481] [Table 6]

[0482] Example 5: Differentiation of edited human embryonic stem cells into pancreatic endoderm cells (PECs) Maintenance of edited human embryonic stem cells (ESCs). Edited human embryonic stem cells at various passage numbers (P38-42) were maintained at 33,000 cells / cm for 4 days of passage in hESM medium (DMEM / F12 + 10% KSR + 10 ng / mL activin A and 10 ng / mL heregulin) with a final 10% human AB serum. 2 or 50,000 cells / cm for 3-day passaging 2 was sown in.

[0483] Aggregation of edited human embryonic stem cells for PEC differentiation. Edited ES cells were dissociated into single cells with ACCUTASE®, followed by centrifugation and resuspending in 2% StemPro (Cat# A1000701, Invitrogen, CA) in DMEM / F12 medium at 1 million cells per ml, for a total of 3.5-4 million cells, using one 850 cm rotation at 8 RPM ± 0.5 RPM. 2ES aggregates derived from edited human embryonic stem cells were seeded in rotor bottles (Cat# 431198, Corning, NY) for 18–20 hours prior to differentiation. ES aggregates derived from edited human embryonic stem cells were differentiated into the pancreatic lineage using rotor bottles as described in Schulz et al. (2012) PLoS ONE 7(5):e37004.

[0484] Example 6: Characterization of differentiated pancreatic endoderm cells (PECs) Flow cytometry for FOXA2 and SOX17 at stage 1 (DE) and CHGA, PDX1, and NKX6.1 at PEC stage. hESC-derived stage 1 aggregates or hESC-derived pancreatic aggregates were washed with PBS and then enzymatically dissociated into single-cell suspensions using ACCUMAX™ (Cat. #A7089, Sigma, MO) at 37°C. MACS separation buffer (Cat. #130-091-221, Miltenyi Biotec, North Rhine-Westphalia, Germany) was added, and the suspension was passed through a 40 μm filter and pelleted. For staining of intracellular markers, cells were fixed in 4% (wt / v) paraformaldehyde for 30 min and washed in FACS buffer (PBS, 0.1% (wt / v) BSA, 0.1% (wt / v) NaN). Subsequently, cells were permeabilized with Perm buffer (PBS, 0.2% (v / v) Triton X-100 (Cat# A16046, Alfa Aesar, MA), 5% (v / v) normal donkey serum, 0.1% (wt / v) NaN) on ice for 30 min, followed by washing with wash buffer (PBS, 1% (wt / v) BSA, 0.1% (wt / v) NaN). Cells were incubated overnight at 4°C with primary antibodies (Table 7) diluted in blocking buffer (PBS, 0.1% (v / v) Triton X-100, 5% (v / v) normal donkey serum, 0.1% (wt / v) NaN3). Cells were washed in IC buffer and then incubated with the appropriate secondary antibody for 60 minutes at 4°C. Cells were washed in IC buffer followed by FACS buffer. Flow cytometry data were acquired using a NovoCyte flow cytometer (ACEA Biosciences, Brussels). Data were analyzed using FlowJo software (Tree Star, Inc.). Untreated cells were identified based on forward (low angle) and side (orthogonal, 90°) light scatter. Background was estimated using antibody controls and undifferentiated cells. In the figures, representative flow cytometry plots are shown for one of the subpopulations. The numbers reported in the figures represent the percentage of total cells from the untreated cell gate.

[0485] [Table 7]

[0486] At the DE stage, the population of FOXA2 and SOX17 double-positive cells exceeded 90% of the total cells in CyT49 wild-type differentiated cells. PD-L1 KI / B2M KO and B2M KO cells showed similar DE percentages compared to wild-type cells (Figures 6 and 7).

[0487] At the PEC stage, flow cytometry was performed for chromogranin (CHGA), PDX1, and NKX6.1. The heterogeneous population at the PEC stage included pancreatic progenitor cells and early endocrine cells (Figure 8). Pie charts of the heterogeneous populations (Figure 9) revealed that the distribution of the differentiated edited cells (PD-L1 KI / B2M KO or B2M KO) was very similar to that of wild-type cells.

[0488] Targeted RNA sequencing. Targeted RNA sequencing for gene expression analysis was performed using an Illumina TruSeq system and a custom panel of oligos targeting 111 genes. The panel primarily contained genes that are markers for developmental stages during pancreatic differentiation. At the end of each differentiation stage, 10 μL of APV (agglomerated pellet volume) was collected and extracted using a Qiagen RNeasy or RNeasy 96 spin column protocol, including on-column deoxyribonuclease treatment. Quantification and quality control were performed using either a TapeStation coupled with Qubit or a Qiagen QIAxcel. 50–200 ng of RNA was processed according to the Illumina TruSeq library preparation protocol, consisting of cDNA synthesis, hybridization of the custom oligo pool, washing of the bound oligos, extension, ligation, PCR amplification of the library, and library purification, before quantification and quality control of the resulting dsDNA libraries using either a TapeStation coupled with Qubit or a Qiagen QIAxcel. Libraries were then diluted to a concentration of 4 nM and pooled, followed by further dilution to 10–12 pM before denaturation, spike-in of PhiX controls, and loading onto an Illumina MiSeq sequencer. After the sequencing run, initial data analysis was performed automatically by BaseSpace to generate raw read counts for each of the custom probes. For each gene, these read counts were then summed across all probes corresponding to that gene by adding one read count (to prevent downstream division by 0). Normalization was performed to the gene SF3B2, and reads were typically visualized as fold change relative to stage 0. When data were processed for principal component analysis, normalization was performed using the DEseq method.

[0489] The expression of selected genes is shown in Figure 10. The dynamic expression patterns of FOXA2, CHGA, PDX1, and NKX6.1 from PD-L1 KI / B2M KO or B2M KO cells were similar to those of wild-type cells.

[0490] Confirmation of B2M and PD-L1 expression at the PEC stage. At the PEC stage, differentiated aggregates were treated with or without interferon-gamma (50 ng / ml) for 48 hours. The aggregates were washed with PBS and then enzymatically dissociated into a single-cell suspension using ACCUMAX™ (Cat. #A7089, Sigma, MO) at 37°C. MACS separation buffer (Cat. #130-091-221, Miltenyi Biotec, North Rhine-Westphalia, Germany) was added, and the suspension was passed through a 40 μm filter and pelleted. For surface marker staining, dissociated cells were incubated with fluorescently conjugated antibodies diluted in MACS separation buffer for 20 minutes, followed by washing in MACS separation buffer. Cells were resuspended in FACS buffer for flow acquisition. Flow cytometry data were obtained using a NovoCyte flow cytometer. As shown in Figures 11A-11F, B2M expression was below the limit of detection in differentiated PECs derived from B2M KO (Figure 11B) or PD-L1 KI / B2M KO (Figure 11C), and PD-L1 was expressed in differentiated PECs derived from PD-L1 KI / B2M KO (Figure 11F). Generally, greater than 90% of PECs expressed PD-L1, indicating a homogeneous population of cells. In many cases, there is a loss of transgene expression over time following differentiation of gene-edited stem cells (Hong et al., Mol. Ther., 2017, 25(1):44-53).

[0491] Immunophenotype of PEC cells. At the PEC stage, differentiated aggregates were treated with or without interferon-gamma (50 ng / ml) for 48 hours. Aggregates were collected for MHC class I and II staining. There was no MHC class II expression in PEC stages derived from wild-type or edited cells (PD-L1 KI / B2M KO and B2M KO) (Figures 12D-12F). HLA-ABC (MHC class I) expression was low (1.3% from wild-type), and it was highly regulated upon IFN-γ stimulation. However, HLA-ABC was not expressed in edited cells (PD-L1 KI / B2M KO and B2M KO) even under IFN-γ stimulation (Figures 12A-12C).

[0492] Example 7: Generation of TXNIP KO human pluripotent stem cells (hPSCs) Guide RNA (gRNA) selection for TXNIP. Ten TXNIP-targeting gRNAs were designed to target exon 1 and exon 2 of the TXNIP coding sequence (Table 8). The PAM sequences are shown in bold font in the target sequences shown in Table 8, and the DNA sequences corresponding to the guide sequences are shown in Table 8. These gRNAs had low off-target scores predicted based on sequence homology prediction using gRNA design software.

[0493] [Table 8]

[0494] TXNIP KO hPSC clone generation and characterization. To evaluate the cleavage efficiency of these gRNAs in hPSCs, TC1133 hiPSC cells were electroporated using a Neon electroporator (Neon transfection system ThermoFisher Cat#MPK5000) with an RNP mixture of Cas9 protein (Biomay) and guide RNA (Synthego) at a molar ratio of 3:1 (gRNA:Cas9), with absolute values ​​of 125 pmol Cas9 and 375 pmol gRNA. To form the RNP complex, gRNA and Cas9 were combined in a single vessel containing R-buffer to a total volume of 25 μL and incubated at room temperature for 15 min. Cells were dissociated using ACCUTASE®, subsequently resuspended in DMEM / F12 medium (Gibco, Cat#11320033), counted using an NC-200 (Chemometec), and centrifuged. A total of 1 × 10 6 Cells were resuspended with the RNP complex and R-buffer was added to a total volume of 125 μL. This mixture was then electroporated using the following parameters: 2 pulses, 30 ms, 1100 V. After electroporation, the cells were pipetted into an Eppendorf tube filled with StemFlex medium containing RevitaCell. This cell suspension was then plated onto tissue culture dishes pre-coated with BIOLAMININ 521 CTG. The cells were cultured for 48 hours in a normoxic incubator (37°C, 8% CO2). After 48 hours, genomic DNA was collected from the cells using QuickExtract.

[0495] PCR was performed for the target TXNIP sequence, and the resulting amplified DNA was sequenced by the Sanger method. TIDE analysis was used to analyze the output sequencing data for indel percentage using Tsunami software. Figure 13 shows the cleavage efficiency for TXNIP gRNA. Next, gRNAs were selected based on their indel frequency in hPSCs.

[0496] The off-target activity of the gRNA with the highest cleavage efficiency was assessed in stem cell-derived DNA using hybrid capture analysis at sites of predicted sequence similarity. Further experiments with the TXNIP gRNA T5 were performed, as it showed high on-target activity without detectable off-target effects.

[0497] TXNIP KO hPSC clone generation and characterization. CyT49 hESCs (Viacyte) were electroporated using TXNIP gRNA T5, and 3 days after electroporation, single cells were sorted into BIOLAMININ 521 CTG 96-well plates containing StemFlex and Revitacell using a WOLF FACS-sorter (Nanocellect). Seeded single cells were grown in a normoxic incubator (37°C, 8% CO2) with medium changes every other day until colonies were large enough to be replated as single cells. Upon confluence, samples were split for maintenance and genomic DNA extraction.

[0498] The TXNIP KO status of the clones was confirmed via PCR and Sanger sequencing. The resulting DNA sequences of the targeted TXNIP region were aligned in Snapgene software to determine indel identity and zygosity. Clones with the desired edits were expanded and further verified via flow cytometry evaluation for TXNIP expression. The karyotype status of the clones was assessed via Cell Line Genetics service, and a normal karyotype was reported (Table 9).

[0499] [Table 9]

[0500] Clones were confirmed to retain pluripotency by intracellular flow cytometry for the pluripotency markers OCT4 and SOX2, and were differentiated into pancreatic endocrine progenitor cells using a previously established method (Schulz et al. (2012) PLoS ONE 7(5):e37004).

[0501] Targeted RNA sequencing for gene expression analysis was performed using Illumina TruSeq and a custom panel of oligos as described above. Selected gene expression is shown in Figure 20. The dynamic expression patterns of FOXA2, CHGA, PDX1, and NKX6.1 from TXNIP KO cells were similar to those of wild-type cells. Flow cytometry for chromogranin (CHGA), PDX1, and NKX6.1 was also performed at the PEC stage. The heterogeneous population at the PEC stage consisted of 30.6% pancreatic progenitor cells (i.e., CHGA - / NKX6.1 + / PDX1 + ) (Figure 21).

[0502] Example 8: Generation of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI human pluripotent stem cells (hPSCs) Design of the B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI strategies. Cells were generated in which the PD-L1 coding sequence was inserted into the B2M locus (thereby knocking out the B2M gene) and the HLA-E coding sequence was inserted into the TXNIP locus (thereby knocking out the TXNIP gene).

[0503] The plasmid design for inserting PD-L1 (CD274) into the B2M locus is shown in Example 3. The donor plasmid contains a CAGGS promoter-driven cDNA of PD-L1 flanked by 800 base pair homology arms with sequence identical to the B2M locus around exon 1. The B2M-2 gRNA was used to facilitate insertion of the PD-L1 transgene at the targeted B2M locus. The PD-L1 donor plasmid was co-transfected with an RNP complex composed of the B2M-targeting gRNA and Cas9 protein. Four micrograms of plasmid DNA was delivered with the RNP per million CyT49 cells (ViaCyte). Electroporation was performed as described in Example 2. Seven days after electroporation, cells were enriched for PD-L1 positive cells via magnetic-assisted cell sorting (MACS) using Miltenyi reagents (anti-mouse IgG microbeads Cat#130-048-401, LS columns Cat#130-042-401 and MidiMACS separators Cat#130-042-302) or Thermofisher reagents (DynaMag™-15 magnet Cat#12301D, CELLection™ pan-mouse IgG kit Cat#11531D, Dynabeads™ pan-mouse IgG Cat#11042).

[0504] After the enriched PD-L1-positive population was expanded, an HLA-E trimer cDNA transgene was inserted into the TXNIP genomic locus via CRISPR-induced HDR using a donor plasmid containing the HLA-E sequence. The HLA-E trimer cDNA consisted of a B2M signal peptide fused to an HLA-G-presenting peptide fused to a B2M membrane protein fused to an HLA-E protein without its signal peptide. This trimer design has been previously published (Gornalusse et al. (2017) Nat. Biotechnol. 35(8):765-772). The HLA-E trimer coding sequence (including the linker) is SEQ ID NO:55 (i.e., SEQ ID NOs:26-31). The donor plasmid for HLA-E delivery contains a CAGGS promoter-driven expression of the HLA-E trimer flanked by 800 base pair homology arms with sequence identical to the TXNIP locus around exon 1 (Figure 14, Table 10, and Table 11). In some embodiments, the donor plasmid comprises SEQ ID NO: 34 or 56.

[0505] [Table 10]

[0506] [Table 11]

[0507] TXNIP-T5 gRNA was used to facilitate insertion of the HLA-E transgene at the targeted TXNIP locus. The HLA-E donor plasmid was co-transfected with an RNP complex composed of TXNIP-T5 gRNA and Cas9 protein. Four micrograms of HLA-E donor plasmid DNA (SEQ ID NO: 56) was delivered with the RNP per million PD-L1+ cells. Alternatively, HLA-E donor plasmid DNA (SEQ ID NO: 34) can be used. Electroporation was performed as described in Example 2. Seven days after electroporation, cells were enriched for HLA-E-positive cells via MACS using Miltenyi or Thermofisher reagents. After HLA-E enrichment, cells were single-cell sorted onto BIOLAMININ 521 CTG-coated 96-well plates containing StemFlex and Revitacell using a WOLF FACS-sorter (Nanocellect). The seeded single cells were grown in a normoxic incubator (37°C, 8% CO2) with medium changes every other day until colonies were large enough to be replated as single cells. Upon confluence, samples were split for maintenance and genomic DNA extraction. Anti-PD-L1 and anti-HLA-E antibodies (Table 4) were used for MACS enrichment and FACS sorting into 96-well plates with gating settings for HLA-E and PD-L1 double-positive cells. For FACS-sorting, unedited cells served as a negative control.

[0508] Correctly targeted clones were identified via PCR using primers amplifying a region from outside the homology arms of the plasmid to the PD-L1 cDNA insert or HLA-E cDNA insert, respectively, allowing amplification of only the KI-integrated DNA for the PD-L1 KI insert and HLA-E KI insert. The on-target inserts were tested for zygosity by PCR to assess whether the KI occurred in a heterozygous or homozygous fashion. If a heterozygous clone was identified, the KI-negative allele was sent for Sanger sequencing to verify that it contained a B2M-disrupting indel or a TXNIP-disrupting indel, respectively. Correct KI clones with complete B2M and TXNIP disruption (either by the KI insert or indel formation) were expanded in an incremental tissue culture format until they reached a population size of 30 million cells. Approximately 10 clones were expanded in this manner and confirmed to be pluripotent by testing for OCT4 and SOX2 via intracellular flow cytometry (Figure 15).

[0509] Clones that passed the above tests were then further tested for karyotype analysis (Cell Line Genetics) as described above. The G-banding results of selected B2M KO / PD-L1 KI+TXNIP KO / HLA-E KI ("V1-B") clones are shown in Table 12. Furthermore, V1-B clones were then tested for their ability to differentiate into pancreatic endoderm progenitor cells (PECs).

[0510] [Table 12]

[0511] PD-L1 and HLA-E continued to be expressed after differentiation to stage 6 cells according to a previously reported pancreatic endocrine protocol (Rezania et al. (2014) Nat. Biotechnol. 32(11):1121-1133) (Figure 16). The differentiated cell population was homogeneous with respect to transgene expression, e.g., 94.4% of cells expressed PD-L1 and 97.0% of cells expressed HLA-E. Figure 22A shows similar morphology of various clonal cells ("S6-V1B-H9," "S6-V1B-3B11," "S6-V1B-1G7," and "S6-V1B-3C2") differentiated to stage 6 compared to wild-type and uncut guide control cells. Selected gene expression of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones is shown in Figures 23A-F. The dynamic expression patterns of INS, NKX6.1, GCK, GCG, and SST from B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clone cells were similar to those of wild-type cells (Figure 23A). The expression levels of stage 6 markers INS (Figure 23B), NKX6.1 (Figure 23C), GCG (Figure 23D), SST (Figure 23E), and GCK (Figure 23F) from various differentiated B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones ("S6-V1B-H9," "S6-V1B-3B11," "S6-V1B-1G7," and "S6-V1B-3C2") were similar to those in stage 6 wild-type cells and wild-type islets. Undifferentiated B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones ("ES-V1B-H9") were used as negative controls.

[0512] Figures 24A-24B show flow cytometry evaluation of INS and GCG expression (Figure 24A) and INS and NKX6.1 expression (Figure 24B) in stage 6 cells differentiated from B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones. Figures 25A-25B show the percentage of INS expression (Figure 25A) and NKX6.1 expression (Figure 25B) in stage 6 cells differentiated from two B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones ("S6-V1B003" and "V1B-H9"). Expression of both was similar to that of wild-type and uncut guide control cells.

[0513] Flow cytometry for chromogranin (CHGA), PDX1, and NKX6.1 was performed at the PEC stage. The heterogeneous population at the PEC stage includes pancreatic progenitor cells and early endocrine cells (Figure 17). Targeted RNA-seq for gene expression analysis was performed as described above. Selected gene expression for the TXNIP KO clone is shown in Figure 18A, and selected gene expression for the V1-B clone is shown in Figure 18B. The dynamic expression patterns of FOXA2, CHGA, PDX1, and NKX6.1 from V1-B or TXNIP KO clone cells were similar to those of wild-type cells.

[0514] Using an HLA-E donor vector containing the nucleotide sequence of SEQ ID NO: 56, cells were generated in which the HLA-E coding sequence was inserted into the TXNIP locus (thereby knocking out the TXNIP gene). Targeted RNA sequencing for gene expression analysis was performed as described above. Selected gene expression for TXNIP KO / HLA-E KI clones is shown in Figure 28. The dynamic expression patterns of FOXA2, CHGA, PDX1, and NKX6.1 from TXNIP KO / HLA-E KI cells were similar to those of wild-type cells.

[0515] Instead, cells were generated in which the HLA-E coding sequence was inserted into the TXNIP locus using an HLA-E donor vector containing the nucleotide sequence of SEQ ID NO: 34. Most edited cells differentiated to the PEC stage, and at least 75% of the population expressed HLA-E (data not shown). Flow cytometry evaluation of PDX1 and NKX6.1 expression in PEC cells differentiated from TXNIP KO cells was similar to that in PEC cells differentiated from wild-type cells (data not shown).

[0516] Example 9: T cell activation / proliferation assay PEC-differentiated cells were tested for their ability to elicit an immune response via an in vitro human T cell activation / proliferation assay. Fresh donor PBMCs were purchased from Hemacare, and CD3+ T cells were purified using a pan T cell isolation kit, human (Miltenyi Cat# 130-096-535). Isolated T cells were labeled with the CellTrace™ CFSE Cell Proliferation Kit protocol (Thermofisher Cat# 34554) according to the manufacturer's instructions and co-incubated with differentiated PECs for 5 days. Dynabeads™ Human T-Activator CD3 / CD28 for T Cell Proliferation and Activation (Thermofisher Cat# 11161D) was used as a positive control for activating T cells. T cells alone, labeled with CFSE, were used as a negative control. The percentage of CD3+ CFSE+ cells was measured to assess the percentage of T cell proliferation (Figures 19A-19B). WT PECs induced T cell proliferation above the T cell-alone control. PECs derived from B2M KO, B2M KO / PD-L1 KI, and B2M KO / PD-L1 KI+TXNIP KO / HLA-E KI CyT49 did not induce T cell proliferation above the T cell-alone control, indicating the hypoimmunogenic nature of the edited cells.

[0517] Example 10: In vivo efficacy testing of gene-targeted clonal lines Pancreatic endoderm cells were generated from clonal hES cell lines derived from CyT49 with the following genetic modifications: 1) targeted deletion of B2M expression and forced expression of PD-L1, 2) targeted deletion of B2M expression and forced expression of HLA-E, or 3) targeted deletion of TXNIP. Additionally, clonal unmodified cell lines were obtained by transfection with non-cleavable guide RNA (NCG).

[0518] Pancreatic endoderm aggregates derived from designated clonal lines were loaded into perforation devices (PDs) to generate test or control articles according to standard procedures. PDs allow direct vascularization upon subcutaneous implantation, and encapsulated pancreatic progenitor cells mature in vivo into functional pancreatic endocrine cells, including glucose-responsive, insulin-producing cells.

[0519] As summarized in Table 13, five groups of athymic nude rats were each administered approximately 7 x 10 cells derived from differentiation of the four clonal lines described above. 6 Two articles containing either pancreatic endoderm cells or wild-type CyT49 hES (ViaCyte) cells were implanted subcutaneously.

[0520] [Table 13]

[0521] Beginning at week 12, all surviving animals underwent efficacy evaluation via glucose-stimulated insulin secretion (GSIS) testing. Blood samples were obtained from non-fasting animals before and after intraperitoneal administration of 3 g / kg glucose. Serum concentrations of human C-peptide were determined via standard enzyme-linked immunosorbent assay.

[0522] GSIS testing was performed at weeks 12, 16, and 20. Results showed no substantial differences between experimental groups, particularly after the 12-week time point. Compared to the C-peptide levels detected in the control group (Group 1, <40 pM–2.0 nM, mean 1.1 nM), C-peptide levels were elevated in two of six animals from Group 3 (TXNIP KO, mean 1.5 nM). The other groups, Group 2 (NCG, mean 0.5 nM), Group 4 (B2M KO / PD-L1 KI, mean 0.5 nM), and Group 5 (B2M KO / HLA-E KI, mean 0.4 nM), showed a similar range of C-peptide levels compared to the control group, but more animals were near the lower end of the range. However, these differences were not statistically significant. These results indicated that neither the introduced genetic modifications nor the manipulations required to generate the clonal line affected the ability of the cell line to differentiate into pancreatic endoderm cells in vitro and subsequently generate functional beta cells in vivo.

[0523] At 20 weeks, after the GSIS test, the animals were euthanized and the implanted test articles were fixed in neutral buffered formalin, processed into slides, and stained by H&E and immunohistochemistry for insulin and glucagon.

[0524] In vivo efficacy evaluation using the GSIS test showed no substantial differences between unedited control articles and edited test articles constructed with pancreatic endoderm cells derived from clonal cell lines, each carrying a subset of genetic modifications. The results suggest that the individual genetic modifications and the process by which they are introduced can be tolerated in vivo.

[0525] Example 11. In vivo efficacy testing of B2M KO / PD-L1 KI, TXNIP KO / HLA-E KI cell lines Four clonal lines were generated essentially as described above in Example 8 and loaded into a perforation device to form the test article. The control article contained unmodified CyT49 cells (ViaCyte). Approximately 7 x 10 6Articles containing pancreatic endoderm cells were implanted subcutaneously into athymic nude rats (2 articles / rat, 8 rats / group).

[0526] At 12, 16, 20, and 24 weeks, all surviving animals underwent glucose-stimulated insulin secretion (GSIS) testing. Blood samples were obtained from fasted animals before and after intraperitoneal administration of 3 g / kg glucose. Serum concentrations of human C-peptide were determined via standard enzyme-linked immunosorbent assay. Serum C-peptide was detected in the majority of animals at 12 weeks after transplantation. Serum C-peptide levels at 16, 20, and 24 weeks after transplantation are shown in Table 14. No statistically significant differences were observed between groups of animals transplanted with gene-edited cells versus control cells.

[0527] [Table 14]

[0528] At 25 weeks, surviving animals are subjected to an insulin challenge (insulin tolerance test, ITT) to assess changes in serum human C-peptide in response to reduced blood glucose in the food-deprived state. Blood samples are obtained from fasted animals before and at multiple time points (15, 30, 60 min) after intraperitoneal administration of 1 unit of insulin per kg body weight. Serum concentrations of human C-peptide are determined via standard enzyme-linked immunosorbent assay.

[0529] At 26 weeks, surviving animals are euthanized and the implanted test articles are processed into slides and stained by H&E and immunohistochemistry (IHC) for insulin and glucagon to identify human pancreatic endocrine cells. Additional IHC for the human-specific nuclear marker NuMA1 is performed to identify the potential location of implant-derived cells outside the lumen of the test article implant.

[0530] Example 12. In vivo efficacy testing of B2M KO / PD-L1 KI, TXNIP KO / HLA-E KI cell lines Aggregates of B2M KO / PD-L1 KI, TXNIP KO / HLA-E KI pancreatic endoderm cells (approximately 7 × 10 6 The cells (containing 100 cells) are compounded into the test article. 46 athymic nude rats are subcutaneously implanted with two test articles each. Animals are evaluated for GSIS, ITT, and non-fasting blood glucose (NFBG) during the study. Ten animals per group are euthanized at scheduled termination points of 13, 17, 26, and 39 weeks, while six additional animals are studied to allow for possible unscheduled early termination. From each animal, two implanted test articles are randomly assigned to either histological evaluation or total C-peptide content evaluation. Table 15 shows the study design.

[0531] [Table 15]

[0532] At 12, 16, 20, 24, 30, and 36 weeks, all surviving animals are subjected to efficacy evaluation via glucose-stimulated insulin secretion (GSIS) testing. Blood samples are obtained from fasting animals before and after intraperitoneal administration of 3 g / kg glucose. Serum concentrations of human C-peptide are determined via standard enzyme-linked immunosorbent assay.

[0533] At 25 and 33 weeks, surviving animals are subjected to an insulin challenge (insulin tolerance test, ITT) to assess changes in serum human C-peptide in response to reduced blood glucose in the food-deprived state. Blood samples are obtained from fasting animals before and at multiple time points (15, 30, 60 min) after intraperitoneal administration of 1 unit of insulin per kg body weight. Serum concentrations of human C-peptide are determined via standard enzyme-linked immunosorbent assay.

[0534] Non-fasting blood glucose (NFBG) will be measured at approximately 12, 16, 20, 24, 25, 30, 33, and 36 weeks prior to the start of fasting for the GSIS and ITT studies.

[0535] Animals will be euthanized at scheduled endpoints specified in Table 13. Euthanasia will be performed by bilateral thoracotomy following CO2 inhalation. Necropsies will be performed at all scheduled and unscheduled terminations and gross abnormalities will be recorded.

[0536] The designated graft is frozen and then homogenized. The total C-peptide content of the homogenate is determined via standard enzyme-linked immunosorbent assay. The total C-peptide content of the graft is used to predict clinical dosing.

[0537] The designated implanted test articles are fixed in neutral buffered formalin, processed into slides, and stained by H&E and immunohistochemistry (IHC) for insulin and glucagon to identify human pancreatic endocrine cells. Additional IHC for the human-specific nuclear marker NuMA1 is performed to identify the potential location of implant-derived cells outside the lumen of the test article implant.

[0538] Example 13: Generation of B2M KO / PD-L1 KI, TXNIP KO / HLA-E KI in human iPSCs Human iPSCs (iPSC 0025) were generated with a PD-L1 coding sequence inserted into the B2M locus. RNP complexes were formed by combining B2M-2 gRNA (SEQ ID NO: 2) and Cas9 at a molar ratio of 3:1 (gRNA:Cas9). To form the RNP complex, gRNA and Cas9 were combined in a single vessel containing R-buffer to a total volume of 25 μL and incubated at room temperature for 15 minutes. Cells were dissociated using ACCUTASE®, then resuspended in DMEM / F12 medium (Gibco, cat#11320033), counted using an NC-200 (Chemometec), and centrifuged. A total of 1 × 10 6Cells were resuspended with the RNP complex. 4μg of B2M-CAGGS-PD-L1 donor plasmid (SEQ ID NO: 33) and R-buffer were added for a total volume of 125μL. This mixture was then electroporated using the following parameters: 2 pulses, 30ms, 1100V. Seven days after electroporation, cells were enriched for PD-L1 positive cells via MACS using Miltenyi or Thermofisher reagents essentially as described above in Example 8.

[0539] After the enriched PD-L1-positive population was expanded, cells were electroporated with an RNP complex containing TXNIP-T5 gRNA (SEQ ID NO: 20) and Cas9 protein at a molar ratio of 3:1 (gRNA:Cas9) and 4 μg of TXNIP-CAGGS-HLA-E donor plasmid 2 (SEQ ID NO: 56), essentially as described above. Seven days after electroporation, cells were enriched for HLA-E-positive cells via MACS using Miltenyi or Thermofisher reagents. After HLA-E enrichment, cells were single-cell sorted onto BIOLAMININ 521 CTG-coated 96-well plates containing StemFlex and Revitacell using a WOLF FACS-sorter (Nanocellect). The seeded single cells were grown in a normoxic incubator (37°C, 8% CO2) with medium changes every other day until colonies were large enough to be replated as single cells. Upon reaching confluence, samples were split for maintenance and genomic DNA extraction. Anti-PD-L1 and anti-HLA-E antibodies (Table 4) were used for MACS enrichment and FACS sorting into 96-well plates with gating settings for HLA-E and PD-L1 double-positive cells. For FACS sorting, unedited cells served as a negative control.

[0540] Correctly targeted clones were identified via PCR using primers amplifying the region from outside the homology arms of the plasmid to the PD-L1 cDNA insert or HLA-E cDNA insert, respectively, allowing amplification of only the KI-integrated DNA for the PD-L1 KI insert and HLA-E KI insert. The on-target inserts were tested for zygosity by PCR to assess whether the KI occurred in a heterozygous or homozygous fashion. If a heterozygous clone was identified, the KI-negative allele was sent for Sanger sequencing to verify that it contained a B2M-disrupting indel or a TXNIP-disrupting indel, respectively. Correct KI clones with complete B2M and TXNIP disruption (either by the KI insert or indel formation) were propagated in an incremental tissue culture format until they reached a population size of 30 million cells. Selected clones were propagated in this manner and confirmed to be pluripotent by testing for OCT4 and SOX2 via intracellular flow cytometry.

[0541] Four edited hiPSC clones (VI-B) were differentiated using the pancreatic endocrine protocol of Rezania et al. (Nat Biotechnol. 2014 Nov;32(11):1121-33). At stage 4, flow cytometry for chromogranin (CHGA), PDX1, and NKX6.1 was performed. The results for PDX1 and NKX6.1 of clone (clone 1) seeded at different representative densities are shown in Figure 26A. CHGA was negative for all four clones. Flow cytometry for PD-L1 and HLA-E was also performed. The results for PD-L1 and HLA-E of clone (clone 1) are shown in Figure 26B.

[0542] Example 14: Process for producing a B2M KO / PD-L1 KI, TXNIP KO / HLA-E KI human pluripotent stem cell (hPSC) frozen cell bank CyT49 hESCs (ViaCyte) were electroporated with an RNP complex containing B2M-2 gRNA (SEQ ID NO: 2) and Cas9 protein at a molar ratio of 3:1 (gRNA:Cas9) and 4 μg of the B2M-CAGGS-PD-L1 donor plasmid (SEQ ID NO: 33) at 1100 V with two 30 ms pulses. After electroporation, the cells were pipetted into an Eppendorf tube filled with RevitaCell StemFlex medium. The cell suspension was then plated onto tissue culture dishes pre-coated with BIOLAMININ 521 CTG at a 1:20 dilution. The cells were cultured in a normoxic incubator (37°C, 8% CO2).

[0543] Seven days after electroporation, cells were enriched for PD-L1-positive cells via MACS using Alexa-488-labeled anti-PD-L1 antibody and magnetic beads (DYNABEADS® pan-mouse IgG; Thermo Fisher Scientific). PD-L1-positive cells were expanded by culturing in XF-KSR growth medium (Gibco) for 7 days.

[0544] The PD-L1-positive cells were then electroporated with an RNP complex containing TXNIP-T5 gRNA (SEQ ID NO: 20) and Cas9 protein at a molar ratio of 3:1 (gRNA:Cas9) and 4 μg of TXNIP-CAGGS-HLA-E donor plasmid 2 (SEQ ID NO: 56) at 1100 V with two 30 ms pulses. After electroporation, the cells were pipetted into an Eppendorf tube filled with RevitaCell StemFlex medium. The cell suspension was then plated onto tissue culture dishes pre-coated with BIOLAMININ 521 CTG at a 1:20 dilution. The cells were cultured in a normoxic incubator (37°C, 8% CO2).

[0545] Seven days after electroporation, cells were enriched for HLA-E-positive cells via MACS using PE-labeled anti-HLA-E antibody and magnetic beads (DYNABEADS® pan-mouse IgG; Thermo Fisher Scientific). PD-L1 and HLA-E double-positive cells were expanded by culturing in XF-KSR growth medium (Gibco) for approximately 5 days.

[0546] PD-L1 and HLA-E double-positive cells were subjected to single-cell sorting. To do this, cells were fed with StemFlex Complete with Revitacell (1x Revitacell final concentration) 3–4 hours before dissociation with ACCUTASE®. After dissociation, single cells were sorted into single wells of a 96-well tissue culture plate coated with BIOLAMININ. Single cells were sorted into wells using the anti-PD-L1 and anti-HLA-E antibodies described above using a WOLF FACS sorter (Nanocellect). Plates were pre-filled with 100–200 μL of StemFlex Complete with Revitacell. Three days after cell seeding, cells were fed with fresh StemFlex and continued to be fed with 100–200 μL of medium every other day. After 10 days of expansion, cells were fed with StemFlex daily until days 12–14. At this point, the plates were dissociated with ACCUTASE® and the collected cell suspension was split 1:2 into two 96-well plates and cultured for approximately 4 days.

[0547] Some cells were collected for visual analysis (morphology) and DNA analysis (zygosity analysis and PCR and DNA sequencing for indel profiles), while the remaining cells were cultured and expanded for further cultivation in T175 flasks. After approximately two weeks of culture, clones were selected for freezing. Cells were characterized before and after freezing for morphology, viability, endotoxin, mycoplasma, karyotype, pluripotency, differentiation potential, on- / off-target analysis, random plasmid integration, and residual Cas9 / plasmid using standard procedures. Cells were frozen in freezing medium and stored in cryovials at -80°C or in liquid nitrogen.

[0548] Specific B2M KO / PD-L1 KI+TXNIP KO / HLA-E KI clones ("seed clones") were generated and isolated by the above process. The seed clones were differentiated to the PEC stage and characterized. Figure 27A shows that the morphology of the seed clones at the PEC stage was similar to that of wild-type cells. Figure 27B shows that the dynamic expression patterns of FOXA2, CHGA, PDX1, and NKX6.1 over the differentiation time course in cells differentiated from the seed clones were similar to that of wild-type cells. Figure 27C shows that the expression patterns of CHGA in the differentiated population were similar to those of wild-type cells. - / NKX6.1 + / PDX1 + The percentage of cells is shown.

Claims

1. 1. A method for generating universal donor cells, comprising: (a) a first site-specific nuclease that targets a site within or near a gene encoding a survival factor; and (b) a first nucleic acid comprising a nucleotide sequence encoding a first tolerogenic factor, flanked by (i) a nucleotide sequence homologous to a region located to the left of the target site in (a) and (ii) a nucleotide sequence homologous to a region located to the right of the target site in (a); wherein the first site-specific nuclease cleaves the target site of (a), and the first nucleic acid of (b) is inserted at a site that partially overlaps, completely overlaps, or is contained within the site of (a), thereby creating a universal donor cell, wherein the universal donor cell has increased cell survival compared to a cell in which the nucleic acid of (b) has not been inserted.

2. 2. The method of claim 1, wherein the survival factor is TXNIP, ZNF143, FOXO1, JNK, or MANF.

3. The method of claim 1 or 2, wherein the first tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

4. The method of any one of claims 1 to 3, wherein the survival factor is TXNIP.

5. The method of claim 4, wherein the first tolerogenic factor is HLA-E.

6. The method of any one of claims 1 to 5, wherein the first site-specific nuclease is a CRISPR system comprising a CRISPR nuclease and a guide RNA (gRNA).

7. 7. The method of claim 6, wherein the CRISPR nuclease is a type II Cas9 nuclease or a type V Cfp1 nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal.

8. The method of claim 6 or 7, wherein the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NOs: 15 to 24.

9. 9. The method of any one of claims 6 to 8, wherein the nucleotide sequence of (b)(i) consists essentially of SEQ ID NO:25, and the nucleotide sequence of (b)(ii) consists essentially of SEQ ID NO:

32.

10. (c) a second site-specific nuclease that targets a site within or near a gene encoding one or more of the MHC-I or MHC-II human leukocyte antigens or components or transcriptional regulators of the MHC-I or MHC-II complexes; and (d) a second nucleic acid comprising a nucleotide sequence encoding a second tolerogenic factor, flanked by (iii) a nucleotide sequence homologous to a region located to the left of the target site in (c) and (iv) a nucleotide sequence homologous to a region located to the right of the target site in (c). to the cell, wherein the second tolerogenic factor of (d) is different from the first tolerogenic factor of (b); 10. The method of any one of claims 1 to 9, wherein the second site-specific nuclease cleaves the target site of (c) and the second nucleic acid of (d) is inserted at a site that partially overlaps, completely overlaps, or is contained within the site of (c), and the universal donor cell has increased immune evasion and / or cell survival compared to a cell in which the second nucleic acid of (d) is not inserted.

11. 11. The method of claim 10, wherein the MHC-I or MHC-II human leukocyte antigen or the component of the MHC-I or MHC-II complex or the transcriptional regulator is HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, B2M, NLRC5, CIITA, RFX5, RFXAP, or RFXANK.

12. The method of claim 10 or 11, wherein the second tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

13. 13. The method of any one of claims 10 to 12, wherein the MHC-I or MHC-II human leukocyte antigen or the component or transcriptional regulator of the MHC-I or MHC-II complex is B2M.

14. The method of claim 13, wherein the second tolerogenic factor is PD-L1.

15. The method of any one of claims 10 to 14, wherein the second site-specific nuclease is a CRISPR system comprising a CRISPR nuclease and a gRNA.

16. 16. The method of claim 15, wherein the CRISPR nuclease is a type II Cas9 nuclease or a type V Cfp1 nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal.

17. The method of claim 15 or 16, wherein the gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NOs: 1 to 3 or 35 to 44.

18. 18. The method of any one of claims 15-17, wherein the nucleotide sequence of (d)(iii) consists essentially of SEQ ID NO:7, and the nucleotide sequence of (d)(iv) consists essentially of SEQ ID NO:

13.

19. 19. The method of any one of claims 6 to 9 or 15 to 18, wherein the CRISPR nuclease and the gRNA are present in a molar ratio of 1:

3.

20. 20. The method of any one of claims 1 to 19, wherein the nucleotide sequence encoding the first tolerogenic factor is operably linked to an exogenous promoter, and the nucleotide sequence encoding the second tolerogenic factor is operably linked to an exogenous promoter.

21. 21. The method of claim 20, wherein the exogenous promoter is a constitutive, inducible, time-specific, tissue-specific or cell-type-specific promoter, optionally the exogenous promoter is a CMV, EF1a, PGK, CAG or UBC promoter.

22. 1. A method for generating universal donor cells, comprising: (a) a first site-specific nuclease that targets a site within or near a gene encoding a survival factor; (b) a first nucleic acid comprising a nucleotide sequence encoding a first tolerogenic factor flanked by (i) a nucleotide sequence homologous to a region located to the left of the target site in (a) and (ii) a nucleotide sequence homologous to a region located to the right of the target site in (a), wherein the first site-specific nuclease cleaves the target site in (a) and, through the process of homologous recombination, the first nucleic acid of (b) inserts the nucleotide sequence encoding the first tolerogenic factor into a site that partially overlaps, completely overlaps, or is contained within the site in (a), thereby serving as a template for disrupting the gene of (a); (c) a second site-specific nuclease that targets a site within or near a gene encoding one or more of the MHC-I or MHC-II human leukocyte antigens or components or transcriptional regulators of the MHC-I or MHC-II complexes; and (d) a second nucleic acid comprising a nucleotide sequence encoding a second tolerogenic factor flanked by (iii) a nucleotide sequence homologous to a region located to the left of the target site in (c) and (iv) a nucleotide sequence homologous to a region located to the right of the target site in (c), wherein the tolerogenic factor in (d) is different from the tolerogenic factor (b), and the second site-specific nuclease cleaves the target site in (c), and through the process of homologous recombination, the second nucleic acid in (d) inserts the nucleotide sequence encoding the second tolerogenic factor into a site that partially overlaps, completely overlaps, or is contained within the site in (c), thereby serving as a template for disrupting the gene in (c). (b) into a cell, thereby creating a universal donor cell, wherein the universal donor cell has increased cell survival compared to a cell not having the first nucleic acid of (b) and the second nucleic acid of (d) inserted therein.

23. 23. The method of claim 22, wherein the survival factor is TXNIP, the first tolerogenic factor is HLA-E, the MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of an MHC-I or MHC-II complex is B2M, and the second tolerogenic factor is PD-L1.

24. The method of any one of claims 1 to 23, wherein the cell is a mammalian cell, optionally wherein the cell is a human cell.

25. The method of any one of claims 1 to 24, wherein the cells are stem cells.

26. The method according to any one of claims 1 to 24, wherein the cells are pluripotent stem cells, embryonic stem cells, adult stem cells, induced pluripotent stem cells or hematopoietic stem cells.

27. The method of any one of claims 1 to 24, wherein the cell is a differentiated cell or a somatic cell.

28. The method of any one of claims 1 to 24, wherein the universal donor cells are capable of differentiating into lineage-restricted progenitor cells or fully differentiated somatic cells.

29. 29. The method of claim 28, wherein the lineage-restricted progenitor cells are pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells.

30. 29. The method of claim 28, wherein the fully differentiated somatic cell is a pancreatic beta cell, an epithelial cell, an endodermal cell, a macrophage, a liver cell, an adipocyte, a kidney cell, a blood cell, a cardiac muscle cell, or a cell of the immune system.

31. A plurality of universal donor cells produced by the method of any one of claims 1 to 27.

32. 32. The plurality of universal donor cells of claim 31, wherein the cells are maintained for a time and under conditions sufficient for the cells to undergo differentiation.

33. 33. A plurality of cells according to claim 31 or 32 for use in treating a subject in need thereof.

34. 34. The plurality of cells for use according to claim 33, wherein the subject is a human having, suspected of having, or at risk of having a disease.

35. A method comprising administering to a subject a plurality of universal donor cells described in claim 31 or 32.

36. 1. A method for treating a subject in need thereof, comprising: (a) obtaining or having obtained a plurality of universal donor cells according to claim 31 following differentiation into lineage-restricted progenitor cells or fully differentiated somatic cells; (b) administering the lineage-restricted progenitor cells or fully differentiated somatic cells to the subject; A method comprising:

37. 1. A method of obtaining cells for administration to a subject in need thereof, comprising: (a) obtaining or having obtained the universal donor cells of claim 31; (b) maintaining the universal donor cells for a time and under conditions sufficient for the cells to differentiate into lineage-restricted progenitor cells or fully differentiated somatic cells; A method comprising:

38. 38. The method of claim 36 or 37, wherein the lineage-restricted progenitor cells are pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells.

39. 38. The method of claim 36 or 37, wherein the fully differentiated somatic cell is a pancreatic beta cell, an epithelial cell, an endodermal cell, a macrophage, a liver cell, an adipocyte, a kidney cell, a blood cell, a cardiac muscle cell or a cell of the immune system.

40. 40. The method of any one of claims 35 to 39, wherein the subject is a human having, suspected of having, or at risk of having a disease.

41. 41. The method of claim 40, wherein the disease is a genetically inherited disease.

42. A guide RNA comprising a spacer sequence corresponding to a target sequence consisting of SEQ ID NOs: 15 to 24.

43. 1. A process for generating universal donor cells, comprising: (a) modifying stem cells by inserting a nucleotide sequence encoding a first tolerogenic factor within or near a gene encoding an MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of an MHC-I or MHC-II complex, thereby generating a first tolerogenic factor-positive cell; (b) enriching for the first tolerogenic factor-positive cells; (c) modifying the first tolerogenic factor-positive cells by inserting a nucleotide sequence encoding a second tolerogenic factor within or near a gene encoding a survival factor, thereby creating first tolerogenic factor-positive / second tolerogenic factor-positive cells; (d) enriching the first tolerogenic factor-positive / second tolerogenic factor-positive cells; (e) performing single cell sorting to select first tolerogenic factor-positive / second tolerogenic factor-positive cells; (f) characterizing the cells from (e) as universal donor cells; (g) freezing the universal donor cells for long-term storage; A process involving:

44. 44. The process of claim 43, wherein the enrichment of first tolerogenic factor-positive cells in (b) comprises magnetic assisted cell sorting (MACS), single cell cloning, expanding the first tolerogenic factor-positive cells, or a combination thereof.

45. 45. The process of claim 43 or 44, wherein the enrichment of first tolerogenic factor-positive / second tolerogenic factor-positive cells in (d) comprises magnetically assisted cell sorting, single cell cloning, expanding the first tolerogenic factor-positive / second tolerogenic factor-positive cells, or a combination thereof.

46. 46. ​​The process of any one of claims 43 to 45, further comprising: (a) expanding the generated first tolerogenic factor-positive cells; (c) expanding the generated first tolerogenic factor-positive / second tolerogenic factor-positive cells; (e) expanding the selected first tolerogenic factor-positive / second tolerogenic factor-positive cells, or a combination thereof.

47. The modification in (a) comprises (1) a first RNA-guided nuclease and a first guide RNA (gRNA) that targets a target site in the locus of an MHC-I or MHC-II human leukocyte antigen, or a component of an MHC-I or MHC-II complex, or a transcriptional regulator; and (2) a first nucleic acid, the first nucleic acid comprising (i) a nucleotide sequence homologous to a region located to the left of the target site in the locus of the MHC-I or MHC-II human leukocyte antigen, or a component of an MHC-I or MHC-II complex, or a transcriptional regulator; (ii) the nucleotide sequence encoding the first tolerogenic factor; and (iii) the target site in the locus of the MHC-I or MHC-II human leukocyte antigen, or a component of an MHC-I or MHC-II complex, or a transcriptional regulator.

47. The process of any one of claims 43 to 46, comprising delivering to the stem cell a first vector comprising a first nucleic acid comprising a nucleotide sequence homologous to a region located to the right of a site, wherein the gene locus of the MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of an MHC-I or MHC-II complex, is cleaved at the target site, and the first nucleic acid comprising the nucleotide sequence encoding the first tolerogenic factor is inserted into the gene locus of the MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of an MHC-I or MHC-II complex, thereby disrupting the gene for the MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of an MHC-I or MHC-II complex.

48. 48. The process of Claim 47, wherein the first RNA-guided nuclease and the first gRNA form a first ribonucleoprotein (RNP) complex.

49. The modification in (a) comprises: (1) a first ribonucleoprotein (RNP) complex comprising a first RNA-guided nuclease and a first guide RNA (gRNA) that targets a target site in the gene locus of an MHC-I or MHC-II human leukocyte antigen, or a component of an MHC-I or MHC-II complex, or a transcriptional regulator; and (2) a first nucleic acid, the first nucleic acid comprising: (i) a nucleotide sequence homologous to a region located to the left of the target site in the gene locus of the MHC-I or MHC-II human leukocyte antigen, or a component of an MHC-I or MHC-II complex, or a transcriptional regulator; (ii) the nucleotide sequence encoding the first tolerogenic factor; and (iii) the MHC-I or MHC-II human leukocyte antigen, or a component of an MHC-I or MHC-II complex, or a transcriptional regulator.

47. The process of any one of claims 43 to 46, comprising delivering to the stem cell a first vector comprising a first nucleic acid comprising a nucleotide sequence homologous to a region located to the right of the target site in a gene locus of the MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of an MHC-I or MHC-II complex, wherein the gene locus of the MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of an MHC-I or MHC-II complex, is cleaved at the target site, and the first nucleic acid comprising the nucleotide sequence encoding a first tolerogenic factor is inserted into the gene locus of the MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of an MHC-I or MHC-II complex, thereby disrupting the gene of the MHC-I or MHC-II human leukocyte antigen, or a component or transcriptional regulator of an MHC-I or MHC-II complex.

50. 50. The process of any one of claims 43 to 49, wherein the gene for an MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of an MHC-I or MHC-II complex is HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, B2M, NLRC5, CIITA, RFX5, RFXAP, or RFXANK.

51. 51. The process of claim 50, wherein the gene for an MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of an MHC-I or MHC-II complex is B2M.

52. 52. The process of claim 51 , wherein the nucleotide sequence of (a)(2)(i) consists essentially of SEQ ID NO:7, and the nucleotide sequence of (a)(2)(iii) consists essentially of SEQ ID NO:

13.

53. 53. The process of claim 51 or 52, wherein the first gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NO:

2.

54. 54. The process of any one of claims 47 to 53, wherein the first RNA-guided nuclease is a Cas9 nuclease.

55. 55. The process of Claim 54, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

56. 56. The process of any one of claims 47 to 55, wherein the first RNP comprises a molar ratio of first gRNA:first RNA-guided nuclease of 3:

1.

57. 57. The process of any one of claims 43 to 56, wherein the first tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4 or CD47.

58. 58. The process of any one of claims 43 to 57, wherein the nucleotide sequence encoding the first tolerogenic factor is operably linked to an exogenous promoter.

59. 59. The process of claim 58, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

60. 60. The process of any one of claims 43 to 59, wherein the first tolerogenic factor is PD-L1.

61. 61. The process of claim 60, wherein the nucleotide sequence encoding PD-L1 consists essentially of SEQ ID NO:

11.

62. 62. The process of claim 61, wherein the nucleotide sequence encoding PD-L1 is operably linked to a CAG promoter.

63. 63. The process of any one of claims 60 to 62, wherein the first vector comprises a nucleotide sequence consisting of SEQ ID NO:

33.

64. 64. The process of any one of claims 43 to 63, wherein the modification in (c) comprises delivering to the stem cell a second vector comprising: (1) a second RNA-guided nuclease and a second guide RNA (gRNA) that target a target site in a survival factor gene locus; and (2) a second nucleic acid, the second nucleic acid comprising: (i) a nucleotide sequence homologous to a region located to the left of the target site in the survival factor gene locus, (ii) the nucleotide sequence encoding the second tolerogenic factor, and (iii) a nucleotide sequence homologous to a region located to the right of the target site in the survival factor gene locus, wherein the survival factor gene locus is cleaved at the target site, and the second nucleic acid comprising the nucleotide sequence encoding the second tolerogenic factor is inserted into the survival factor gene locus, thereby disrupting the survival factor gene.

65. 65. The process of Claim 64, wherein the second RNA-guided nuclease and the second gRNA form a second ribonucleoprotein (RNP) complex.

66. 64. The process of any one of claims 43 to 63, wherein the modification in (c) comprises delivering to the first tolerogenic factor-positive cells a second vector comprising: (1) a second ribonucleoprotein (RNP) complex comprising a second RNA-guided nuclease and a second guide RNA (gRNA) that targets a target site in a survival factor gene locus; and (2) a second nucleic acid, the second nucleic acid comprising: (i) a nucleotide sequence homologous to a region located to the left of the target site in the survival factor gene locus, (ii) the nucleotide sequence encoding the second tolerogenic factor, and (iii) a nucleotide sequence homologous to a region located to the right of the target site in the second survival factor gene locus, wherein the survival factor gene locus is cleaved at the target site, and the second nucleic acid comprising the nucleotide sequence encoding the second tolerogenic factor is inserted into the survival factor gene locus, thereby disrupting the survival factor gene.

67. 67. The process of any one of claims 43 to 66, wherein the survival gene is TXNIP, ZNF143, FOXO1, JNK or MANF.

68. 68. The process of claim 67, wherein the survival gene is TXNIP.

69. 69. The process of Claim 68, wherein the second gRNA comprises a spacer sequence corresponding to a target sequence consisting of SEQ ID NO:

20.

70. 70. The process of claim 68 or 69, wherein the nucleotide sequence of (c)(2)(i) consists essentially of SEQ ID NO:25, and the nucleotide sequence of (c)(2)(iii) consists essentially of SEQ ID NO:

32.

71. 71. The process of any one of claims 64 to 70, wherein the second RNA-guided nuclease is a Cas9 nuclease.

72. 72. The process of Claim 71, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

73. 73. The process of any one of claims 64-72, wherein the second RNP comprises a molar ratio of second gRNA:second RNA-guided nuclease of 3:

1.

74. 74. The process of any one of claims 43 to 73, wherein the second tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4 or CD47.

75. 75. The process of any one of claims 43 to 74, wherein the nucleotide sequence encoding the second tolerogenic factor is operably linked to an exogenous promoter.

76. 76. The process of claim 75, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

77. 77. The process of any one of claims 43 to 76, wherein the second tolerogenic factor is HLA-E.

78. 78. The process of claim 77, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide.

79. 79. The process of claim 78, wherein the sequence encoding the HLA-E trimer consists essentially of SEQ ID NO:

55.

80. 80. The process of claim 78 or 79, wherein the nucleotide sequence encoding HLA-E is operably linked to a CAG promoter.

81. 81. The process of any one of claims 77 to 80, wherein the second vector comprises a nucleotide sequence consisting of SEQ ID NO: 34 or 56.

82. 82. The process of any one of claims 43 to 81, wherein the single-cell sorting in (e) comprises fluorescence-activated cell sorting (FACS), single-cell cloning, expanding the single-cell sorted cells, or a combination thereof.

83. 83. The process of any one of claims 43 to 82, wherein the characterization in (f) comprises DNA analysis for zygosity and / or indel profile.

84. 84. The process of any one of claims 43-83, wherein the characterization in (f) comprises analyzing cells for morphology, viability, karyotype analysis, endotoxin levels, mycoplasma levels, on / off target analysis, random vector insertions, residual Cas9, residual vector, pluripotent state, differentiation potential, or a combination thereof.

85. 85. The process of any one of claims 43 to 84, further comprising freezing prior to said characterizing in (f).

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