Universal donor cells

Abstract

Provided herein is a genetically modified cell, such as a universal donor cell, that is compatible with multiple subjects, and a method for generating said genetically modified cell.The universal donor cell comprises at least one genetic modification in or near a gene that encodes one or more MHC-I or MHC-II human leukocyte antigens or a component or transcription regulator of the MHC-I or MHC-II complex, wherein said genetic modification comprises the insertion of a polynucleotide that encodes a tolerogenic factor and / or a survival factor.The universal donor cell may further comprise at least one genetic modification in or near a gene that encodes a survival factor, wherein said genetic modification comprises the insertion of a polynucleotide that encodes a second tolerogenic factor and / or a different survival factor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 132,890, filed December 31, 2020, U.S. Provisional Application No. 63 / 234,997, filed August 19, 2021, and U.S. Provisional Application No. 63 / 288,356, filed December 10, 2021, the entire disclosures of each of which are incorporated herein by reference.

[0002] Incorporation by reference to sequence listing This application contains a Sequence Listing that was submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on December 22, 2021, is named CT154_Sequence_Listing_ST25.txt, and is approximately 131,000 bytes in size.

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

[0004] background Various approaches have been proposed to overcome allogeneic rejection of transplanted or engrafted cells, including HLA matching, antibody-mediated blockade of pathways that trigger T cell activation, the use of immunosuppressive drug cocktails, and autologous cell therapy. Another strategy for attenuating graft rejection involves minimizing allogeneic differences between the transplanted or engrafted cells and the recipient. Human leukocyte antigens (HLA), 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 between a single HLA gene and the recipient can trigger robust immune responses (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 divided 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 cell 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 elicit robust cellular immune responses directed against these cells, ultimately resulting in the elimination of the transplanted or engrafted cells by activated cytolytic CD8+ T cells. MHC-I proteins are closely associated with beta2-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 MHC-I genes, which are expressed in a wide range of cells, MHC-II gene expression 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). Generating "universal donor" cells compatible with all HLA genotypes offers an alternative strategy that could eliminate the immune rejection and associated economic costs of current immune evasion methods. One approach previously taken to generate such universal donor cell lines has been to functionally disrupt the expression of MHC-I and MHC-II class genes. This can be achieved, for example, by genetically disrupting both genetic alleles encoding the MHC-I light chain, B2M. The resulting B2M-null cell lines and their derivatives are predicted to exhibit a significant reduction in surface MHC-I and, therefore, reduced immunogenicity to allogeneic CD8+ T cells. B2M-deficient hESC lines have been generated by deleting a few nucleotides in exon 2 of the B2M gene using a transcription activator-like effector nuclease (TALEN) targeting approach (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 lack surface HLA-I, they were found to still contain mRNA specific for B2M and MHC-I. B2M and MHC-I mRNAs were expressed at levels equivalent to those in untargeted hESCs (both constitutive and IFN-γ-induced). Thus, there is concern that these TALEN-targeted B2M hESC lines may express sufficient residual cell surface MHC-I to induce immune rejection, as observed in 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-targeted hESC lines to B2M were not investigated for off-target cleavage events, non-specific cleavage using TALENs remains a significant problem that raises major concerns about the safety of their clinical use (Grau, J. et al. "TALENoffer: 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, another report generated iPS cells that escape allogeneic recognition by knocking out the first B2M allele and knocking in the HLA-E gene into the second B2M allele, resulting in surface expression of HLA-E dimers or trimers in the absence of HLA-A, HLA-B, or HLA-C surface expression (Gornalusse, GG et al., "HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells," Nature Biotechnology, 2017, 35, 765-773). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] 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 [Non-patent document 2] Rubinstein P., "HLA matching for bone marrow transplantation--how much is enough?" N Engl J Med., 2001, 345: 1842-1844 [Non-patent document 3] Lu, P. et al., "Generating hypoimmunogenic human embryonic stem cells by the disruption of beta 2-microglobulin," Stem Cell Rev. 2013, 9: 806-813 [Non-patent document 4] Gross, R. and Rappuoli, R. "Pertussis toxin promoter sequences involved in modulation," Proc Natl Acad Sci, 1993, 90: 3913-3917 [Non-Patent Document 5] Grau, J. et al. "TALENoffer: genome-wide TALEN off-target prediction," Bioinformatics, 2013, 29: 2931-2932 [Non-patent document 6] Guilinger JP et al. "Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity," Nat Methods 2014, 11: 429-435 [Non-Patent Document 7] Gornalusse, GG 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 strategy is that MHC class I-negative cells are susceptible to lysis by natural killer (NK) cells, because HLA molecules function as major ligand inhibitors for NK cells. Host NK cells have been shown to reject 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 on previous methods to generate universal donor cells that can avoid immune responses and generate cells that can survive after engraftment. As described herein, cell survival after engraftment or transplantation can be mediated by many other pathways unrelated to allogeneic rejection, such as hypoxia, reactive oxygen species, nutrient deprivation, and oxidative stress. As also described herein, gene transfer of survival factors (genes and / or proteins) can aid in 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] Abstract In some aspects, the disclosure encompasses genetically modified cells comprising: (a) a first exogenous polynucleotide insertion encoding midbrain astrocyte-derived neurotrophic factor (MANF), a second exogenous polynucleotide insertion encoding tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a third exogenous polynucleotide insertion encoding cluster of differentiation 39 (CD39), and / or a fourth exogenous polynucleotide insertion encoding cluster of differentiation 73 (CD73), wherein the genetically modified cells express CD39, MANF, TNFAIP3, and / or CD73; and / or (b) a disruption of a gene encoding transforming growth factor beta (TGFβ), beta 2-microglobulin (B2M), thioredoxin-interacting protein (TXNIP), and / or class II transactivator of differentiation (CIITA), wherein the genetically modified cells have disrupted expression of TGFβ, B2M, TXNIP, and / or CIITA.

[0009] In some aspects, the genetically modified cells of the present disclosure are stem cells, hematopoietic stem cells, lineage-restricted progenitor cells, and / or fully differentiated somatic cells.

[0010] In some aspects, the disclosure encompasses a plurality of genetically modified cells comprising one or more exogenous polynucleotide insertions comprising MANF, TNFAIP3, CD39, CD73, and / or one or more disruptions of genes encoding transforming growth factor beta (TGFβ), protein, beta2-microglobulin (B2M) protein, thioredoxin-interacting protein (TXNIP) protein, and / or class II transactivator of TGFβ (CIITA) protein, wherein expression of the TGFβ protein, B2M protein, TXNIP protein, and / or CIITA protein is disrupted. In some aspects, the disclosure encompasses a plurality of genetically modified cells, wherein at least about 50% of the cells express MANF, HLA-E, TNFAIP3, PD-L-1, CD39, and / or CD73.

[0011] In some further aspects, the present disclosure encompasses a population of lineage-restricted progenitor cells (including, but not limited to, endoderm cells, primitive intestinal cells, posterior foregut cells, pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, pancreatic endocrine cells, or immature beta cells; a fully differentiated somatic cell is a pancreatic beta cell) or fully differentiated somatic cells derived from a plurality of genetically modified cells. In some aspects, the present disclosure further encompasses a population of lineage-restricted progenitor cells (including, but not limited to, endoderm cells, primitive intestinal cells, posterior foregut cells, pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, pancreatic endocrine cells, or immature beta cells; a fully differentiated somatic cell is a pancreatic beta cell) or fully differentiated somatic cells derived from a plurality of genetically modified cells, wherein at least about 50% of the cells express MANF, HLA-E, TNFAIP3, PD-L-1, CD39, and / or CD73.

[0012] In some aspects, the disclosure provides a composition comprising a plurality of genetically modified cells or a cell population of genetically modified cells, the composition comprising: (a) a first exogenous polynucleotide insertion encoding midbrain astrocyte-derived neurotrophic factor (MANF), a second exogenous polynucleotide insertion encoding tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a third exogenous polynucleotide insertion encoding cluster of differentiation 39 (CD39), and / or a fourth exogenous polynucleotide insertion encoding cluster of differentiation 73 (CD73), wherein the genetically modified cells are and / or (b) expressing MANF, TNFAIP3, and / or CD73; and / or comprising a disruption of a gene encoding a transforming growth factor beta (TGFβ) protein, a beta 2-microglobulin (B2M) protein, a thioredoxin interacting protein (TXNIP) protein, and / or a class II transactivator of TGFβ (CIITA) protein, wherein the genetically modified cells have disrupted expression of a TGFβ protein, a B2M protein, a TXNIP protein, and / or a CIITA protein.

[0013] In some aspects, the present disclosure also encompasses methods for treating a subject in need thereof, the methods comprising: (a) obtaining or obtaining a plurality of genetically modified cells disclosed herein, wherein the plurality of genetically modified cells comprises stem cells; (b) differentiating the genetically modified cells into pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells; and (c) administering the pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells to the subject.

[0014] In a further aspect, the present disclosure encompasses a method for treating a subject in need thereof, the method comprising: (a) obtaining or obtaining a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from a plurality of genetically modified cells disclosed herein, wherein the lineage-restricted progenitor cells or fully differentiated somatic cells comprise pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells; and (b) administering the pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, and / or beta cells to the subject.

[0015] In some aspects, the present disclosure encompasses methods for treating a subject in need thereof, the method comprising: (a) obtaining or obtaining a plurality of genetically modified cells, wherein the plurality of genetically modified cells disclosed herein comprises stem cells; (b) differentiating the genetically modified cells into pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells; and (c) administering the pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells to the subject.

[0016] 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 presented herein are not intended to limit the disclosure to the particular embodiments disclosed, but on the contrary, are intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0017] Other features and advantages of the present disclosure will become apparent in the following detailed description of the embodiments of the invention, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 shows the plasmid map of the B2M-CAGGS-MANF-P2A-TNFAIP3-P2A-PD-L-1 donor vector.

[0019] [Figure 2] Figure 2 shows the plasmid map of the CIITA-CAGGS-CD39 donor vector.

[0020] [Figure 3] Figure 3 shows the plasmid map of the B2M-CAGGS-CD39-P2A-PD-L-1 donor vector.

[0021] [Figure 4] Figure 4 shows the plasmid map of the B2M-CAGGS-TNFAIP3-P2A-PD-L-1 donor vector.

[0022] [Figure 5] Figure 5 shows the plasmid map of the TXNIP-CAGGS-MANF-P2A-HLA-E donor vector.

[0023] [Figure 6] FIG. 6 shows flow cytometry of CD39 expression in L3V003B and L3V004B cell lines.

[0024] [Figure 7] Figure 7 shows the morphology of PEC cells differentiated from wild-type (WT) cells (upper panel) or X1 cells (i.e., TXNIP KO / MANF-P2A-HLA-E KI&B2M KO / TNFAIP3(A20)-P2A-PD-L-1 KI) and cells at stage 6 (S6).

[0025] [Figure 8]Figure 8 shows selected gene expression in PEC cells and stage 6 (S6) cells differentiated from wild-type (WT) cells (top panel) or X1 (“X1”) cells (i.e., TXNIP KO / MANF-P2A-HLA-E KI&B2M KO / TNFAIP3(A20)-P2A-PD-L-1 KI).

[0026] [Figure 9] Figure 9 shows flow cytometry for CHGA, PDX1, and NKX6.1 in PEC cells differentiated from wild-type (WT) cells or TXNIP KO / MANF-P2A-HLA-E KI&B2M KO / TNFAIP3(A20)-P2A-PD-L-1 KI cells (L1V028-C3, L1V028-24).

[0027] [Figure 10A] FIG. 10A shows flow cytometry for CHGA, PDX1, and NKX6.1 in stage 6 (S6) cells differentiated from wild-type (WT) cells.

[0028] [Figure 10B] Figure 10B shows flow cytometry of CHGA, PDX1, and NKX6.1 in stage 6 (S6) cells differentiated from X1 cells (i.e., TXNIP KO / MANF-P2A-HLA-E KI&B2M KO / TNFAIP3(A20)-P2A-PD-L-1 KI).

[0029] [Figure 11] Figure 11 shows C-peptide levels at 12, 16, 20, and 24 weeks in blood samples obtained from athymic nude rats transplanted with cell aggregates of L1V009B bulk cells (GRP1) or L1V008 clonal isolates (B2M KO / MANF-P2A-TNFAIP3-P2A-PD-L-1 KI; GRP2 and GRP3) or control cells (GRP4), at 90 minutes after intraperitoneal administration of 3 g / kg glucose for GRP1-3 and 60 minutes after administration for GRP4.

[0030] [Figure 12] Figure 12 shows C-peptide levels at 12, 16, 20, and 24 weeks in blood samples obtained 90 minutes (60 minutes for the 24 week reading) after intraperitoneal administration of 3 g / kg glucose from athymic nude rats transplanted with cell aggregates derived from B2M KO / CD39-P2A-PD-L-1 KI or control cells.

[0031] [Figure 13] Figure 13 shows C-peptide levels in blood samples obtained after glucose stimulation at 12 and 16 weeks from NSG mice implanted with capsules containing unmodified (NCG) or B2M KO / TNFAIP3-P2A-PD-L-1 KI&TXNIP KO / MANF-P2A-HLA-E KI(X1) cell aggregates differentiated to the PEC stage or stage 6 (S6).

[0032] [Figure 14] Figure 14 shows the mean C-peptide levels in PEC-control (NCG) and PEC-X1 (B2M KO / TNFAIP3-P2A-PD-L-1 KI and TXNIP KO / MANF-P2A-HLA-E KI) groups of mice at 12, 16, 20, and 24 weeks after glucose stimulation.

[0033] [Figure 15] Figure 15 shows C-peptide levels at 12, 16, and 20 weeks in NSG mice implanted with capsules containing cells differentiated from unmodified (CON) or clones (i.e., 6D09, 6H07, and 5C10) of B2M KO / TNFAIP3-P2A-PD-L-1 KI&TXNIP KO / MANF-P2A-HLA-E KI(X1) cells.

[0034] [Figure 16]Figure 16 shows the plasmid map of the B2M-CAGGS-CD39-P2A-CD73-P2A-PD-L-1 donor vector.

[0035] [Figure 17] Figure 17 shows flow cytometry analysis of SOX17 and FOXA2 expression in L1V017B cells (i.e., CD39-P2A-PD-L-1 KI and B2M KO), L1V018B cells (i.e., CD39-P2A-CD73-P2A-PD-L-1 KI and B2M KO), and L1V019B cells (i.e., TNFAIP3(A20)-P2A-PD-L-1 KI and B2M KO).

[0036] [Figure 18] Figure 18 shows flow cytometry analysis of CHGA, NKX6.1, and PDX1 expression in L1V017B cells (i.e., CD39-P2A-PD-L-1 KI and B2M KO), L1V018B cells (i.e., CD39-P2A-CD73-P2A-PD-L-1 KI and B2M KO), and L1V019B cells (i.e., TNFAIP3(A20)-P2A-PD-L-1 KI and B2M KO).

[0037] [Figure 19] Figure 19 shows the time course of gene expression of various markers in L1V017B cells (i.e., CD39-P2A-PD-L-1 KI and B2M KO), L1V018B cells (i.e., CD39-P2A-CD73-P2A-PD-L-1 KI and B2M KO), and L1V019B cells (i.e., TNFAIP3(A20)-P2A-PD-L-1 KI and B2M KO).

[0038] [Figure 20A]Figure 20A shows TIDE analysis demonstrating 90% KO of the TGF-β2 gene in X1 (B2M KO / TNFAIP3-P2A-PD-L-1 KI&TXNIP KO / MANF-P2A-HLA-E KI)+TGF-β2 KO cells ("L3V002B"), with significant editing at +1 and -7 causing a frameshift in the coding region.

[0039] [Figure 20B] Figure 20B shows TIDE analysis demonstrating 90% KO of the TGF-β2 gene in X4 (B2M KO / TNFAIP3-P2A-PD-L-1 KI&TXNIP KO / MANF-P2A-HLA-E KI&CIITA KO / CD39 KI)+TGF-β2 cells ("L3V004B"), with significant editing at +1 and -7 causing a frameshift in the coding region.

[0040] [Figure 21] Figure 21 shows immune evasion assay data in the presence of X1 and B2M KO edited cells with or without TGF-β blocker in the media using a peripheral blood mononuclear cell proliferation assay.

[0041] [Figure 22A] Figure 22A shows data from an ELISA assay for secreted TGF-β2 in conditioned medium from differentiated wild-type, V1B (HLA-E KI, TXNIP KO, PD-L-1 KI, B2M KO), and TGF-β2 KO PEC cells for 72 hours.

[0042] [Figure 22B] Figure 22B shows data from an ELISA assay for secreted TGF-β1 in conditioned medium from differentiated wild-type, V1B (HLA-E KI, TXNIP KO, PD-L-1 KI, B2M KO), and TGF-β2 KO PEC cells for 72 hours.

[0043] [Figure 23A]FIG. 23A presents data for TGF-β secreted from V1B and TGF-β2 KO PEC cells.

[0044] [Figure 23B] FIG. 23B presents data for GDF-9 secreted from V1B and TGF-β2 KO PEC cells.

[0045] [Figure 23C] FIG. 23C presents data for PDGF-AA secreted from V1B and TGF-β2 KO PEC cells.

[0046] [Figures 24A-24B] Figures 24A-24B show fibroblast migration assays using human lung fibroblast (MRC-5) cells with conditioned medium from WT, V1B, and X1 PEC cells (Figure 24A), and conditioned medium from WT and TGF-β2 KO PEC cells (Figure 24B).

[0047] [Figures 25A-25B] Figures 25A-25C show fibroblast migration assays using human fibrosarcoma (HT1080) cells with conditioned medium from WT, V1B, and X1 PEC cells (Figure 25A), conditioned medium from WT and TGF-β2 KO PEC cells (Figure 25B), and conditioned medium from WT, X4 (L3V003B), and X4 + TGF-β2 KO (L3V004B) PEC cells (Figure 25C). [Figure 25C] Figures 25A-25C show fibroblast migration assays using human fibrosarcoma (HT1080) cells with conditioned medium from WT, V1B, and X1 PEC cells (Figure 25A), conditioned medium from WT and TGF-β2 KO PEC cells (Figure 25B), and conditioned medium from WT, X4 (L3V003B), and X4 + TGF-β2 KO (L3V004B) PEC cells (Figure 25C). DETAILED DESCRIPTION OF THE INVENTION

[0048] Detailed Description I. Definition Deletion: As used herein, the term "deletion" may be used interchangeably with the terms "gene deletion" or "knockout" and generally refers to a genetic modification in which a site or region of genomic DNA is removed by any molecular biological method, such as the methods described herein, for example, by delivering an endonuclease and at least one gRNA to the site. Any number of nucleotides can be deleted. In some embodiments, the deletion involves the removal of at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, or at least 25 nucleotides. In some embodiments, the deletion involves the removal of 10-50 nucleotides, 25-75 nucleotides, 50-100 nucleotides, 50-200 nucleotides, or more than 100 nucleotides. In some embodiments, the deletion involves the removal of part or all of a single target gene, such as the B2M gene, the TXNIP gene, the CIITA gene, or the TGF-β2 gene. In some embodiments, the deletion involves the removal of part or all of two target genes, three target genes, or four target genes. In some embodiments, the removal of part of a target gene refers to the removal of all or part of the promoter and / or coding sequence of the gene. In some embodiments, the deletion involves the removal of a transcriptional regulator of the target gene, such as the promoter region. In some embodiments, the deletion involves the removal of all or part of the 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 leads to reduced expression of the gene compared to unmodified cells. In some embodiments, the deletion leads to the loss of expression of the gene compared to unmodified cells.

[0049] Disruption: As used herein, the terms "disruption," "disrupting," or "disrupted" refer to a genetic modification that alters the level of expression of a target gene. In some aspects, the disruption can be due to the deletion of at least one nucleotide within or near the target gene or the deletion of part or all of the target gene, as described above. In other aspects, the disruption can also be due to the substitution of at least one nucleotide within or near the target gene and / or the insertion of at least one nucleotide. In further aspects, the disruption can be due to the insertion of one or more exogenous polynucleotides within or near the target gene. Generally, as used herein, disruption of expression refers to the reduction or elimination of expression of a target gene. In some embodiments, the disruption can be a reduction in the level of expression (e.g., expression of less than 30%, less than 25%, less than 20%, less than 10%, or less than 5% of the level of an unmodified cell). In some embodiments, the disruption can be the elimination of expression (e.g., no expression or undetectable levels of RNA and / or protein expression). Expression can be measured using any standard RNA-based, protein-based, and / or antibody-based detection method (e.g., RT-PCR, ELISA, flow cytometry, immunocytochemistry, etc.) A detectable level is defined as a level greater than the limit of detection (LOD), which is the lowest concentration that can be measured (detected) with statistical significance by a given detection method.

[0050] Endonuclease: As used herein, the term "endonuclease" generally refers to an enzyme that cleaves phosphodiester bonds in polynucleotides. In some embodiments, the endonuclease specifically cleaves phosphodiester bonds in DNA polynucleotides. 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, for example, a type II CRISPR Cas9 endonuclease or a type V CRISPR Cpf1 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, Cmr3, 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 an engineered version thereof, or a combination thereof. In some embodiments, the endonuclease can introduce one or more single-strand breaks (SSBs) and / or one or more double-strand breaks (DSBs).

[0051] Exogenous: As used herein, the term "exogenous" refers to a polynucleotide sequence that originates outside of a recipient cell or organism, that is assembled outside of a recipient cell or organism, or that originates from a recipient cell or organism but is integrated into the recipient genome at a location other than its naturally occurring location. An exogenous polynucleotide sequence may include a gene sequence, may include the coding sequence (CDS) of a gene, may include coding sequences from more than one gene, may include promoter sequences, enhancer sequences, and / or other regulatory elements, may include ribosomal skipping sequences, and / or may include artificial sequences. An exogenous polynucleotide may be codon-optimized to ensure efficient translation in the recipient cell or organism.

[0052] 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 genomic DNA. Examples of genetic modifications include insertions, deletions, duplications, inversions, and translocations, as well as 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), which shifts the reading frame of the target gene, thereby leading to an altered or absent gene product.

[0053] Guide RNA (gRNA): As used herein, the term "guide RNA" or "gRNA" generally refers to a short ribonucleic acid that can interact with an endonuclease, 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 comprising 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 can be pre-complexed with a DNA endonuclease.

[0054] 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 the methods described herein, for example, by delivering an endonuclease and at least one gRNA to a site in the genomic DNA. In some embodiments, the insertion of the exogenous polynucleotide occurs within or near a target gene. In some embodiments, the insertion of the exogenous polynucleotide may occur within or near a site in the genomic DNA that was the site of a previous genetic modification, such as a deletion or insertion-deletion mutation. In some embodiments, the insertion occurs at a site in the genomic DNA that partially overlaps, completely overlaps, or is contained within the site of a previous genetic modification, such as a deletion or insertion-deletion mutation. In some embodiments, the insertion simultaneously leads to disruption of a gene at the targeted site of insertion. In some embodiments, the insertion occurs at a safe harbor locus. In some embodiments, the insertion involves the introduction of a polynucleotide encoding a protein of interest. In some embodiments, the insertion involves the introduction of a polynucleotide encoding a tolerogenic factor. In some embodiments, the insertion involves the introduction of a polynucleotide encoding a survival factor. In some embodiments, the insertion involves the introduction of a polynucleotide encoding MANF, TNFAIP3, CD39, CD73, PD-L-1, and / or HLA-E. In some embodiments, the insertion involves the introduction of an exogenous promoter, e.g., a constitutive promoter, e.g., a CAG or CAGGS promoter. In some embodiments, the insertion involves the introduction of a polynucleotide encoding a non-coding gene. Generally, the inserted polynucleotide is flanked by sequences (e.g., homology arms) that have substantial sequence homology to genomic DNA at or near the insertion site.

[0055] 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 biomolecules found on the cell surface of all nucleated cells in vertebrates, including mammals, e.g., humans, that function to display intracellularly derived (i.e., cytosolic) non-self or foreign antigens, e.g., peptides of proteins, to cytotoxic T cells, e.g., CD8+ T cells, to stimulate an immune response. In some embodiments, the MHC-I biomolecule is an MHC-I gene or MHC-I protein. Cell surface expression of all MHC-I proteins requires complex formation of the MHC-I protein with the beta 2 microglobulin (B2M) protein. In some embodiments, reducing MHC-I human leukocyte antigen (HLA) expression relative to unmodified cells is accompanied by reduced (or decreased) expression of the MHC-I gene. In some embodiments, the reduced expression of MHC-I human leukocyte antigen (HLA) relative to unmodified cells is accompanied by reduced (or decreased) cell surface expression of MHC-I proteins. 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).

[0056] 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 that are typically found on the cell surface of antigen-presenting cells in vertebrates, including mammals, e.g., humans, and function to display non-self or foreign antigens, e.g., peptides of proteins, derived from outside the cell (extracellular) 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, the reduced expression of MHC-II human leukocyte antigen (HLA) compared to unmodified cells is accompanied by reduced (or decreased) expression of the MHC-II gene. In some embodiments, the reduced expression of MHC-II human leukocyte antigen (HLA) compared to unmodified cells is accompanied by reduced (or decreased) cell surface expression of the MHC-II protein. In some embodiments, the MHC-II biomolecules include 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).

[0057] Polynucleotide: As used herein, the term "polynucleotide" may be used interchangeably with the term "nucleic acid" and generally refers to a biological molecule containing two or more nucleotides. In some embodiments, a polynucleotide contains at least 2 nucleotides, at least 5 nucleotides, at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 500 nucleotides, or any number of nucleotides. For example, a polynucleotide may contain 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 may be a DNA or RNA molecule or a hybrid DNA / RNA molecule. A polynucleotide may 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 in the genome of unmodified cells or universal donor cells.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 adeno-associated virus vector.In some embodiments, the polynucleotide is a circular or linear molecule.

[0058] Safe harbor locus: As used herein, the term "safe harbor locus" generally refers to any position, site, or region of genomic DNA that may be adaptable without adverse effects on cells. 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 generally not transcribed. In some embodiments, a safe harbor locus is the AAVS1 (PPP1 R12C), 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.

[0059] Safety switch: As used herein, the term "safety switch" generally refers to a biological molecule that directs 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), directs a cell to undergo apoptosis by metabolizing a prodrug, e.g., ganciclovir. In some embodiments, overexpression of the presence of the safety switch itself directs a cell to undergo apoptosis. In some embodiments, the safety switch is a p53-based molecule, HSV-tk, or inducible caspase-9.

[0060] 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 a 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.

[0061] Survival Factor: As used herein, the term "survival factor" generally refers to a protein (e.g., expressed by a polynucleotide 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 compared 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 is one closely related to 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 after engraftment than the unmodified cell, 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 MANF (NCBI Gene ID No: 7873), ZNF143 (NCBI Gene ID No: 7702), TXNIP (NCBI Gene ID No: 10628), FOXO1 (NCBI Gene ID No: 2308), or JNK (NCBI Gene ID No: 5599). 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., a universal donor cell, to survive at a higher rate after transplantation or engraftment into a host subject compared to an unmodified cell. In some embodiments, a deletion or insertion-deletion mutation within or near the TXNIP gene enables the cell, e.g., a universal donor cell, to survive at a higher rate after transplantation or engraftment into a host subject compared to an unmodified cell.

[0062] Tolerogenic factor: As used herein, the term "tolerogenic factor" generally refers to a protein (e.g., expressed by a polynucleotide described herein) that, when increased or decreased in a cell, enables the cell, e.g., a universal donor cell, to inhibit or avoid immune rejection at a higher rate than an unmodified cell after transplantation or engraftment into a host subject. 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., a 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 an unmodified cell after engraftment. In some embodiments, the tolerogenic factor is TNFAIP3 (NCBI Gene ID No: 7128), CD39 (NCBI Gene ID No: 953), CD73 (NCBI Gene ID No: 4907), PD-L-1 (NCBI Gene ID No: 29126), HLA-E (NCBI Gene ID No: 3133), HLA-G (NCBI Gene ID No: 3135), CTLA-4 (NCBI Gene ID No: 1493), or CD47 (NCBI Gene ID No: 961). In some embodiments, the tolerogenic factor is inserted into a cell, e.g., a universal donor cell. In some embodiments, the tolerogenic factor is deleted from a cell, e.g., a universal donor cell. In some embodiments, insertion of a polynucleotide encoding TNFAIP3, CD39, CD73, HLA-E, PD-L-1, HLA-G, CTLA-4, and / or CD47 enables the cells, e.g., universal donor cells, to inhibit or avoid immune rejection following transplantation or engraftment into a host subject.

[0063] MHC-I or MHC-II transcriptional regulator: As used herein, the term "MHC-I or MHC-II transcriptional regulator" generally refers to a biomolecule that modulates, e.g., increases or decreases, expression of MHC-I and / or MHC-II human leukocyte antigens. In some embodiments, the biomolecule 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, deletion or reduction of expression of CIITA or NLRC5 reduces expression of at least one MHC-I or MHC-II gene.

[0064] Universal donor cells: As used herein, the term "universal donor cells" generally refers to genetically modified cells that are less susceptible to allogeneic rejection during cell transplantation and / or exhibit increased survival after transplantation compared to unmodified cells. In some embodiments, the genetically modified cells described herein are universal donor cells. In some embodiments, the universal donor cells have increased immune evasion and / or post-transplant survival compared to unmodified cells. In some embodiments, the universal donor cells have increased cell survival compared to unmodified cells. In some embodiments, the universal donor cells can be stem cells. In some embodiments, the universal donor cells can be embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), or hematopoietic stem or progenitor cells (HSPCs) (also referred to as hematopoietic stem cells (HSCs)). In some embodiments, the universal donor cells can be differentiated cells. In some embodiments, the universal donor cells can be somatic cells (e.g., immune system cells). In some embodiments, the 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 are capable of differentiating into lineage-restricted progenitor cells or fully differentiated somatic cells. In some embodiments, the lineage-restricted progenitor cells are pancreatic endoderm progenitors, pancreatic endocrine progenitors, 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.

[0065] Unmodified cell: As used herein, the term "unmodified cell" refers to a cell that has not been subjected to genetic modification involving 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, the unmodified cell can be a stem cell. In some embodiments, the 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, the unmodified cell can be a differentiated cell. In some embodiments, the unmodified cell can be selected from somatic cells (e.g., immune system cells, e.g., T cells, e.g., CD8+ T cells). When universal donor cells are compared "against unmodified cells," the universal donor cell and the unmodified cell are the same cell type or share a common parental cell line, e.g., universal donor iPSCs are compared to unmodified iPSCs.

[0066] Within or near a gene: As used herein, the term "inside or near a gene" refers to a site or region of genomic DNA that is an intron or exon component of the gene, or that is located proximal 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 may be at the 5' or 3' end of the gene (e.g., at the 5' or 3' end of the coding region of the gene). In some embodiments, a site of genomic DNA located near a gene may be a promoter region or a repressor region that modulates expression of the gene. In some embodiments, a site of genomic DNA located near a gene may 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, within 40 Kb, within 30 Kb, within 20 Kb, within 10 Kb, within 5 Kb, within 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).

[0067] As used herein, the terms "comprising" or "comprises" are inclusive or open-ended and do not exclude additional, unrecited elements, components, or method steps; the phrases "consisting of" or "consists of" are closed and exclude any unspecified elements, steps, or components; and the phrases "consisting essentially of" or "consists essentially" mean that certain additional components may be present, i.e., components that do not substantially affect the essential characteristics of the compound, composition, or method. When used in reference to a sequence, the phrases "consisting essentially of" or "consists essentially" mean that the sequence may include substitutions and / or additional sequences that do not alter the essential function or properties of the sequence. II. Strategies to Evade the Immune Response and Increase Survival

[0068] Described herein are strategies for enabling 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 enable 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 polynucleotide 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 complex, wherein the genetic modification comprises the insertion of a polynucleotide encoding a second tolerogenic factor.

[0069] In some embodiments, the genetically modified cells comprise the introduction within or near at least one gene of at least one genetic modification that reduces expression of one or more MHC-I and MHC-II human leukocyte antigens compared to unmodified cells; at least one genetic modification that increases expression of at least one polynucleotide encoding a tolerogenic factor compared to unmodified cells; and at least one genetic modification that alters expression of at least one gene encoding a survival factor compared to unmodified cells. In other embodiments, the genetically modified cells comprise the introduction within or near at least one gene of at least one deletion or insertion-deletion mutation that alters expression of one or more MHC-I and MHC-II human leukocyte antigens compared 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 deletion site of the gene that alters 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 alters expression of at least one gene encoding a survival factor compared to unmodified cells.

[0070] The 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 divided 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 cell types and present peptides processed by "non-self" antigens 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 robust cellular immune response directed against these cells, ultimately leading to the elimination of the transplanted or engrafted cells by activated cytolytic CD8+ T cells. MHC-I proteins are closely associated with B2M in the endoplasmic reticulum, which is essential for the formation of functional MHC-I molecules on the cell surface. Furthermore, there are three non-classical MHC-Ib molecules (HLA-E, HLA-F, and HLA-G), which 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 engraftment of non-host cells, for example, for regenerative medicine purposes.

[0071] MHC-I cell surface molecules are composed of an MHC-encoded heavy chain (HLA-A, HLA-B, or HLA-C) and the invariant subunit B2M. Therefore, reducing the concentration of intracellular B2M provides an effective method for reducing cell surface expression of MHC-I cell surface molecules.

[0072] In some embodiments, the cell comprises a genomic modification of one or more MHC-I or MHC-II genes. In some embodiments, the cell comprises a genomic modification of one or more polynucleotide sequences that regulate the expression of MHC-I and / or MHC-II. In some embodiments, the genetic modification of the present disclosure is performed using any gene editing method, including but not limited to, the methods described herein.

[0073] 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 at least one base pair in the MHC-I and / or MHC-II gene via gene deletion and / or insertion. 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 the CIITA gene via gene deletion. 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 at least one transcriptional regulator of MHC-I or MHC-II via gene deletion. 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.

[0074] In some embodiments, the genome of the cell has been modified to delete all or part of the HLA-A, HLA-B, and / or HLA-C genes. In some embodiments, the genome of the cell has been modified to delete all or part of the promoter regions of the HLA-A, HLA-B, and / or HLA-C genes. In some embodiments, the genome of the cell has been modified to delete all or part of the genes encoding transcriptional regulators of MHC-I or MHC-II. In some embodiments, the genome of the cell has been modified to delete all or part of the promoter regions of genes encoding transcriptional regulators of MHC-I or MHC-II.

[0075] In some embodiments, the cell's genome is modified to disrupt or reduce expression of beta2-microglobulin (B2M), also known as β2-microglobulin, B2 microglobulin, or IMD43. B2M is a non-polymorphic gene encoding a common protein subunit required for surface expression of all polymorphic MHC class I heavy chains. HLA-I proteins are closely associated with B2M in the endoplasmic reticulum, where it is essential for the formation of functional cell-surface-expressed HLA-I molecules. In some embodiments, the gRNA targets a site in the B2M gene comprising the sequence 5'-GCTACTCTCTCTTTCTGGCC-3' (SEQ ID NO: 1). In some embodiments, the gRNA targets a site in the B2M gene comprising the sequence 5'-GGCCGAGATGTCTCGCTCCG-3' (SEQ ID NO: 2). In some embodiments, the gRNA targets a site in the B2M gene comprising the sequence 5'-CGCGAGCACAGCTAAGGCCA-3' (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: 4), 5'-GAGTAGCGCGAGCACAGCTA-3' (SEQ ID NO: 5), 5'-ACTGGACGCGTCGCGCTGGC-3' (SEQ ID NO: 6), 5'-AAGTGGAGGCGTCGCGCTGG-3' (SEQ ID NO: 7), 5-GGCCACGGAGCGAGACATCT-3' (SEQ ID NO: 8), 5'-GCCCGAATGCTGTCAGCTTC-3' (SEQ ID NO: 9), 5'-CTCGCGCTACTCTCTCTTTC-3' (SEQ ID NO: 10), 5'-TCCTGAAGCTGACAGCATTC-3' (SEQ ID NO: 11), 5'-TTCCTGAAGCTGACAGCATT-3' (SEQ ID NO: 12), or 5'-ACTCTCTCTTTCTGGCCTGG-3' (SEQ ID NO: 13). In some embodiments, the gRNA comprises an RNA version of the polynucleotide sequence of SEQ ID NO: 2. In other embodiments, the gRNA comprises an RNA version of any of SEQ ID NOs: 1 or 3-13. The gRNA / CRISPR nuclease complex targets and cleaves the target site in the B2M locus.The repair of double-strand break by NHEJ results in the deletion of at least one nucleotide and / or the insertion of at least one nucleotide, thereby disrupting or eliminating the expression of B2M. Alternatively, the B2M locus can be targeted by at least two CRISPR systems, each containing a different gRNA, thereby cutting two sites in the B2M locus, leading to the deletion of the sequence between the two cuts, thereby eliminating the expression of B2M.

[0076] In some embodiments, the genetically modified cells contain at least one genetic modification that disrupts the expression of at least one gene encoding a survival factor, such as TXNIP, compared to unmodified cells. In some embodiments, the genome of the cells is modified to reduce expression of thioredoxin-interacting protein (TXNIP), also known as EST01027, HHCPA78, THIF, VDUP1, or ARRDC6. TXNIP is a metabolic gene involved in regulating redox and may also function as a tumor suppressor. Downregulation or knockout of TXNIP can protect cells from metabolic stress. In some embodiments, the gRNA targets a site in the TXNIP gene that contains the sequence 5'-GAAGCGTGTCTTCATAGCGC-3' (SEQ ID NO: 32). In some embodiments, the gRNA targets a site in the TXNIP gene that contains the sequence 5'-TTACTCGTGTCAAAGCCGTT-3' (SEQ ID NO: 33). In some embodiments, the gRNA targets a site in the TXNIP gene comprising the sequence 5'-TGTCAAAGCCGTTAGGATCC-3' (SEQ ID NO: 34). In some embodiments, the gRNA targets a site in the TXNIP gene comprising the sequence 5'-GCCGTTAGGATCCTGGCTTG-3' (SEQ ID NO: 35). In some embodiments, the gRNA targets a site in the TXNIP gene comprising the sequence 5'-GCGGAGTGGCTAAAGTGCTT-3' (SEQ ID NO: 36). In some embodiments, the gRNA targets a site in the TXNIP gene comprising the sequence 5'-TCCGCAAGCCAGGATCCTAA-3' (SEQ ID NO: 37). In some embodiments, the gRNA targets a site in the TXNIP gene comprising the sequence 5'-GTTCGGCTTTGAGCTTCCTC-3' (SEQ ID NO: 38). In some embodiments, the gRNA targets a site in the TXNIP gene comprising the sequence 5'-GAGATGGTGATCATGAGACC-3' (SEQ ID NO: 39). In some embodiments, the gRNA targets a site in the TXNIP gene that includes the sequence 5'-TTGTACTCATATTTGTTTCC-3' (SEQ ID NO: 40).In some embodiments, the gRNA targets a site in the TXNIP gene containing the sequence 5'-AACAAATATGAGTACAAGTT-3' (SEQ ID NO: 41). In some embodiments, the gRNA comprises an RNA version of the polynucleotide sequence of SEQ ID NO: 37. In other embodiments, the gRNA comprises an RNA version of any one of SEQ ID NOs: 32-36 or 38-41. The gRNA / CRISPR nuclease complex targets and cleaves the target site in the TXNIP locus. Repair of the double-strand break by NHEJ results in the deletion of at least one nucleotide and / or the insertion of at least one nucleotide, thereby disrupting or eliminating TXNIP expression. Alternatively, TXNIP expression can be disrupted or eliminated by inserting a polynucleotide encoding an exogenous gene into the TXNIP locus.

[0077] In some embodiments, the cell's genome is modified to disrupt expression of class II transactivator (CIITA), also known as C2TA, CIITAIV, MHC2TA, NLRA, or class II major histocompatibility complex transactivator. CIITA is a master regulator of major histocompatibility complex (MHC) gene expression. CIITA is a member of the nucleotide-binding domain (NBD) leucine-rich repeat (LRR) family of proteins and 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. In some embodiments, the gRNA targets a site in the CIITA gene comprising 5'-GGTCCATCTGGTCATAGAAG-3' (SEQ ID NO: 25). In some embodiments, the gRNA targets a site in the CIITA gene comprising 5'-GCTCCAGGTAGCCACCTTCT-3' (SEQ ID NO: 48). In some embodiments, the gRNA targets a site in the CIITA gene comprising 5'-TAGGGGCCCCAACTCCATGG-3' (SEQ ID NO: 49). In some embodiments, the gRNA targets a site in the CIITA gene comprising 5'-GGCTTATGCCAATATCGGTG-3' (SEQ ID NO: 50). In some embodiments, the gRNA targets a site in the CIITA gene comprising 5'-AGGTGATGAAGAGACCAGGG-3' (SEQ ID NO: 51). In some embodiments, the gRNA comprises an RNA version of the sequence of SEQ ID NO: 25. The gRNA / CRISPR nuclease complex targets and cleaves the target site in the CIITA locus. Repair of the double-strand break by NHEJ results in the deletion of at least one nucleotide and / or the insertion of at least one nucleotide, thereby disrupting or eliminating expression of CIITA. Alternatively, expression of CIITA can be disrupted or eliminated by inserting a polynucleotide encoding an exogenous gene into the CIITA locus.

[0078] In some embodiments, the genome of the cells is modified to disrupt expression of TGF-β2, also known as TGFB2, transforming growth factor beta 2, glioblastoma-derived T-cell suppressor factor, transforming growth factor beta 2 proprotein prepro-transforming growth factor beta 2, cetermin, G-TSF, transforming growth factor beta 2, BSC-1 cell growth inhibitory factor 3, TGF-beta 2, polyergin, and LDS4. The gene encodes a secreted ligand of the TGF-β2 superfamily of proteins. TGF-β2 is a key activator of fibroblasts, a central effector of the fibrotic response, and promotes a fibrogenic phenotype in immune and vascular cells. Disruption of TGF-β2 expression can improve long-term survival of engrafted universal donor cells. In some embodiments, the genome of the cells is modified to disrupt the TGF-β2 gene. The gRNA targets a site in the TGF-β2 gene that contains 5'-GTTCATGCGCAAGAGGATCG-3' (SEQ ID NO: 57). The gRNA / CRISPR nuclease complex targets and cleaves the target site in the TGF-β2 locus. Repair of the double-strand break by NHEJ results in the deletion of at least one nucleotide and / or the insertion of at least one nucleotide, thereby disrupting or eliminating the expression of TGF-β2. Alternatively, the expression of TGF-β2 can be disrupted or eliminated by inserting a polynucleotide encoding an exogenous gene into the TGF-β2 locus.

[0079] In some embodiments, the genome of the cell is modified to reduce the expression of NLR family, CARD domain-containing 5 (NLRC5). NLRC5 is a crucial regulator of MHC-I-mediated immune responses, and like CIITA, NLRC5 is highly inducible by IFN-γ and can be translocated to the nucleus. NLRC5 activates the promoter of MHC-I genes and induces the transcription of MHC-I and related genes involved in MHC-I antigen presentation.

[0080] In some embodiments, polynucleotides encoding one or more tolerogenic factors can be inserted into cells, e.g., genetically modified or non-genetically modified cells, to create immune-privileged universal donor cells. Exemplary tolerogenic factors include, but are not limited to, one or more of TNFAIP3, CD39, PD-L-1, HLA-E, CD73, HLA-C, HLA-F, HLA-G, CTLA-4-Ig, CD47, CI inhibitor, and IL-35. In some embodiments, the tolerogenic factor is TNFA1P3 or A20, also known as OTUD7C, TNFA1P2, AISBL, or TNF-alpha-induced protein 3. TNFA1P3 or A20 is a key regulator of inflammation and immunity and is known to inhibit NF-kappaB activation and TNF-mediated apoptosis. In some embodiments, the tolerogenic factor is CD39, which is also known as ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1), NTPDase1, ATPDase, or SPG64. CD39 is a tolerogenic factor, but may also provide benefits through increased angiogenesis, anti-inflammatory activity, and / or other means. In some embodiments, the tolerogenic factor is PD-L-1 (programmed death-ligand 1), which is also known as cluster of differentiation 274 (CD274), B7 homolog (B7-H, B7H1), PDCD1L1, PDCD1LG1, or PDL1. PD-L-1 appears to play a major role in suppressing the adaptive arm of the immune system and is thought to be a co-inhibitor of the immune response. In some embodiments, the tolerogenic factor is HLA-E, which is also known as EA1.2, EA2.1, HLA-6.2, MHC, QA1, or major histocompatibility complex, class I, E. HLA-E is a key modulator of natural killer (NK) and cytotoxic T lymphocyte (CTL) activation and inhibitory function.In some embodiments, the tolerogenic factor is CD73, also known as 5'-nucleotidase ecto (NT5E), 5'-nucleotidase (5'-NT), ecto-5'-nucleotidase, ENT, EN, NT5, NTE, or E5NT. CD73 is a plasma membrane protein that catalyzes the conversion of AMP to adenosine. CD73-derived adenosine promotes ectopic differentiation of dendritic cells (DCs) by activating the A2b receptor on DCs, thereby promoting a tolerogenic phenotype characterized by increased production of IL-6, IL-10, VEGF, and IL-8 and expression of immunosuppressive proteins such as IDO, TGF-β, arginase 2, and COX2. In some embodiments, the genetic modification, e.g., the insertion of at least one polynucleotide encoding at least one tolerogenic factor, enables the universal donor cells to inhibit or avoid immune rejection after engraftment at a rate that is 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 greater than that of unmodified cells. In some embodiments, the insertion of polynucleotides encoding TNFAIP3, CD39, PD-L-1, HLA-E, CD73, HLA-G, CTLA-4, and / or CD47 enables the universal donor cells to inhibit or avoid immune rejection after transplantation or engraftment into a host subject.

[0081] In some embodiments, a polynucleotide encoding one or more survival factors, such as MANF, can be inserted into genetically modified or unmodified cells to create universal donor cells with increased survival. In some embodiments, the survival factor is MANF, also known as arginine-rich mutated in primary tumors (ARMET), arginine-rich protein (ARP), or midbrain astrocyte-derived neurotrophic factor. MANF is an endoplasmic reticulum (ER) stress-induced neurotrophic factor that promotes the proliferation and survival of pancreatic beta cells and the survival of dopaminergic neurons. In some embodiments, insertion of a polynucleotide encoding one or more survival factors, such as MANF, enables universal donor cells to survive after transplantation or engraftment into a host subject 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 transplantation or engraftment.

[0082] Polynucleotides encoding tolerogenic and / or survival factors generally contain a left homologous arm and a right homologous arm flanking the nucleotide sequence encoding the tolerogenic factor. The homologous arms have substantial sequence homology to genomic DNA at or near the targeted insertion site. For example, the left homologous arm can be a nucleotide sequence homologous to a region located to the left of or upstream of the target or cut site, and the right homologous arm can be a nucleotide sequence homologous to a region located to the right of or downstream of the target or cut site. The proximal end of each homologous arm can be homologous to a genomic DNA sequence adjacent to the cut site. Alternatively, the proximal end of each homologous 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 cut site. Thus, a polynucleotide encoding a tolerogenic factor can be inserted into or replace a targeted locus within about 10, 20, 30, 40, 50, 60, or 70 base pairs of the cut site, and additional genomic DNA flanking the cut site (and not sharing homology with the homologous arm) can be deleted. The homologous arm can range in length from about 50 nucleotides to several thousand nucleotides. In some embodiments, the homologous arm can range in length from about 500 to about 1,000 nucleic acid bases. In some embodiments, the homologous arm is about 700, about 800, or about 900 nucleic acid bases in length. In some embodiments, the homologous arm is about 800 nucleic acid bases in length. The substantial sequence homology between the homologous arm 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%. In some embodiments, the homologous arm is identical to the genomic DNA.

[0083] In some embodiments, homology arms are used with B2M guides (e.g., gRNAs comprising RNA versions of SEQ ID NOs: 1-13). In some embodiments, homology arms are designed for use with any B2M guide that eliminates the start site of the B2M gene. In some embodiments, the B2M homology arm can comprise or essentially consist of the nucleotide sequence of SEQ ID NO: 15 or 22, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 15 or 22. In some embodiments, the left B2M homology arm can comprise or essentially consist of the nucleotide sequence of SEQ ID NO: 15, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the right B2M homology arm can comprise or essentially consist of the nucleotide sequence of SEQ ID NO: 22, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 22.

[0084] In some embodiments, homology arms are used with TXNIP guides (e.g., gRNAs containing RNA versions of SEQ ID NOs: 32-41). In some embodiments, homology arms are designed for use with any TXNIP guide that targets exon 1 of TXNIP (e.g., gRNAs containing RNA versions of SEQ ID NOs: 32-41). In some embodiments, a TXNIP homology arm can comprise or consist essentially of the nucleotide sequence of SEQ ID NO: 26 or 28, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 26 or 28. In some embodiments, the left TXNIP homology arm can comprise or consist essentially of the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 26. In some embodiments, the right TXNIP homology arm may comprise or essentially consist of the nucleotide sequence of SEQ ID NO:28, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:28.

[0085] In some embodiments, homology arms are used with CIITA guides (e.g., gRNAs comprising RNA versions of SEQ ID NOs: 25 or 48-51). In some embodiments, the CIITA homology arm may comprise or essentially consist of the nucleotide sequence of SEQ ID NO: 42 or 44, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 42 or 44. In some embodiments, the left CIITA homology arm may comprise or essentially consist of the nucleotide sequence of SEQ ID NO: 42, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 42. In some embodiments, the right CIITA homology arm may comprise or essentially consist of the nucleotide sequence of SEQ ID NO: 44, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 44.

[0086] In some embodiments, homology arms are used with a TGF-β2 guide (e.g., a gRNA targeting a target sequence comprising SEQ ID NO: 57).

[0087] At least one polynucleotide encoding at least one tolerogenic factor and / or survival factor may contain one or more ribosomal skip-encoding sequences, such that upon expression, a single transcript is produced, but two or more separate proteins are produced due to ribosomal skipping during translation. In some embodiments, the ribosomal skip may be a short peptide (approximately 20 amino acids) that prevents the ribosome from creating a peptide bond between glycine and proline at the C-terminus of a growing polypeptide chain. The ribosome pauses after the glycine, resulting in the release of the nascent polypeptide chain. Translation resumes, and proline becomes the first amino acid of the second polypeptide chain. This mechanism results in apparent co-translational cleavage of the polypeptide. A highly conserved sequence at the C-terminus of the ribosomal skip peptide contributes to steric hindrance and ribosomal skipping. In some embodiments, the ribosomal skip peptide is a member of the 2A sequence family. Suitable 2A sequence family members include F2A, T2A, E2A, and P2A, where F2A is derived from foot-and-mouth disease virus 2A, T2A is derived from thosea asigna virus 2A, E2A is derived from equine rhinitis A virus, and P2A is derived from porcine teschovirus-1 2A. In some embodiments, the ribosomal skip peptide is P2A. In some embodiments, the sequence encoding ribosomal skip P2A comprises or consists of the nucleotide sequence of SEQ ID NO: 18. In other embodiments, the ribosomal skip can be an internal ribosome entry sequence (IRES), which is an RNA element that allows translation initiation in a cap-independent manner. Thus, the IRES allows the production of two separate proteins from a single transcription unit. IRES elements are well known in the art and can be derived, for example, from viral genomes (e.g., picornavirus, aphthovirus, pestivirus IRES) or cellular mRNAs (e.g., various growth factors, transcription factors, oncogenes, etc.).

[0088] At least one polynucleotide encoding at least one tolerogenic factor and / or survival factor can be operably linked to an exogenous promoter. The exogenous promoter can be a constitutive promoter, an inducible promoter, a time-specific promoter, a tissue-specific promoter, or a cell-type-specific promoter. In some embodiments, the exogenous promoter is a CMV, EF1a, PGK, CAG / CAGGS, or UBC promoter. Generally, the CAG or CAGGS promoter comprises a CMV enhancer, a chicken β-actin promoter, and a chimeric intron. In some embodiments, the CAG or CAGGS promoter comprises or essentially consists of the nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 16.

[0089] In some embodiments, at least one polynucleotide encoding at least one tolerogenic factor and / or survival factor can be inserted into a safe harbor locus, e.g., the AAVS 1 locus. In some embodiments, at least one polynucleotide encoding at least one tolerogenic factor and / or survival 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, a transcriptional regulator of MHC-I or MHC-II, or a survival factor gene.

[0090] In certain embodiments, the present disclosure contemplates universal donor cells having one or more insertions of exogenous polynucleotides corresponding to any of the genes listed as knock-ins in Table 1, and / or disruption of expression of one or more of the genes listed as knock-outs in Table 1. The engineered universal donor cells may include an insertion of one polynucleotide, any two polynucleotides, any three polynucleotides, any four polynucleotides, any five polynucleotides, or all six polynucleotides corresponding to the genes listed in Table 1 at any target genomic location (e.g., a safe harbor location), and / or the engineered universal donor cells may include disruption of expression (e.g., reduced or eliminated expression) of one, two, three, or four of the target genes listed in Table 1. The cells may include any possible combination of the listed gene knock-ins and gene knock-outs. In some embodiments, two or more inserted polynucleotides can be linked via one or more sequences encoding ribosomal skips, such as the 2A peptide, thus allowing two or more separate proteins to be expressed from a single RNA transcript. In some embodiments, the polynucleotide or polynucleotides to be inserted into the genome of a cell are operably linked to an exogenous promoter. [Table 1]

[0091] In some embodiments, the polynucleotide encoding PD-L-1 is inserted at a site within or near the B2M locus, within or near the TXNIP locus, within or near the CIITA locus, or within or near the TGF-β2 locus. In some embodiments, the polynucleotide encoding PD-L-1 is inserted at a site within or near the B2M locus, within or near the TXNIP locus, or within or near the CIITA locus. In some embodiments, the polynucleotide encoding PD-L-1 is inserted at a site within or near the B2M locus. In some embodiments, the polynucleotide encoding PD-L-1 is inserted at a site within or near the B2M locus simultaneously with or after deletion of all or a portion of the B2M gene or promoter. In some embodiments, the polynucleotide encoding PD-L-1 is inserted at a site within or near the TXNIP locus simultaneously with or after deletion of all or a portion of the TXNIP gene or promoter. In some embodiments, a polynucleotide encoding PD-L-1 is inserted into a site within or near the CIITA locus simultaneously with or after deletion of all or a portion of the CIITA gene or promoter. In other embodiments, a polynucleotide encoding PD-L-1 is inserted into a site within or near the TGF-β2 locus simultaneously with or after deletion of all or a portion of the TGF-β2 gene or promoter. The polynucleotide encoding PD-L-1 is operably linked to an exogenous promoter. The exogenous promoter may be a CAG or CAGGS promoter. In some embodiments, the polynucleotide encoding PD-L-1 comprises the nucleotide sequence of SEQ ID NO:20, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:20.

[0092] In some embodiments, a polynucleotide encoding TNFAIP3 is inserted at a site within or near the B2M locus, within or near the TXNIP locus, within or near the CIITA locus, or within or near the TGF-β2 locus. In some embodiments, a polynucleotide encoding TNFAIP3 is inserted at a site within or near the B2M locus, within or near the TXNIP locus, or within or near the CIITA locus. In some embodiments, a polynucleotide encoding TNFAIP3 is inserted at a site within or near the B2M locus. In some embodiments, a polynucleotide encoding TNFAIP3 is inserted at a site within or near the B2M locus simultaneously with or after deletion of all or a portion of the B2M gene or promoter. In some embodiments, a polynucleotide encoding TNFAIP3 is inserted at a site within or near the TXNIP locus simultaneously with or after deletion of all or a portion of the TXNIP gene or promoter. In some embodiments, a polynucleotide encoding TNFAIP3 is inserted into a site within or near the CIITA locus simultaneously with or after deletion of all or part of the CIITA gene or promoter. In other embodiments, a polynucleotide encoding TNFAIP3 is inserted into a site within or near the TGF-β2 locus simultaneously with or after deletion of all or part of the TGF-β2 gene or promoter. The polynucleotide encoding TNFAIP3 is operably linked to an exogenous promoter. The exogenous promoter can be a CAG or CAGGS promoter. In some embodiments, the polynucleotide encoding TNFAIP3 comprises the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 19.

[0093] In some embodiments, a polynucleotide encoding MANF is inserted into a site within or near the B2M locus, within or near the TXNIP locus, within or near the CIITA locus, or within or near the TGF-β2 locus. In some embodiments, a polynucleotide encoding MANF is inserted into a site within or near the B2M locus, within or near the TXNIP locus, or within or near the CIITA locus. In some embodiments, a polynucleotide encoding MANF is inserted into a site within or near the B2M locus. In some embodiments, a polynucleotide encoding MANF is inserted into a site within or near the B2M locus simultaneously with or after deletion of all or a portion of the B2M gene or promoter. In other embodiments, a polynucleotide encoding MANF is inserted into a site within or near the TXNIP locus simultaneously with or after deletion of all or a portion of the TXNIP gene or promoter. In some embodiments, a polynucleotide encoding MANF is inserted into a site within or near the CIITA locus simultaneously with or after the deletion of all or part of the CIITA gene or promoter. In other embodiments, a polynucleotide encoding MANF is inserted into a site within or near the TGF-β2 locus simultaneously with or after the deletion of all or part of the TGF-β2 gene or promoter. The polynucleotide encoding MANF is operably linked to an exogenous promoter. The exogenous promoter can be a CAG or CAGGS promoter. In some embodiments, the polynucleotide encoding MANF comprises the nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 17.

[0094] In some embodiments, a polynucleotide encoding CD39 is inserted at a site within or near the B2M locus, within or near the TXNIP locus, within or near the CIITA locus, or within or near the TGF-β2 locus. In some embodiments, a polynucleotide encoding CD39 is inserted at a site within or near the B2M locus, within or near the TXNIP locus, or within or near the CIITA locus. In some embodiments, a polynucleotide encoding CD39 is inserted at a site within or near the CIITA locus or within or near the B2M locus. In some embodiments, a polynucleotide encoding CD39 is inserted at a site within or near the B2M locus simultaneously with or after deletion of all or a portion of the B2M gene or promoter. In other embodiments, a polynucleotide encoding CD39 is inserted at a site within or near the TXNIP locus simultaneously with or after deletion of all or a portion of the TXNIP gene or promoter. In some embodiments, a polynucleotide encoding CD39 is inserted into a site within or near the CIITA locus simultaneously with or after deletion of the CIITA gene or promoter. In other embodiments, a polynucleotide encoding CD39 is inserted into a site within or near the TGF-β2 locus simultaneously with or after deletion of all or part of the TGF-β2 gene or promoter. The polynucleotide encoding CD39 is operably linked to an exogenous promoter. The exogenous promoter can be a CAG or CAGGS promoter. In some embodiments, the polynucleotide encoding CD39 comprises the nucleotide sequence of SEQ ID NO:27 or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:27.

[0095] In some embodiments, a polynucleotide encoding HLA-E is inserted into a site within or near the B2M locus, within or near the TXNIP locus, within or near the CIITA locus, or within or near the TGF-β2 locus. In some embodiments, a polynucleotide encoding HLA-E is inserted into a site within or near the B2M locus, within or near the TXNIP locus, or within or near the CIITA locus. In some embodiments, a polynucleotide encoding HLA-E is inserted into a site within or near the TXNIP locus. In some embodiments, a polynucleotide encoding HLA-E is inserted into a site within or near the B2M locus simultaneously with or after deletion of all or a portion of the B2M gene or promoter. In other embodiments, a polynucleotide encoding HLA-E is inserted into a site within or near the TXNIP locus simultaneously with or after deletion of all or a portion of the TXNIP gene or promoter. In some embodiments, a polynucleotide encoding HLA-E is inserted into a site within or near the CIITA locus simultaneously with or after deletion of the CIITA gene or promoter. In other embodiments, a polynucleotide encoding HLA-E is inserted into a site within or near the TGF-β2 locus simultaneously with or after deletion of all or part of the TGF-β2 gene or promoter. The polynucleotide encoding HLA-E comprises a sequence encoding an HLA-E trimer, the HLA-E trimer comprising a B2M membrane protein fused to HLA-E without a signal peptide, an HLA-G-presenting peptide fused thereto, and a B2M signal peptide fused thereto. The polynucleotide encoding HLA-E is operably linked to an exogenous promoter. The exogenous promoter may be a CMV promoter. In some embodiments, the polynucleotide encoding HLA-E comprises the nucleotide sequence of SEQ ID NO: 43 or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 43.

[0096] In some embodiments, a polynucleotide encoding CD73 is inserted at a site within or near the B2M locus, within or near the TXNIP locus, within or near the CIITA locus, or within or near the TGF-β2 locus. In some embodiments, a polynucleotide encoding CD73 is inserted at a site within or near the B2M locus, within or near the TXNIP locus, or within or near the CIITA locus. In some embodiments, a polynucleotide encoding CD73 is inserted at a site within or near the B2M locus or within or near the CIITA locus. In some embodiments, a polynucleotide encoding CD73 is inserted at a site within or near the B2M locus simultaneously with or after deletion of the B2M gene or promoter. In some embodiments, a polynucleotide encoding CD73 is inserted at a site within or near the TXNIP locus simultaneously with or after deletion of the TXNIP gene or promoter. In some embodiments, a polynucleotide encoding CD73 is inserted into a site within or near the CIITA locus simultaneously with or after deletion of the CIITA gene or promoter. In other embodiments, a polynucleotide encoding CD73 is inserted into a site within or near the TGF-β2 locus simultaneously with or after deletion of all or part of the TGF-β2 gene or promoter. The polynucleotide encoding CD73 is operably linked to an exogenous promoter. The exogenous promoter can be a CAG or CAGGS promoter. In some embodiments, the polynucleotide encoding CD73 comprises the nucleotide sequence of SEQ ID NO:46, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:46.

[0097] In some embodiments, polynucleotides encoding MANF, TNFAIP3, and PD-L-1 are inserted into a site within or near the B2M locus, within or near the TXNIP locus, within or near the CIITA locus, or within or near the TGF-β2 locus. In some embodiments, polynucleotides encoding MANF, TNFAIP3, and PD-L-1 are inserted into a site within or near the B2M locus, within or near the TXNIP locus, or within or near the CIITA locus. In some embodiments, polynucleotides encoding MANF, TNFAIP3, and PD-L-1 are inserted into a site within or near the B2M locus. In some embodiments, polynucleotides encoding MANF, TNFAIP3, and PD-L-1 are inserted into a site within or near the B2M locus simultaneously with or after deletion of all or part of the B2M gene or promoter. In some embodiments, polynucleotides encoding MANF, TNFAIP3, and PD-L-1 are inserted into a site within or near the TXNIP locus simultaneously with or after deletion of all or a portion of the TXNIP gene or promoter. In some embodiments, polynucleotides encoding MANF, TNFAIP3, and PD-L-1 are inserted into a site within or near the CIITA locus simultaneously with or after deletion of all or a portion of the CIITA gene or promoter. In other embodiments, polynucleotides encoding MANF, TNFAIP3, and PD-L-1 are inserted into a site within or near the TGF-β2 locus simultaneously with or after deletion of all or a portion of the TGF-β2 gene or promoter. The polynucleotides encoding MANF, TNFAIP3, and PD-L-1 comprise a sequence encoding MANF linked to a sequence encoding a first ribosomal skip linked to a sequence encoding TNFAIP3, and a sequence encoding a second ribosomal skip linked to a sequence encoding PD-L-1. The first and second ribosomal skips can be 2A sequence family members, for example, both can be P2A.In some embodiments, the polynucleotide comprises a MANF-P2A-TNFAIP3-P2A-PD-L-1 coding sequence. In some embodiments, the polynucleotide encoding MANF-P2A-TNFAIP3-P2A-PD-L-1 comprises or consists of the nucleotide sequence of SEQ ID NO:52, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:52. In some embodiments, the polynucleotide encoding MANF-P2A-TNFAIP3-P2A-PD-L-1 is operably linked to an exogenous promoter. The exogenous promoter may be a CAG or CAGGS promoter. In some embodiments, the donor vector encoding MANF-P2A-TNFAIP3-P2A-PD-L-1 and comprising B2M homology arms has the nucleotide sequence of SEQ ID NO:24. In some embodiments, the donor vector encodes MANF-P2A-TNFAIP3-P2A-PD-L-1 and comprises a TXNIP homology arm. In some embodiments, the donor vector encodes MANF-P2A-TNFAIP3-P2A-PD-L-1 and comprises a CTIIA homology arm. In some embodiments, the donor vector encodes MANF-P2A-TNFAIP3-P2A-PD-L-1 and comprises a TGF-β2 homology arm.

[0098] In some embodiments, polynucleotides encoding TNFAIP3 and PD-L-1 are inserted at a site within or near the B2M locus, within or near the TXNIP locus, within or near the CIITA locus, or within or near the TGF-β2 locus. In some embodiments, polynucleotides encoding TNFAIP3 and PD-L-1 are inserted at a site within or near the B2M locus, within or near the TXNIP locus, or within or near the CIITA locus. In some embodiments, polynucleotides encoding TNFAIP3 and PD-L-1 are inserted at a site within or near the B2M locus. In some embodiments, polynucleotides encoding TNFAIP3 and PD-L-1 are inserted at a site within or near the B2M locus simultaneously with or after deletion of all or part of the B2M gene or promoter. In some embodiments, polynucleotides encoding TNFAIP3 and PD-L-1 are inserted into a site within or near the TXNIP locus simultaneously with or after deletion of all or a portion of the TXNIP gene or promoter. In some embodiments, polynucleotides encoding TNFAIP3 and PD-L-1 are inserted into a site within or near the CIITA locus simultaneously with or after deletion of all or a portion of the CIITA gene or promoter. In other embodiments, polynucleotides encoding TNFAIP3 and PD-L-1 are inserted into a site within or near the TGF-β2 locus simultaneously with or after deletion of all or a portion of the TGF-β2 gene or promoter. The polynucleotides encoding TNFAIP3 and PD-L-1 comprise a sequence encoding TNFAIP3 linked to a sequence encoding a ribosomal skip linked to a sequence encoding PD-L-1. The ribosomal skip can be a 2A sequence family member, such as P2A. In some embodiments, the polynucleotide comprises a TNFAIP3-P2A-PD-L-1 coding sequence.In some embodiments, the polynucleotide encoding TNFAIP3-P2A-PD-L-1 comprises or consists of the nucleotide sequence of SEQ ID NO:54, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:54. In some embodiments, the polynucleotide encoding TNFAIP3-P2A-PD-L-1 is operably linked to an exogenous promoter. The exogenous promoter may be a CAG or CAGGS promoter. In some embodiments, the donor plasmid encoding TNFAIP3-P2A-PD-L-1 and comprising B2M homology arms has the nucleotide sequence of SEQ ID NO:31. In some embodiments, the donor plasmid encoding TNFAIP3-P2A-PD-L-1 and comprising TXNIP homology arms. In some embodiments, the donor plasmid encoding TNFAIP3-P2A-PD-L-1 and comprising CIITA homology arms. In some embodiments, the donor vector encodes TNFAIP3-P2A-PD-L-1 and contains TGF-β2 homology arms.

[0099] In some embodiments, polynucleotides encoding MANF and HLA-E are inserted into a site within or near the B2M locus, within or near the TXNIP locus, within or near the CIITA locus, or within or near the TGF-β2 locus. In some embodiments, polynucleotides encoding MANF and HLA-E are inserted into a site within or near the B2M locus or within or near the TXNIP locus. In some embodiments, polynucleotides encoding MANF and HLA-E are inserted into a site within or near the TXNIP locus. In some embodiments, polynucleotides encoding MANF and HLA-E are inserted into a site within or near the B2M locus simultaneously with or after deletion of all or a portion of the B2M gene or promoter. In some embodiments, polynucleotides encoding MANF and HLA-E are inserted into a site within or near the TXNIP locus simultaneously with or after deletion of all or a portion of the TXNIP gene or promoter. In some embodiments, polynucleotides encoding MANF and HLA-E are inserted into a site within or near the CIITA locus simultaneously with or after deletion of all or part of the CIITA gene or promoter. In other embodiments, polynucleotides encoding HLA-E are inserted into a site within or near the TGF-β2 locus simultaneously with or after deletion of all or part of the TGF-β2 gene or promoter. The polynucleotides encoding MANF and HLA-E comprise a sequence encoding MANF linked to a sequence encoding a ribosomal skip linked to a sequence encoding HLA-E. The ribosomal skip may be a 2A sequence family member, such as P2A. The sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer, which comprises a B2M membrane protein fused to HLA-E without a signal peptide, an HLA-G-presenting peptide fused to it, and a B2M signal peptide fused to it. In some embodiments, the polynucleotide comprises a MANF-P2A-HLA-E coding sequence.In some embodiments, the polynucleotide encoding MANF-P2A-HLA-E comprises or consists of the nucleotide sequence of SEQ ID NO: 55 or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 55. In some embodiments, the polynucleotide encoding MANF-P2A-HLA-E is operably linked to an exogenous promoter. The exogenous promoter may be a CAG or CAGGS promoter. In some embodiments, the donor plasmid encoding MANF-P2A-HLA-E and comprising TXNIP homology arms has the nucleotide sequence of SEQ ID NO: 45. In some embodiments, the donor plasmid encoding MANF-P2A-HLA-E and comprising B2M homology arms. In some embodiments, the donor plasmid encoding MANF-P2A-HLA-E and comprising CIITA homology arms. In some embodiments, the donor plasmid encoding MANF-P2A-HLA-E and comprising TGF-β2 homology arms.

[0100] In some embodiments, polynucleotides encoding CD39 and PD-L-1 are inserted at a site within or near the B2M locus, within or near the TXNIP locus, within or near the CIITA locus, or within or near the TGF-β2 locus. In some embodiments, polynucleotides encoding CD39 and PD-L-1 are inserted at a site within or near the B2M locus, within or near the TXNIP locus, or within or near the CIITA locus. In some embodiments, polynucleotides encoding CD39 and PD-L-1 are inserted at a site within or near the B2M locus. In some embodiments, polynucleotides encoding CD39 and PD-L-1 are inserted at a site within or near the B2M locus simultaneously with or after deletion of all or a portion of the B2M gene or promoter. In some embodiments, polynucleotides encoding CD39 and PD-L-1 are inserted at a site within or near the TXNIP locus simultaneously with or after deletion of all or a portion of the TXNIP gene or promoter. In some embodiments, polynucleotides encoding CD39 and PD-L-1 are inserted into a site within or near the CIITA locus, simultaneously with or after deletion of all or part of the CIITA gene or promoter. In other embodiments, polynucleotides encoding CD39 and PD-L-1 are inserted into a site within or near the TGF-β2 locus, simultaneously with or after deletion of all or part of the TGF-β2 gene or promoter. The polynucleotides encoding CD39 and PD-L-1 comprise a sequence encoding CD39 linked to a sequence encoding a ribosomal skip linked to a sequence encoding PD-L-1. The ribosomal skip may be a 2A sequence family member, such as P2A. In some embodiments, the polynucleotide comprises a CD39-P2A-PD-L-1 coding sequence.In some embodiments, the polynucleotide encoding CD39-P2A-PD-L-1 comprises or consists of the nucleotide sequence of SEQ ID NO:53, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:53. In some embodiments, the polynucleotide encoding CD39-P2A-PD-L-1 is operably linked to an exogenous promoter. The exogenous promoter may be a CAG or CAGGS promoter. In some embodiments, the donor plasmid encoding CD39-P2A-PD-L-1 and comprising B2M homology arms has the nucleotide sequence of SEQ ID NO:30. In some embodiments, the donor plasmid encoding CD39-P2A-PD-L-1 and comprising TXNIP homology arms. In some embodiments, the donor plasmid encoding CD39-P2A-PD-L-1 and comprising CIITA homology arms. In some embodiments, the donor plasmid encodes CD39-P2A-PD-L-1 and contains TGF-β2 homology arms.

[0101] In some embodiments, polynucleotides encoding CD39, CD73, and PD-L-1 are inserted at a site within or near the B2M locus, within or near the TXNIP locus, within or near the CIITA locus, or within or near the TGF-β2 locus. In some embodiments, polynucleotides encoding CD39, CD73, and PD-L-1 are inserted at a site within or near the B2M locus, within or near the TXNIP locus, or within or near the CIITA locus. In some embodiments, polynucleotides encoding CD39, CD73, and PD-L-1 are inserted at a site within or near the B2M locus. In some embodiments, polynucleotides encoding CD39, CD73, and PD-L-1 are inserted at a site within or near the B2M locus simultaneously with or after deletion of all or part of the B2M gene or promoter. In some embodiments, polynucleotides encoding CD39, CD73, and PD-L-1 are inserted into a site within or near the TXNIP locus simultaneously with or after deletion of all or a portion of the TXNIP gene or promoter. In some embodiments, polynucleotides encoding CD39, CD73, and PD-L-1 are inserted into a site within or near the CIITA locus simultaneously with or after deletion of all or a portion of the CIITA gene or promoter. In other embodiments, polynucleotides encoding CD39, CD73, and PD-L-1 are inserted into a site within or near the TGF-β2 locus simultaneously with or after deletion of all or a portion of the TGF-β2 gene or promoter. The polynucleotides encoding CD39, CD73, and PD-L-1 comprise a sequence encoding CD39 linked to a sequence encoding a ribosomal skip linked to a sequence encoding CD73, and a sequence encoding a ribosomal skip linked to a sequence encoding PD-L-1. The ribosomal skip can be a 2A sequence family member, such as P2A. In some embodiments, the polynucleotide comprises a CD39-P2A-CD73-P2A-PD-L-1 coding sequence.In some embodiments, the polynucleotide encoding CD39-P2A-CD73-P2A-PD-L-1 comprises or consists of the nucleotide sequence of SEQ ID NO: 56, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 56. In some embodiments, the polynucleotide encoding CD39-P2A-CD73-P2A-PD-L-1 is operably linked to an exogenous promoter. The exogenous promoter may be a CAG or CAGGS promoter. In some embodiments, the donor plasmid encoding CD39-P2A-CD73-P2A-PD-L-1 and comprising B2M homology arms has the nucleotide sequence of SEQ ID NO: 47. In some embodiments, the donor plasmid encoding CD39-P2A-CD73-P2A-PD-L-1 and comprising TXNIP homology arms. In some embodiments, the donor plasmid encodes CD39-P2A-CD73-P2A-PD-L-1 and comprises a CIITA homology arm.In some embodiments, the donor plasmid encodes CD39-P2A-CD73-P2A-PD-L-1 and comprises a TGF-β2 homology arm.

[0102] In some embodiments, polynucleotides encoding CD39 and CD73 are inserted into a site within or near the B2M locus, within or near the TXNIP locus, within or near the CIITA locus, or within or near the TGF-β2 locus. In some embodiments, polynucleotides encoding CD39 and CD73 are inserted into a site within or near the B2M locus, within or near the TXNIP locus, or within or near the CIITA locus. In some embodiments, polynucleotides encoding CD39 and CD73 are inserted into a site within or near the B2M locus. In some embodiments, polynucleotides encoding CD39 and CD73 are inserted into a site within or near the B2M locus simultaneously with or after deletion of all or a portion of the B2M gene or promoter. In some embodiments, polynucleotides encoding CD39 and CD73 are inserted into a site within or near the TXNIP locus simultaneously with or after deletion of all or a portion of the TXNIP gene or promoter. In some embodiments, polynucleotides encoding CD39 and CD73 are inserted into a site within or near the CIITA locus simultaneously with or after deletion of all or part of the CIITA gene or promoter. In other embodiments, polynucleotides encoding CD39 and CD73 are inserted into a site within or near the TGF-β2 locus simultaneously with or after deletion of all or part of the TGF-β2 gene or promoter. The polynucleotides encoding CD39 and CD73 comprise a sequence encoding CD39 linked to a sequence encoding a ribosomal skip linked to a sequence encoding CD73. The ribosomal skip can be a 2A sequence family member, such as P2A. In some embodiments, the polynucleotide comprises a CD39-P2A-CD73 coding sequence.In some embodiments, the polynucleotide encoding CD39-P2A-CD73 comprises or consists of the nucleotide sequence of SEQ ID NO:58, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO:58. In some embodiments, the polynucleotide encoding CD39-P2A-CD73 is operably linked to an exogenous promoter. The exogenous promoter may be a CAG or CAGGS promoter. In some embodiments, the donor plasmid encodes CD39-P2A-CD73 and comprises B2M homology arms. In some embodiments, the donor plasmid encodes CD39-P2A-CD73-P2A-PD-L-1 and comprises TXNIP homology arms. In some embodiments, the donor plasmid encodes CD39-P2A-CD73-P2A-PD-L-1 and comprises CIITA homology arms. In some embodiments, the donor plasmid encodes CD39-P2A-CD73-P2A-PD-L-1 and contains TGF-β2 homology arms.

[0103] In some embodiments, at least one polynucleotide encoding at least one tolerogenic factor and / or survival factor can be delivered to cells as part of a vector. For example, the vector can be a plasmid vector. In various embodiments, the amount of plasmid vector delivered to cells can range from about 0.5 μg to about 10 μg (per about 10 cells). 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 cells can be about 4 μg.

[0104] In certain embodiments, cells with no MHC-II expression and moderate MHC-I expression are genetically modified to have no surface expression of MHC-I or MHC-II. In another embodiment, cells with no MHC-I / II surface expression are further edited to have PD-L-1 expression, e.g., insertion of a polynucleotide encoding PD-L-1, MANF expression, e.g., insertion of a polynucleotide encoding MANF, CD39 expression, e.g., insertion of a polynucleotide encoding CD39, CD73 expression, e.g., insertion of a polynucleotide encoding CD73, HLA-E expression, e.g., insertion of a polynucleotide encoding HLA-E, TNFAIP3 expression, e.g., insertion of a polynucleotide encoding TNFAIP, and / or any combination thereof. In another embodiment, cells with no MHC-I / II surface expression are further edited to have PD-L-1 expression, e.g., insertion of a polynucleotide encoding PD-L-1. In yet another embodiment, cells that do not have surface expression of MHC-I / II are further edited to have expression of PD-L-1, e.g., an insertion of a polynucleotide encoding PD-L-1, and are also genetically modified to have increased or decreased expression of at least one gene encoding a survival factor relative to the unmodified cell.

[0105] In some embodiments, the cells further comprise, by genetic modification, increased or decreased expression of one or more additional genes not necessarily related to immune evasion or cell survival, e.g., after engraftment or transplantation. In some embodiments, the cells further comprise increased expression of one or more safety switch proteins compared 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., into 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; 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, homing, viability, self-renewal, persistence, and / or survival, which are integrated with the construct.

[0106] One aspect of the present invention provides a method for generating a genomically engineered universal donor cell containing at least one targeted genome modification at one or more selected sites in the genome, the method comprising the steps of: genetically engineering a cell type described herein by introducing one or more constructs into the cell to enable targeted modification at the selected site; introducing one or more double-strand breaks at the selected site of the cell using one or more endonucleases capable of recognizing the selected site; and culturing the edited cell to allow endogenous DNA repair to generate targeted insertions or deletions at the selected site; thereby obtaining a genomically engineered universal donor cell. Targeted gene knockdown or knockout can be performed before, simultaneously with, or after the targeted polynucleotide insertion. The genomically engineered universal donor cell can be subjected to successive rounds of genome modification, thus targeting and modifying multiple sites. The genomically engineered cells can be cultured, characterized, selected, and expanded using techniques well known in the art. The universal donor cells generated by this method contain at least one functional targeted genomic modification and, if the genomically modified cell is a stem cell, are capable of differentiating into a progenitor cell or a fully differentiated cell.

[0107] In some other embodiments, the genomically engineered universal donor cells comprise the introduction or increase in expression of at least one of HLA-E, HLA-G, CD47, PD-L-1, TNFAIP3, MANF, CD73, and / or CD39. In some embodiments, the genomically engineered universal donor cells comprise the introduction or increase in expression of HLA-E, PD-L-1, TNFAIP3, and / or MANF. In some embodiments, the genomically engineered universal donor cells comprise the introduction or increase in expression of HLA-E, PD-L-1, TNFAIP3, MANF, and / or CD39. In some embodiments, the genomically engineered universal donor cells comprise the introduction or increase in expression of PD-L-1 and CD39, and / or the introduction or increase in expression of PD-L-1, CD73, and CD39. 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 low B2M. In some embodiments, the genomically engineered universal donor cells comprise B2M null or low B2M and TXNIP null or low TXNIP. In some embodiments, the genomically engineered universal donor cells comprise B2M null or low B2M, TXNIP null or low TXNIP, and CIITA null or low CIITA. In some embodiments, the genomically engineered universal donor cells comprise B2M null or low B2M, TXNIP null or low TXNIP, CIITA null or low CIITA, and TGF-β2 null or low TGF-β2.

[0108] 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, CD47, PD-L-1, TNFAIP3, MANF, CD73, and / or CD39. In some embodiments, the genomically engineered universal donor cells comprise an integrated or non-integrated exogenous polynucleotide encoding one or more of HLA-E, PD-L-1, TNFAIP3, MANF, CD73, and / or CD39. In some embodiments, the introduced expression is increased expression from any of the non-expressed or under-expressed genes contained in the cells. In some embodiments, the non-integrated exogenous polynucleotide is introduced using Sendai virus, AAV, episome, or plasmid. In some embodiments, the universal donor cells are B2M-null and TXNIP-null, with the introduced expression of TNFAIP3, PD-L-1, MANF, and HLA-E. In some embodiments, the universal donor cells are CIITA-null. In some embodiments, the universal donor cells are TGF-β2-null. In some embodiments, the universal donor cells are (i) B2M-null, with polynucleotides encoding TNFAIP3 and PD-L-1 inserted within or near the B2M locus, and (ii) TXNIP-null, with polynucleotides encoding MANF and HLA-E inserted within or near the TXNIP locus. In some embodiments, the universal donor cells are (i) B2M-null, with polynucleotides encoding TNFAIP3 and PD-L-1 inserted within or near the B2M locus, (ii) TXNIP-null, with polynucleotides encoding MANF and HLA-E inserted within or near the TXNIP locus, and (iii) CIITA-null, with a polynucleotide encoding CD39 inserted within or near the CIITA locus.In some embodiments, the universal donor cell is (i) B2M-null, with polynucleotides encoding TNFAIP3 and PD-L-1 inserted within or near the B2M locus, (ii) TXNIP-null, with polynucleotides encoding MANF and HLA-E inserted within or near the TXNIP locus, (iii) CIITA-null, with a polynucleotide encoding CD39 inserted within or near the CIITA locus, and (iv) TGF-β2-null. In some embodiments, the universal donor cell is (i) B2M-null, with a first polynucleotide encoding TNFAIP3 and PD-L-1 inserted within or near a first site in the B2M locus, and a second polynucleotide encoding CD39 and PD-L-1 inserted within or near a second site in the B2M locus, and (ii) TXNIP-null, with polynucleotides encoding MANF and HLA-E inserted within or near the TXNIP locus. In some embodiments, the universal donor cell is (i) B2M-null, with a first polynucleotide encoding TNFAIP3 and PD-L-1 inserted within or near a first site in the B2M locus, and a second polynucleotide encoding CD39 and PD-L-1 inserted within or near a second site in the B2M locus, (ii) TXNIP-null, with polynucleotides encoding MANF and HLA-E inserted within or near the TXNIP locus, and (iii) TGF-β2-null. In further embodiments, the universal donor cell is B2M-null, with polynucleotides encoding MANF, TNFAIP3, and PD-L-1 inserted within or near the B2M locus.In some embodiments, the universal donor cells are (i) B2M-null and have a polynucleotide encoding PD-L-1 inserted within or near the B2M locus, (ii) TXNIP-null and have a polynucleotide encoding HLA-E inserted within or near the TXNIP locus, (iii) CIITA-null and have a polynucleotide encoding CD39 inserted within or near the CIITA locus, and (iv) TGF-β2-null. In still other embodiments, the universal donor cells are B2M-null and have polynucleotides encoding MANF, TNFAIP3, and PD-L-1 inserted within or near the B2M locus, and optionally are CIITA-null and have a polynucleotide encoding CD39 inserted within or near the CIITA locus. In yet other embodiments, the universal donor cell is B2M-null and has a first polynucleotide encoding MANF, TNFAIP3, and PD-L-1 inserted at or near a first site in the B2M locus, and a second polynucleotide encoding CD39 and PD-L-1 inserted at or near a second site in the B2M locus, and is optionally TGF-β2-null. In other embodiments, the universal donor cell is (i) B2M-null and has polynucleotides encoding MANF, TNFAIP3, and PD-L-1 inserted at or near the B2M locus, (ii) CIITA-null and has a polynucleotide encoding CD39 inserted at or near the CIITA locus, and (iii) TGF-β2-null. In further embodiments, the universal donor cells are (i) B2M null and have a polynucleotide encoding PD-L-1 inserted within or near the B2M locus, (ii) TXNIP null and have a polynucleotide encoding HLA-E inserted within or near the TXNIP locus, (iii) CIITA null and have a polynucleotide encoding CD39 inserted within or near the CIITA locus, and (iv) TGF-β2 null.In still other embodiments, the universal donor cell is (i) B2M-null, with a first polynucleotide encoding PD-L-1 inserted at or near a first site in the B2M locus and a second polynucleotide encoding CD39 inserted at or near a second site in the B2M locus, and (ii) TXNIP-null, with a polynucleotide encoding HLA-E inserted at or near the TXNIP locus. In some embodiments, the universal donor cell is B2M-null, with polynucleotides encoding CD39 and PD-L-1 inserted at or near the B2M locus. In some embodiments, the universal donor cell is B2M-null, with polynucleotides encoding CD39, CD73, and PD-L-1 inserted at or near the B2M locus, and optionally is TGF-β2-null.

[0109] In certain embodiments, the universal donor cell further comprises increased or decreased expression of at least one safety switch protein. It is contemplated that the methods of generating any of the genetically modified cells described herein are carried out using at least any of the gene editing methods described herein. III. Genome Editing Methods

[0110] Genome editing generally refers to the process of modifying the nucleotide sequence of genome, preferably in a precise or predetermined manner.In some embodiments, the genome editing method described herein, for example, CRISPR-endonuclease system, can be used to genetically modify the cells described herein, for example, to create universal donor cells.In some embodiments, the genome editing method described herein, for example, CRISPR-endonuclease system, can be used to genetically modify the cells described herein, for example, to introduce at least one genetic modification into or near at least one gene, which reduces the expression of one or more MHC-I and / or MHC-II human leukocyte antigens or other components of MHC-I or MHC-II complexes compared to unmodified cells; to introduce at least one genetic modification that increases the expression of at least one polynucleotide encoding an immune tolerogenic factor compared to unmodified cells; and / or to introduce at least one genetic modification that increases or decreases the expression of at least one gene encoding a survival factor compared to unmodified cells.

[0111] An example of a genome editing method described herein is a method using site-specific nucleases to cut deoxyribonucleic acid (DNA) at precise target locations within the genome, thereby creating single- or double-stranded DNA breaks at specific locations within the genome. Such breaks can be, and usually are, 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. In NHEJ, the DNA ends resulting from the double-stranded break are directly joined, sometimes with the loss or addition of nucleotide sequences, which can disrupt or enhance gene expression. In HDR, a homologous or donor sequence is used as a template to insert a defined DNA sequence at the breakpoint. Homologous sequence can be, for example, sister chromatid, etc., in endogenous genome. Alternatively, donor sequence can be an exogenous polynucleotide, such as a plasmid, single-stranded oligonucleotide, double-stranded oligonucleotide, duplex oligonucleotide or virus, which has a region (for example, left and right homologous arm) that is highly homologous to the locus that is cut by nuclease, but can also contain additional sequence or sequence changes, including deletion, that can be integrated into the cut target locus.The third repair mechanism can be microhomology-mediated end joining (MMEJ), also referred to as " alternative NHEJ ", and its genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cut site.MMEJ can use a few base pairs of homologous sequences flanking the DNA break site to drive more favorable DNA end-joining repair outcomes, and recent reports have further elucidated the molecular mechanisms of this process; see, for example, 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 likely repair outcomes based on analysis of potential microhomologies at the DNA break site.

[0112] Each of these genome editing mechanisms can be used to create desired genetic modification.The step of genome editing process can be to create one or two DNA breaks in the vicinity of the intended mutation site in the target gene locus, the latter being double-strand break or two single-strand breaks.This can be achieved by using the endonuclease described and exemplified herein.

[0113] Generally, the genome editing methods described herein can be in vitro or ex vivo methods. In some embodiments, the genome editing methods disclosed herein are not methods for therapeutic treatment of the human or animal body and / or are not processes for modifying the genetic identity of human germline. CRISPR endonuclease system

[0114] CRISPR-endonuclease system is a defense mechanism that naturally exists in prokaryotes, and is repurposed as an RNA-guided DNA targeting platform used for gene editing.CRISPR system includes type I, type II, type III, type IV, type V and type VI system.In some embodiments, CRISPR system is type II CRISPR / Cas9 system.In other embodiments, CRISPR system is type V CRISPR / Cpf system.CRISPR system relies on DNA endonuclease, such as Cas9, and two non-coding RNAs-crisprRNA (crRNA) and transactivating RNA (tracrRNA) for targeting DNA cleavage.

[0115] The crRNA drives the sequence recognition and specificity of the CRISPR-endonuclease complex through Watson-Crick base pairing with a sequence typically within approximately 20 nucleotides (nt) of target DNA. Varying the sequence of the 5' 20 nt of the crRNA allows the CRISPR-endonuclease complex to be targeted to specific loci. When the target sequence is followed by a specific short DNA motif (with the sequence NGG) called a protospacer adjacent motif (PAM), the CRISPR-endonuclease complex will only bind to DNA sequences that contain a sequence match with the first 20 nt of the single guide RNA (sgRNA).

[0116] The tracrRNA hybridizes to the 3' end of the crRNA to form an RNA-duplex structure to which the endonuclease binds to form a catalytically active CRISPR-endonuclease complex, which can then cleave the target DNA.

[0117] Once the CRISPR-endonuclease complex binds to DNA at the target site, each of the two independent nuclease domains within the endonuclease cleaves one of the DNA strands three bases upstream of the PAM site, leaving a double-strand break (DSB) in which both strands of DNA are terminated by base pairs (blunt ends).

[0118] 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 can be used, such as S. aureus Cas9, N. meningitidis Cas9, S. thermophilus CRISPR 1 Cas9, S. thermophilus CRISPR 3 Cas9, or T. denticola Cas9. 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, Csm 2, 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 can be used. In some embodiments, modified versions of the aforementioned endonucleases (e.g., homologs thereof, recombinant naturally occurring molecules thereof, codon-optimized versions thereof, or modified versions thereof) can be used.

[0119] The CRISPR nuclease may be linked to at least one nuclear localization signal (NLS), and the at least one NLS may be located at or within 50 amino acids from the amino terminus of the CRISPR nuclease, and / or the at least one NLS may be located at or within 50 amino acids from the carboxy terminus of the CRISPR nuclease.

[0120] Exemplary CRISPR / Cas polypeptides include the Cas9 polypeptides 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. Since the discovery of Cas genes, the CRISPR / Cas gene nomenclature system has been extensively rewritten. Fonfara et al. also provide PAM sequences for Cas9 polypeptides from various species. Zinc finger nuclease

[0121] 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 engineered to bind cognate 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 itself has no sequence specificity, generates a DNA double-strand break between the ZFN half-sites, the initiating step in genome editing.

[0122] The DNA-binding domain of each ZFN generally consists of three to six zinc fingers with a Cys2-His2-rich architecture, each of which 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. Altering the amino acids in the fingers that make critical contacts with DNA alters the sequence specificity of a given finger. Thus, a four-finger zinc finger protein selectively recognizes a 12-bp target sequence, where the target sequence is a composite of the triplet preferences contributed by each finger, but triplet preferences may be influenced to varying degrees by neighboring fingers. An important aspect of ZFNs is their ease of retargeting to almost any genomic address by simply modifying individual fingers. The vast majority of ZFN applications use proteins with four to six fingers, each recognizing 12 to 18 bp. Thus, a pair of ZFNs generally recognizes a combined target sequence of 24–36 bp, not including the typical 5–7 bp spacer between half sites. The binding sites can be further separated by a larger spacer, including 15–17 bp. Assuming that repetitive sequences or gene homologs are eliminated during the design process, target sequences of this length are likely unique in the human genome. Nevertheless, ZFN protein-DNA interactions are not absolute in their specificity, and off-target binding and cleavage events can occur as heterodimers between two ZFNs or as homodimers of one or the other ZFN. The latter possibility has been effectively eliminated by engineering the dimerization interface of the FokI domain to create "plus" and "minus" variants, also known as obligate heterodimer variants, which can only dimerize with each other and not with themselves. Enforcing obligate heterodimerization prevents homodimer formation. This significantly enhances the specificity of ZFNs, as well as any other nucleases in which these FokI variants are employed.

[0123] Various ZFN-based systems have been described in the art, and modifications thereof are regularly reported, and numerous references describe the rules and parameters used to guide the design of ZFNs; see, for example, 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. Transcription activator-like effector nucleases (TALENs)

[0124] TALENs represent another format of modular nucleases in which, like ZFNs, an engineered DNA-binding domain is linked to a FokI nuclease domain, and the TALEN pair operates in tandem to achieve targeted DNA cleavage. The key difference between TALENs and ZFNs is the nature of the DNA-binding domain and its associated target DNA sequence recognition properties. TALEN DNA-binding domains are derived from TALE proteins, which were first described for the plant bacterial pathogen Xanthomonas sp. TALEs consist of a tandem array of 33-35 amino acid repeats, each of which recognizes a single base pair within a target DNA sequence, typically up to 20 bp long, for a total target sequence length of up to 40 bp. The nucleotide specificity of each repeat is determined by a repeat variable diresidue (RVD), which contains only 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 constitutes a much simpler recognition code than zinc fingers, thus offering advantages over zinc fingers in nuclease design. Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and for TALENs, the use of obligate heterodimer variants of the FokI domain is beneficial to reduce off-target activity.

[0125] Additional variants of the FokI domain with catalytically inactivated functions have been created. When either a TALEN or ZFN pair contains an inactive FokI domain, only single-strand DNA breaks (nicks) occur at the target site, rather than DSBs. The results are comparable to those achieved using CRISPR / Cas9 or CRISPR / Cpf1 "nickase" mutants 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 benefit is that off-target nicks are repaired quickly and precisely, unlike DSBs, which are prone to NHEJ-mediated misrepair.

[0126] Various TALEN-based systems have been described in the art, and modifications thereof are regularly reported; see, for example, 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. Homing endonucleases

[0127] Homing endonucleases (HEs) are sequence-specific endonucleases that have long recognition sequences (14–44 base pairs) and cleave DNA with high specificity, often at unique sites within 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, which are 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 create DSBs at target loci as the first step in genome editing. Furthermore, some natural and engineered HEs only cut single-stranded DNA, thereby functioning as site-specific nickases. The large target sequences and specificity conferred by HEs make them attractive candidates for creating site-specific DSBs.

[0128] 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. MegaTAL / Tev-mTALEN / MegaTev

[0129] As further examples of hybrid nucleases, the MegaTAL and Tev-mTALEN platforms use fusions of a TALE DNA-binding domain with a catalytically active HE, leveraging 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.

[0130] In a further variation, the MegaTev architecture is a fusion of Meganuclease (Mega) with the nuclease domain from 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 useful for achieving the targeted genome modification described herein. dCas9-FokI or dCpf1-Fok1 and other nucleases

[0131] Combining the structural and functional properties of the above nuclease platforms provides additional approaches for genome editing that potentially overcome some of their inherent deficiencies. For example, CRISPR genome editing systems typically use a single Cas9 endonuclease to create DSBs. Targeting specificity is driven by a 20- or 24-nucleotide sequence within the guide RNA that Watson-Crick base-pairs with the target DNA (plus, in the case of S. pyogenes-derived Cas9, two additional bases within the adjacent NAG or NGG PAM sequence). While such sequences are long enough to be unique to the human genome, the specificity of the RNA / DNA interaction is not absolute; significant promiscuity is sometimes tolerated, particularly for the 5' half of the target sequence, effectively reducing the number of bases driving specificity. One solution to this problem has been 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, for example, Tsai et al., Nature Biotech, 2014, 32: 569-76; and Guilinger et al., Nature Biotech., 2014, 32: 577-82. Because FokI must form a dimer to be catalytically active, two guide RNAs are required to tether two FokI fusions in close proximity to form a dimer and cleave the DNA. This essentially doubles the number of bases at the combined target site, thereby increasing the targeting stringency by the CRISPR-based system.

[0132] As a further example, fusion of a TALE DNA binding domain with a catalytically active HE, such as I-TevI, exploits both the tunable DNA binding and specificity of TALEs and the cleavage sequence specificity of I-TevI, and is expected to further reduce off-target cleavage. Base Editing

[0133] In some embodiments, base editing is used to edit genes in cells. Base editing is a technique that allows for the conversion of one nucleotide to another without causing double-strand breaks in DNA. Base editing allows for the conversion of C to T, G to A, or vice versa. An example of a cytosine editor is rAPOBEC1 fused to a catalytically inactive form of Cas9. Cas9 assists in binding to the desired site, and rAPOBEC1 cytidine deaminase induces a point mutation. As described herein, the conversion of adenine requires a mutant transfer RNA adenosine deaminase (TadA), Cas9 nickase, and sgRNA. The construct allows for the introduction of site-specific mutations without introducing strand breaks. In some embodiments, base editing is used to introduce one or more mutations into the cells described herein. RNA-guided endonucleases

[0134] An RNA-guided endonuclease system, as used herein, 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 100% amino acid sequence identity to a wild-type exemplary endonuclease, e.g., Cas9 from S. pyogenes, SEQ ID NO: 8 of US2014 / 0068797 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 within 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 within 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 consecutive amino acids within 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 consecutive amino acids within the RuvC nuclease domain of the endonuclease.

[0135] 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, as described above). The modified form of the endonuclease may not have substantial nucleic acid cleavage activity. When the endonuclease is a modified form that does not have substantial nucleic acid cleavage activity, it is referred to herein as "enzymatically inactive."

[0136] Contemplated mutations may include substitutions, additions, and 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 mimetic (e.g., a phosphomimetic). The mutation may be a conservative mutation. For example, the mutation converts the mutated amino acid to an amino acid of similar size, shape, charge, polarity, conformation, and / or a rotamer of the mutated amino acid (e.g., a cysteine / serine mutation, a lysine / asparagine mutation, a histidine / phenylalanine mutation). Mutations can cause a shift in the reading frame and / or the creation of a premature stop codon. Mutations can cause changes in the regulatory regions of a gene or locus that affect the expression of one or more genes. guide RNA

[0137] 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 may include at least a spacer sequence that hybridizes with the target nucleic acid sequence of interest and a CRISPR repeat sequence. In a CRISPR type II system, the gRNA also includes a second RNA called a tracrRNA sequence. In a CRISPR type II guide RNA (gRNA), the CRISPR repeat sequence and the tracrRNA sequence hybridize with each other to form a duplex. In a CRISPR type V system, the gRNA includes a crRNA that forms a duplex. In some embodiments, the gRNA can bind an endonuclease, thus forming a complex with the gRNA and the endonuclease. The gRNA can provide target specificity to the complex through its association with the endonuclease. Thus, the activity of the endonuclease can be directed by a genome-targeting nucleic acid.

[0138] Exemplary guide RNAs include a spacer sequence containing 15-200 nucleotides, where 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.

[0139] The gRNA can be a dual-molecule guide RNA. The gRNA can be a single-molecule guide RNA.

[0140] The dual-molecule 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.

[0141] A single-molecule guide RNA (sgRNA) can include, from 5' to 3', an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single-molecule guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension can include elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker can link the minimal CRISPR repeat and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can include one or more hairpins.

[0142] In some embodiments, the sgRNA comprises a 20-nucleotide spacer sequence 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 of more than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA comprises a spacer sequence of variable length, between 17 and 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 nucleotide, more than 5 nucleotides, more than 10 nucleotides, more than 15 nucleotides, more than 20 nucleotides, more than 25 nucleotides, more than 30 nucleotides, more than 35 nucleotides, more than 40 nucleotides, more than 45 nucleotides, more than 50 nucleotides, more than 60 nucleotides, more than 70 nucleotides, more than 80 nucleotides, more than 90 nucleotides, more than 100 nucleotides, more than 120 nucleotides, more than 140 nucleotides, more than 160 nucleotides, more than 180 nucleotides, or more than 200 nucleotides. In some embodiments, the sgRNA comprises a spacer extension sequence having a length of less than 3 nucleotides, less than 5 nucleotides, less than 10 nucleotides, less than 15 nucleotides, less than 20 nucleotides, less than 25 nucleotides, less than 30 nucleotides, less than 35 nucleotides, less than 40 nucleotides, less than 45 nucleotides, less than 50 nucleotides, less than 60 nucleotides, less than 70 nucleotides, less than 80 nucleotides, less than 90 nucleotides, or less than 100 nucleotides.

[0143] 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 may decrease or increase the stability of the nucleic acid targeting nucleic acid. The moiety may be a transcription terminator segment (i.e., a transcription termination sequence). The moiety may function in eukaryotic cells. The moiety may function in prokaryotic cells. The moiety may 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., to allow 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, chloroplasts, etc.), modifications or sequences that provide tracking (e.g., direct conjugation with a fluorescent molecule, conjugation with a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.), and / or modifications or sequences that provide binding sites for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.).

[0144] In some embodiments, the sgRNA comprises a spacer sequence that hybridizes with a sequence within 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.

[0145] In the CRISPR-endonuclease system, spacer sequence can be designed to hybridize with the target polynucleotide located at the 5' side of the PAM of the endonuclease used in the system.Spacer can be perfectly matched with the target sequence, or can have mismatches.Each endonuclease, for example, Cas9 nuclease, has a specific PAM sequence that it recognizes in target DNA.For example, S.pyogenes Cas9 recognizes PAM that comprises the sequence 5'-NRG-3', where R comprises either A or G, and N is any nucleotide, and N is located immediately 3' from the target nucleic acid sequence that is targeted by the spacer sequence.

[0146] The target polynucleotide sequence may comprise 20 nucleotides. The target polynucleotide may comprise less 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' to the first nucleotide of the PAM.

[0147] The spacer sequence that hybridizes to the target polynucleotide can have a length of at least about 6 nucleotides (nt). The spacer sequence can 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, from about 10 nt to about 40 nt, or from about The spacer sequence may be from 10 nt to about 35 nt, from about 10 nt to about 30 nt, from about 10 nt to about 25 nt, from about 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some examples, the spacer sequence may comprise 20 nucleotides. In some examples, the spacer may comprise 19 nucleotides. In some examples, the spacer may comprise 18 nucleotides. In some examples, the spacer may comprise 22 nucleotides.

[0148] In some examples, 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 examples, 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 6 contiguous nucleotides 5'-most to the target sequence on the complementary strand of the target nucleic acid. The percent complementarity between the spacer sequence and the target nucleic acid can be at least 60% over approximately 20 contiguous nucleotides. The length of the spacer sequence and the target nucleic acid can differ by 1 to 6 nucleotides, which can be considered a bulge(s).

[0149] The tracrRNA sequence may contain nucleotides that hybridize with the minimal CRISPR repeat sequence in cells. The minimal tracrRNA sequence and the minimal CRISPR repeat sequence may form a duplex, i.e., a base-paired, double-stranded structure. The minimal tracrRNA sequence and the minimal CRISPR repeat may bind together to an RNA-guided endonuclease. At least a portion of the minimal tracrRNA sequence may hybridize with 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.

[0150] The minimal tracrRNA sequence can be from about 7 nucleotides to about 100 nucleotides in length. For example, the minimal tracrRNA sequence can be from about 7 nucleotides (nt) to about 50 nt, from about 7 nt to about 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25 nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, or from 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 nts 23-48 as described in Jinek et al., supra.

[0151] The minimal tracrRNA sequence can be at least about 60% identical to a reference minimal tracrRNA (e.g., a wild-type tracrRNA from S. pyogenes) 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 a reference minimal tracrRNA sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0152] The duplex between minimal CRISPR RNA and minimal tracrRNA can comprise a double helix.The duplex between minimal CRISPR RNA and minimal tracrRNA can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more.The duplex between minimal CRISPR RNA and minimal tracrRNA can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more.

[0153] 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 or mismatches. A duplex may contain up to about 1, 2, 3, 4, or 5 or mismatches. A duplex may contain no more than two mismatches.

[0154] 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).

[0155] In some embodiments, the gRNA may include a tracrRNA extension sequence. The tracrRNA extension sequence may have a length of about 1 to about 400 nucleotides. The tracrRNA extension sequence may have a length of more than 1 nucleotide, more than 5 nucleotides, more than 10 nucleotides, more than 15 nucleotides, more than 20 nucleotides, more than 25 nucleotides, more than 30 nucleotides, more than 35 nucleotides, more than 40 nucleotides, more than 45 nucleotides, more than 50 nucleotides, more than 60 nucleotides, more than 70 nucleotides, more than 80 nucleotides, more than 90 nucleotides, more than 100 nucleotides, more than 120 nucleotides, more than 140 nucleotides, more than 160 nucleotides, more than 180 nucleotides, or more than 200 nucleotides. The tracrRNA extension sequence may have a length of about 20 nucleotides to about 5000 nucleotides or more. The tracrRNA extension sequence can have a length of less than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides. The tracrRNA extension sequence can comprise a length of less than 10 nucleotides. The tracrRNA extension sequence can be 10-30 nucleotides in length. The tracrRNA extension sequence can be 30-70 nucleotides in length.

[0156] 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 an overall 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, or 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.

[0157] In some embodiments, the sgRNA may include a linker sequence having a length of about 3 to about 100 nucleotides. Jinek et al., supra, for example, used a simple four-nucleotide "tetraloop" (-GAAA-) (Jinek et al., Science, 2012, 337 (6096): 816-821). Exemplary linkers have a length 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 can 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 can be between 4 and 40 nucleotides. The linker can be at least about or more than 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 nucleotides. The linker can be at most about or more than 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 nucleotides.

[0158] The linker may comprise any of a variety of sequences, but in some instances, the linker does not comprise a sequence with extensive regions of homology to other portions of the guide RNA, which could result in intramolecular binding that could interfere with other functional regions of the guide. Jinek et al., supra, used the simple four-nucleotide sequence -GAAA- (Jinek et al., Science, 2012, 337 (6096): 816-821), but many other sequences, including longer sequences, can be used as well.

[0159] Linker sequence can comprise functional part.For example, linker sequence can comprise one or more features, including aptamer, ribozyme, hairpin that interacts with protein, protein binding site, CRISPR array, intron or exon.Linker sequence can comprise at least about 1, 2, 3, 4, or 5 or more functional part.In some examples, linker sequence can comprise at most about 1, 2, 3, 4, or 5 or more functional part.

[0160] In some embodiments, the sgRNA does not contain, for example, a uracil at the 3' end of the sgRNA sequence. In some embodiments, the sgRNA contains, for example, one or more uracils 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.

[0161] The sgRNA may 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 severity of an innate immune response, and / or enhances other properties described in the art.

[0162] 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.

[0163] Morpholino-based 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.

[0164] Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 2000, 122: 8595-8602.

[0165] In some embodiments, the modified gRNA may include one or more substituted sugar moieties at the 2' position, for example, one of the following: OH, SH, SCH, F, OCN, OCH, OCH O(CH) CH, O(CH) NH, or O(CH) CH, where 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; SOCH; SOCH; ONO; NO; N; NH; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving group; reporter group; intercalator; 2'-O-(2-methoxyethyl); 2'-methoxy(2'-O-CH); 2'-propoxy(2'-OCH) CH2CH3); and 2'-fluoro (2'-F). Similar modifications may be made at other positions in the gRNA, particularly the 3' position of the sugar of 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 unit, may be replaced with novel groups.

[0166] Guide RNAs may also, or instead, 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 transiently in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also called 5-methyl-2'deoxycytosine, often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, 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, WH 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, can also be included. 5-Me-C substitution has been shown to increase the stability of nucleic acid duplexes by 0.6-1.2°C (Sanghvi, YS, in Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and is an example of base substitution.

[0167] Modified nucleobases include other synthetic and natural nucleobases, such as 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 These may include thymine, 5-uracil (pseudouracil), 4-thiouracil, 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-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Genome-targeting nucleic acid and endonuclease complexes

[0168] The gRNA interacts with an endonuclease (e.g., an RNA-guided nuclease such as Cas9), thereby forming a complex, and the gRNA guides the endonuclease to the target polynucleotide.

[0169] The endonuclease and gRNA can be administered separately to a cell or a subject. In some embodiments, the endonuclease can be pre-complexed with one or more guide RNAs, or with one or more crRNAs together with a tracrRNA. 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 within the RNP can be, for example, a Cas9 endonuclease or a Cpfl endonuclease. The endonuclease can be flanked by one or more nuclear localization signals (NLSs) at the N-terminus, C-terminus, or both the N-terminus and C-terminus. For example, the Cas9 endonuclease can be flanked by two NLSs, one at the N-terminus and the second 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 within the RNP can range from about 1: 1 to about 10: 1. For example, the molar ratio of sgRNA to Cas9 endonuclease within the RNP can be 3: 1. Nucleic acids encoding components of the system

[0170] The present disclosure provides nucleic acids comprising nucleotide sequences encoding genome-targeting nucleic acids of the present disclosure, 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.

[0171] Nucleic acids 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 may be comprised in a vector (e.g., a recombinant expression vector).

[0172] 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.

[0173] In some instances, vectors may be 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 equivalent functions.

[0174] The term "operably linked" means that the nucleotide sequence of interest is linked to a regulatory sequence(s) 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). Those skilled in the art will understand that the design of an expression vector can depend on factors such as the selection of target cells and the desired expression level.

[0175] Contemplated expression vectors include, but are not limited to, vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, viral vectors based on retroviruses (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 can also be used as long as they are compatible with the host cell.

[0176] In some examples, vector can comprise one or more transcription and / or translation control elements.Depending on the host / vector system used, any of several suitable transcription and translation control elements can be used in expression vector, including constitutive promoter and inducible promoter, transcription enhancer element, transcription terminator, etc.Vector can be a self-inactivating vector that inactivates viral sequence or CRISPR mechanism components or other elements.

[0177] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional 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 the cytomegalovirus enhancer fused to the chicken beta-actin promoter, a hybrid construct comprising the 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.

[0178] 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 or CAGGS promoter). In some cases, the promoter may be a spatially restricted and / or temporally restricted promoter (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.).

[0179] Introduction of the complexes, polypeptides, and nucleic acids of the present disclosure into cells can be achieved 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. IV.Cell type

[0180] The cells described herein, e.g., universal donor cells (and the corresponding unmodified cells), can belong to any possible class of cell type. In some embodiments, the cells, e.g., universal donor cells (and the corresponding unmodified cells), can be mammalian cells. In some embodiments, the cells, e.g., universal donor cells (and the corresponding unmodified cells), can be human cells. In some embodiments, the cells, e.g., universal donor cells (and the corresponding unmodified cells), can be stem cells. In some embodiments, the cells, e.g., universal donor cells (and the corresponding unmodified cells), can be pluripotent stem cells (PSCs). In some embodiments, the cells, e.g., universal donor cells (and the corresponding unmodified cells), can be embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), or hematopoietic stem or progenitor cells (HSPCs) (also referred to as hematopoietic stem cells (HSCs)). In some embodiments, the cells, e.g., universal donor cells (and the corresponding unmodified cells), can be differentiated cells. In some embodiments, the cells, e.g., universal donor cells (and the corresponding unmodified cells), can be somatic cells, e.g., immune system cells, pancreatic cells, or contractile cells, e.g., skeletal muscle cells.

[0181] The cells described herein, such as universal donor stem cells, can be differentiated into relevant cell types to assess HLA expression and evaluate the immunogenicity of universal stem cell lines.Generally, differentiation involves maintaining the desired cells for a sufficient period and under sufficient conditions to differentiate the cells into the desired differentiated cells.For example, the universal stem cells disclosed herein can be differentiated into mesenchymal progenitor cells (MPCs), low immunogenic cardiomyocytes, muscle progenitor cells, blast cells, endothelial cells (ECs), macrophages, hepatocytes, beta cells (e.g., pancreatic beta cells), pancreatic endoderm precursors, pancreatic endocrine precursors, pancreatic endocrine cells, hematopoietic progenitor cells, or neural progenitor cells (NPCs). In some embodiments, universal donor cells can be differentiated into definitive endoderm cells, undifferentiated intestinal cells, posterior foregut cells, pancreatic endoderm cells (PECs), pancreatic endocrine cells, immature beta cells, or maturing beta cells.

[0182] Stem cells are capable of both proliferating and giving rise to more progenitor cells, which in turn have the capacity to generate a large number of mother cells, which in turn can give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and subsequently produce progeny that differentiate into one or more mature cell types, but also retain one or more cells with the developmental potential of the parent. Thus, the term "stem cell" refers to a cell that, under certain circumstances, has the ability or potential to differentiate into a more specialized or differentiated phenotype and, under certain circumstances, retains the ability to proliferate without substantial differentiation. In one aspect, the term progeny or stem cell refers to a generalized mother cell, whose descendants often specialize in different directions by differentiation, acquiring entirely individual characteristics, similar to, for example, the progressive diversification of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. For example, differentiated cells are derived from pluripotent cells that are themselves derived from pluripotent cells. Each of these multipotent 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 greater developmental potential. Such ability can be natural or can be artificially induced by treatment with various factors.

[0183] For example, human embryonic stem cells (hESCs) can be artificially differentiated into insulin-producing cells through a seven-step process that requires the addition of specific growth factors and small molecules. These seven steps include 1) definitive endoderm, 2) undifferentiated gut, 3) posterior foregut, 4) pancreatic endoderm, 5) pancreatic endoderm precursor, 6) immature beta cell, and 7) maturing beta cell. For example, human pluripotent stem cells can be differentiated into the pancreatic lineage as described in Schulz et al. (2012) PLoS ONE 7 (5): e37004, Rezania et al. (2014) Nat. Biotechnol. 32 (11): 1121-1133, and / or US20200208116. In many biological instances, stem cells can also be "pluripotent" because they can produce progeny of more than one distinct cell type, although this is not required for "stemness."

[0184] A "differentiated cell" is a cell that has progressed further down the developmental pathway than the cell to which it is being compared. Thus, a stem cell can differentiate into a lineage-restricted precursor cell (e.g., a myocyte progenitor cell), which in turn can differentiate into other types of precursor cells further down the pathway (e.g., a myocyte precursor), and then into a terminally differentiated cell, e.g., a myocyte, which may or may not retain the ability to fulfill a characteristic role in a particular tissue type and further proliferate. In some embodiments, the differentiated cell can be a pancreatic beta cell. embryonic stem cells

[0185] The cells described herein can be embryonic stem cells (ESCs). ESCs are derived from the blastocysts of mammalian embryos and can be differentiated into any cell type and rapidly expanded. ESCs are also believed to have a normal karyotype, maintain high telomerase activity, and exhibit remarkable long-term proliferation potential, making them excellent candidates for use as universal donor cells. adult stem cells

[0186] The cells described herein may be adult stem cells (ASCs). ASCs are undifferentiated cells that can be found in mammals, such as humans. ASCs are defined by their ability to self-renew, for example, by their ability to maintain their undifferentiated state while undergoing several rounds of cell replication, and by their ability to differentiate into several distinct cell types, such as glial cells. Adult stem cells are a broad class of stem cells and can include hematopoietic stem cells, breast 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. induced pluripotent stem cells

[0187] The cells described herein may be induced pluripotent stem cells (iPSCs). iPSCs can be generated directly from adult human cells by introducing genes encoding crucial transcription factors involved in pluripotency, such as OCT4, SOX2, cMYC, and KLF4. iPSCs may be derived from the same subject who will subsequently receive progenitor cells. That is, somatic cells can be obtained from the subject, reprogrammed into induced pluripotent stem cells, and then redifferentiated into progenitor cells (e.g., autologous cells) that are administered to the subject. However, autologous cells carry the risk of immune responses and low survival rates after engraftment. In some embodiments, iPSCs can be obtained from commercial sources. In some embodiments, iPSCs are gene-edited prior to differentiation into lineage-restricted progenitor cells or fully differentiated somatic cells. Human hematopoietic stem and progenitor cells

[0188] 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 the erythroid (erythrocytes 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) lineages. Blood cells are produced by the proliferation and differentiation of a very small population of multipotent hematopoietic stem cells (HSCs), which also have the ability to replenish themselves through self-renewal. During differentiation, HSC progeny progress through various intermediate maturation stages, generating multipotential and lineage-committed progenitor cells before reaching maturity. The bone marrow (BM) is the primary site of hematopoiesis in humans, and under normal conditions, only a small number of hematopoietic stem and progenitor cells (HSPCs) can be found in peripheral blood (PB). Treatment with cytokines, some myelosuppressive drugs used in cancer treatment, and compounds that disrupt the interaction between hematopoietic cells and BM stromal cells can rapidly mobilize large numbers of stem and progenitor cells into the circulation. Differentiation of cells into other cell types

[0189] Another step of the method of the present disclosure may include differentiating cells into differentiated cells. The differentiation step can be performed according to any method known in the art. For example, human iPSCs are differentiated into definitive endoderm using various treatments, including activin and B27 supplementation (Life Technologies). The definitive endoderm is further differentiated into hepatocytes, and treatments include 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 can be performed according to Sawitza et al., Sci Rep. 2015; 5: 13320. The differentiated cells can be any somatic cells of a mammal, for example, a human. In some embodiments, the somatic cell may 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, a gastrointestinal cell, a pancreatic islet), a keratinizing epithelial cell (e.g., an epidermal keratinocyte), a stratified barrier epithelial cell of the wet zone, a sensory transducer cell (e.g., a photoreceptor), an autonomic nervous system neuron cell, 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.

[0190] Generally, populations of universal donor cells disclosed herein maintain expression of the inserted one or more 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 the inserted one or more tolerogenic and / or survival factors. Furthermore, populations of lineage-restricted or fully differentiated cells derived from the universal donor cells disclosed herein maintain expression of the inserted one or more 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 and / or survival factors. V. Formulation and Administration Formulation and delivery for gene editing

[0191] The guide RNAs, polynucleotides described herein, such as polynucleotides encoding tolerogenic and / or survival factors, or polynucleotides encoding endonucleases, and endonucleases can be formulated and delivered to cells in any manner known in the art.

[0192] The guide RNA and / or polynucleotide can be formulated with pharmaceutically acceptable excipients, such as carriers, solvents, stabilizers, adjuvants, diluents, etc., depending on the particular mode of administration and dosage form. The guide RNA and / or polynucleotide composition can be formulated to be physiologically compatible and achieve a pH ranging from about pH 3 to about pH 11, or from about pH 3 to about pH 7, depending on the formulation and route of administration. In some cases, the pH can be adjusted to a range from about pH 5.0 to about pH 8. In some cases, the composition can comprise a therapeutically effective amount of at least one compound described herein together with one or more pharmaceutically acceptable excipients. Optionally, the composition can comprise a combination of compounds described herein, or can comprise a second active ingredient useful for treating or preventing bacterial growth (e.g., without limitation, an antibacterial or antimicrobial agent), or can comprise a combination of reagents of the present disclosure.

[0193] Suitable excipients include, for example, large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and carrier molecules, including inactive virus particles. Other exemplary excipients may include antioxidants (such as and without limitation ascorbic acid), chelating agents (such as and without limitation EDTA), carbohydrates (such as and without limitation dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (such as and without limitation oil, water, saline, glycerol, and ethanol), wetting or emulsifying agents, pH buffering substances, etc.

[0194] The guide RNA polynucleotide (RNA or DNA) and / or endonuclease polynucleotide(s) (RNA or DNA) can be delivered by viral or non-viral delivery vehicles known in the art. Alternatively, the endonuclease polypeptide(s) 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 guide RNAs, or one or more crRNAs together with tracrRNA.

[0195] Polynucleotide can be delivered by non-viral delivery vehicle, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule 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 (this focuses on the non-viral delivery vehicle for siRNA, which is also useful for the delivery of other polynucleotides).

[0196] For polynucleotides of the present disclosure, formulations can be selected from, for example, any of those taught in International Application No. PCT / US2012 / 069610.

[0197] Polynucleotides such as guide RNAs, sgRNAs, and mRNAs encoding endonucleases can be delivered to cells or subjects by lipid nanoparticles (LNPs).

[0198] LNP refers to any particle with a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, nanoparticles are in the size ranges of 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

[0199] LNPs can be made of cationic, anionic, or neutral lipids. Neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Disadvantages of cationic lipids include poor stability and rapid clearance, resulting in low efficacy, as well as the generation of inflammatory or anti-inflammatory responses.

[0200] LNPs may also be composed of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0201] Any lipid or lipid combination known in the art can be used to prepare LNPs. Examples of lipids used to prepare LNPs are DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids are 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0202] Lipids can be combined in any number of molar ratios to make LNPs. Additionally, polynucleotide(s) can be combined with lipid(s) in a wide range of molar ratios to make LNPs.

[0203] Recombinant adeno-associated virus (AAV) vectors can be used for delivery. The techniques for producing rAAV particles, in which a cell is supplied with the packaged AAV genome, including the polynucleotide to be delivered, the rep and cap genes, and helper virus functions, are standard in the art. The production of rAAV generally 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 the rAAV genome (i.e., not present in the rAAV genome), and helper virus functions. The AAV rep and cap genes may be derived from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, the AAV serotypes described herein, and may be derived from an AAV serotype different from the rAAV genome ITRs. The production of pseudotyped rAAV is disclosed, for example, in International Patent Application Publication No. WO01 / 83692. Formulation and Administration of Cells, E.g., Universal Donor Cells

[0204] The genetically modified cells described herein, e.g., universal donor cells, can be formulated and administered to a subject in any manner known in the art.

[0205] The terms "administering," "introducing," "implanting," "engrafting," and "transplanting" are used interchangeably in reference to placing 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 a 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 within a subject where at least a portion of the implanted cells or cellular components remain viable. The period of cell viability after administration to a subject can be as short as a few hours, e.g., 24 hours, to several days, to as long as several years, or even for the life of the subject, i.e., long-term engraftment.

[0206] Genetically modified cells described herein, e.g., universal donor cells, can be viable for a longer period of time after administration to a subject than unmodified cells.

[0207] In some embodiments, compositions comprising the cells described herein can be administered by a suitable route, which may include intravenous administration, for example, as a bolus or by continuous infusion over a period of time. In some embodiments, intravenous administration can be performed by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, or intrathecal routes. In some embodiments, the compositions can be in solid, aqueous, or liquid form. In some embodiments, aqueous or liquid forms can be atomized or lyophilized. In some embodiments, atomized or lyophilized forms can be reconstituted with an aqueous or liquid solution.

[0208] The cell composition can 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 can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients, in amounts suitable for use in the therapeutic methods described herein.

[0209] The additional agent contained in the cell composition can include the pharmaceutically acceptable salt of the component in the cell composition.Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino group of polypeptide) formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, tartaric acid, mandelic acid, etc. The salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0210] Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that do not contain any material other than active ingredients and water, or contain buffers such as sodium phosphate at physiological pH values, physiological saline, or both, such as phosphate-buffered saline. In addition, aqueous carriers can contain more than one buffer salt, and salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol, and other solutes. Liquid compositions can also contain liquid phases in addition to and other than water. Exemplary 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 is effective for treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0211] In some embodiments, the composition comprising the cells can be administered to a subject, e.g., a human subject, who has, is suspected of having, or is at risk for a disease or disorder. In some embodiments, the composition can be administered to a subject who does not have, is not suspected of having, or is not at risk for a disease or disorder. In some embodiments, the subject is a healthy human. In some embodiments, the subject, e.g., a human subject, has, is suspected of having, or is at risk for a genetic disease or disorder. In some embodiments, the subject is suffering from or at risk for developing symptoms indicative of a disease or disorder. In some embodiments, the disease is diabetes, e.g., type I diabetes or type II diabetes. In some embodiments, the disorder is pancreatic resection. VI. CERTAIN COMPOSITIONS AND METHODS OF THE DISCLOSURE

[0212] Accordingly, the present disclosure specifically relates to the following non-limiting compositions and methods:

[0213] In a first composition, Composition 1, the present disclosure provides a composition comprising genetically modified cells comprising: (a) a disrupted B2M gene and a first insertion of a first polynucleotide encoding midbrain astrocyte-derived neurotrophic factor (MANF) into the disrupted B2M gene; and (b) a disrupted TXNIP gene and a second insertion of a second polynucleotide encoding tumor necrosis factor alpha-induced protein 3 (TNFAIP3) into the disrupted TXNIP gene, wherein the cells express MANF and TNFAIP3 and have disrupted expression of B2M and TXNIP.

[0214] In another composition, Composition 2, the present disclosure provides a composition according to Composition 1, wherein the first insert further comprises a third polynucleotide encoding an HLA class I histocompatibility antigen, alpha chain E (HLA-E).

[0215] In another composition, Composition 3, the present disclosure provides a composition according to Composition 1 or 2, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer, the HLA-E trimer comprising a B2M membrane protein fused to HLA-E without a signal peptide, an HLA-G-presenting peptide fused thereto, and a B2M signal peptide fused thereto.

[0216] In another composition, composition 4, the present disclosure provides a composition according to any one of compositions 1 to 3, wherein a first polynucleotide encoding MANF is linked to a third polynucleotide encoding HLA-E by a polynucleotide encoding a P2A peptide, such that the first insert comprises a MANF-P2A-HLA-E construct.

[0217] In another composition, Composition 5, the present disclosure provides a composition according to Composition 4, wherein the MANF-P2A-HLA-E construct comprises a polynucleotide sequence consisting essentially of SEQ ID NO:55.

[0218] In another composition, composition 6, the present disclosure provides a composition according to composition 4 or 5, wherein the MANF-P2A-HLA-E construct is operably linked to an exogenous promoter.

[0219] In another composition, Composition 7, the present disclosure provides a composition according to any one of Compositions 1 to 6, wherein the second insert further comprises a fourth polynucleotide encoding programmed death-ligand 1 (PD-L-1).

[0220] In another composition, Composition 8, the present disclosure provides a composition according to any one of Compositions 1 to 7, wherein a second nucleotide sequence encoding TNFAIP3 is linked to a fourth nucleotide sequence encoding PD-L-1 by a nucleotide sequence encoding a P2A peptide, such that the second insert comprises a TNFAIP3-P2A-PD-L-1 construct.

[0221] In another composition, Composition 9, the present disclosure provides a composition according to Composition 8, wherein the TNFAIP3-P2A-PD-L-1 construct comprises a nucleotide sequence consisting essentially of SEQ ID NO:54.

[0222] In another composition, composition 10, the present disclosure provides a composition according to composition 8 or 9, wherein the TNFAIP3-P2A-PD-L-1 construct is operably linked to an exogenous promoter.

[0223] In another composition, composition 11, the present disclosure provides a composition according to any one of compositions 1 to 10, wherein disrupting expression of B2M and TXNIP includes reducing or eliminating expression of B2M and / or TXNIP.

[0224] In another composition, composition 12, the present disclosure provides a composition according to any one of compositions 1 to 11, wherein the universal donor cells have increased immune evasion and / or post-transplant survival compared to comparable cells without the polynucleotide insertion and gene disruption.

[0225] In another composition, composition 13, the present disclosure provides a composition according to any one of compositions 1 to 12, wherein the cells are stem cells.

[0226] In another composition, composition 14, the present disclosure provides a composition according to composition 13, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0227] In another composition, composition 15, the present disclosure provides a composition according to any one of compositions 1 to 12, wherein the cell is a differentiated cell or a somatic cell.

[0228] In another composition, composition 16, the present disclosure provides a composition according to composition 15, wherein the cells are differentiated into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0229] In another composition, composition 17, the present disclosure provides a composition according to composition 16, wherein the lineage-restricted progenitor cell is a definitive endoderm cell, an undifferentiated intestinal cell, a posterior foregut cell, a pancreatic endoderm precursor, a pancreatic endocrine precursor, or an immature beta cell, and the fully differentiated somatic cell is a beta cell.

[0230] In another composition, composition 18, the present disclosure provides a composition comprising a plurality of genetically modified cells according to any one of compositions 1 through 17.

[0231] In another composition, composition 19, the present disclosure provides a composition comprising a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from a plurality of genetically modified cells of composition 18.

[0232] In another composition, composition 20, the present disclosure provides a composition according to composition 19, wherein the population comprises definitive endoderm cells, undifferentiated intestinal cells, posterior foregut cells, pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells.

[0233] In another composition, composition 21, the present disclosure provides a composition comprising a plurality of cells of composition 18 or a population of cells of composition 19, and at least one pharmaceutically acceptable excipient.

[0234] In a first method, Method 22, the present disclosure provides a method, Method 22, for treating a pancreatic disease or disorder in a subject in need thereof, comprising: (a) obtaining or obtaining a population of lineage-restricted progenitor cells or fully differentiated somatic cells according to claim 19, wherein the lineage-restricted progenitor cells or fully differentiated somatic cells comprise pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells; and (b) administering the pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, and / or beta cells to the subject.

[0235] In another method, Method 23, the present disclosure provides a method, Method 23, for treating a pancreatic disease or disorder in a subject in need thereof, comprising: (a) obtaining or obtaining a plurality of genetically modified cells of claim 18, wherein the plurality of genetically modified cells comprises stem cells; (b) differentiating the genetically modified cells into pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells; and (c) administering the pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells to the subject.

[0236] In another method, method 24, the present disclosure provides the method provided in method 22 or 23, wherein the pancreatic disease or disorder is type I diabetes, type II diabetes, or pancreatectomy.

[0237] In another composition, Composition 25, the present disclosure provides a composition comprising genetically modified cells: (a) comprising a first exogenous polynucleotide insertion encoding midbrain astrocyte-derived neurotrophic factor (MANF), a second exogenous polynucleotide insertion encoding tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a third exogenous polynucleotide insertion encoding cluster of differentiation 39 (CD39), and / or a fourth exogenous polynucleotide insertion encoding cluster of differentiation 73 (CD73), wherein the genetically modified cells express CD39, MANF, TNFAIP3, and / or CD73; and / or (b) comprising a disruption in a gene encoding transforming growth factor beta (TGFβ) protein, beta2-microglobulin (B2M) protein, thioredoxin-interacting protein (TXNIP) protein, and / or class II transactivator of differentiation (CIITA) protein, wherein the disruption in expression of the TGFβ protein, B2M protein, TXNIP protein, and / or CIITA protein is present.

[0238] In another composition, composition 26, the present disclosure provides a composition according to composition 25, wherein the genetically modified cell comprises a first exogenous polynucleotide encoding MANF and expresses MANF.

[0239] In another composition, composition 27, the present disclosure provides a composition according to composition 25 or 26, wherein the first exogenous polynucleotide is operably linked to an exogenous promoter.

[0240] In another composition, composition 28, the present disclosure provides a composition according to any one of compositions 25 to 27, wherein the first exogenous polynucleotide comprises a nucleotide sequence consisting essentially of SEQ ID NO:17.

[0241] In another composition, composition 29, the present disclosure provides a composition according to any one of compositions 25 to 28, wherein the genetically modified cell comprises a second exogenous polynucleotide encoding TNFAIP3 and expresses TNFAIP3.

[0242] In another composition, composition 30, the present disclosure provides a composition according to any one of compositions 25 to 29, wherein the second exogenous polynucleotide is operably linked to an exogenous promoter.

[0243] In another composition, composition 31, the present disclosure provides a composition according to any one of compositions 25 to 30, wherein the second exogenous polynucleotide comprises a nucleotide sequence consisting essentially of SEQ ID NO:19.

[0244] In another composition, composition 32, the present disclosure provides a composition according to any one of compositions 25 to 31, wherein the genetically modified cells comprise a third exogenous polynucleotide encoding CD39 and express CD39.

[0245] In another composition, composition 33, the present disclosure provides a composition according to any one of compositions 25 to 32, wherein the third exogenous polynucleotide is operably linked to an exogenous promoter.

[0246] In another composition, composition 34, the present disclosure provides a composition according to any one of compositions 25 to 33, wherein the third exogenous polynucleotide comprises a nucleotide sequence consisting essentially of SEQ ID NO:27.

[0247] In another composition, composition 35, the present disclosure provides a composition according to any one of compositions 25 to 34, wherein the genetically modified cells comprise a fourth exogenous polynucleotide encoding CD73 and express CD73.

[0248] In another composition, composition 36, the present disclosure provides a composition according to any one of compositions 25 to 35, wherein a fourth exogenous polynucleotide encoding CD73 is operably linked to an exogenous promoter.

[0249] In another composition, composition 37, the present disclosure provides a composition according to any one of compositions 25 to 36, wherein the fourth exogenous polynucleotide encoding CD73 comprises a nucleotide sequence consisting essentially of SEQ ID NO:46.

[0250] In another composition, composition 38, the present disclosure provides a composition according to any one of compositions 25 to 37, wherein the genetically modified cells comprise a third exogenous polynucleotide encoding CD39 and a fourth exogenous polynucleotide encoding CD73, and express CD39 and CD73, and the third exogenous polynucleotide encoding CD39 is linked to the fourth exogenous polynucleotide encoding CD73 by a polynucleotide encoding a P2A peptide, such that the third exogenous polynucleotide encoding CD39, the polynucleotide encoding the P2A peptide, and the fourth exogenous polynucleotide encoding CD73 form a CD39-P2A-CD73 construct.

[0251] In another composition, composition 39, the present disclosure provides a composition according to composition 38, wherein the CD39-P2A-CD73 construct is operably linked to an exogenous promoter.

[0252] In another composition, composition 40, the present disclosure provides a composition according to composition 38 or 39, wherein the CD39-P2A-CD73 construct comprises a nucleotide sequence consisting essentially of SEQ ID NO:58.

[0253] In another composition, composition 41, the present disclosure provides a composition according to any one of compositions 25 to 40, further comprising a fifth exogenous polynucleotide encoding an HLA class I histocompatibility antigen, alpha chain E (HLA-E), wherein the genetically modified cells express HLA-E.

[0254] In another composition, composition 42, the present disclosure provides a composition according to composition 41, wherein the fifth exogenous polynucleotide encoding HLA-E comprises a polynucleotide encoding an HLA-E trimer, the HLA-E trimer comprising a B2M membrane protein fused to HLA-E without a signal peptide, an HLA-G-presenting peptide fused thereto, and a B2M signal peptide fused thereto.

[0255] In another composition, composition 43, the present disclosure provides a composition according to composition 41 or 42, wherein a fifth exogenous polynucleotide encoding HLA-E is operably linked to an exogenous promoter.

[0256] In another composition, composition 44, the present disclosure provides a composition according to any one of compositions 41 to 43, wherein the fifth exogenous polynucleotide encoding HLA-E comprises a nucleotide sequence consisting essentially of SEQ ID NO: 43.

[0257] In another composition, composition 45, the present disclosure provides a composition according to any one of compositions 41 to 44, comprising a first exogenous polynucleotide encoding MANF and a fifth exogenous polynucleotide encoding HLA-E, wherein the first exogenous polynucleotide encoding MANF is linked to the fifth exogenous polynucleotide encoding HLA-E by a polynucleotide encoding a P2A peptide, such that the first exogenous polynucleotide, the polynucleotide encoding the P2A peptide, and the fifth exogenous polynucleotide form a MANF-P2A-HLA-E construct.

[0258] In another composition, composition 46, the present disclosure provides a composition according to composition 45, wherein the MANF-P2A-HLA-E construct is operably linked to an exogenous promoter.

[0259] In another composition, composition 47, the present disclosure provides a composition according to composition 45 or 46, wherein the MANF-P2A-HLA-E construct comprises a polynucleotide sequence consisting essentially of SEQ ID NO:55.

[0260] In another composition, composition 48, the present disclosure provides a composition according to any one of compositions 25 to 47, further comprising a sixth exogenous polynucleotide encoding programmed death-ligand 1 (PD-L-1), wherein the genetically modified cells express PD-L-1.

[0261] In another composition, composition 49, the present disclosure provides a composition according to composition 48, wherein a sixth exogenous polynucleotide encoding PD-L-1 is operably linked to an exogenous promoter.

[0262] In another composition, composition 50, the present disclosure provides a composition according to composition 48 or 49, wherein the sixth exogenous polynucleotide comprises a nucleotide sequence consisting essentially of SEQ ID NO:20.

[0263] In another composition, composition 51, the present disclosure provides a composition according to any one of compositions 48 to 50, comprising a second exogenous polynucleotide encoding TNFAIP3 and a sixth exogenous polynucleotide encoding PD-L-1, wherein the second exogenous polynucleotide encoding TNFAIP3 is linked to the sixth exogenous polynucleotide sequence encoding PD-L-1 by a polynucleotide encoding a P2A peptide, such that the second exogenous polynucleotide, the polynucleotide encoding the P2A peptide, and the sixth exogenous polynucleotide form a TNFAIP3-P2A-PD-L-1 construct.

[0264] In another composition, composition 52, the present disclosure provides a composition according to composition 51, wherein the TNFAIP3-P2A-PD-L-1 construct is operably linked to an exogenous promoter.

[0265] In another composition, composition 53, the present disclosure provides a composition according to composition 51 or 52, wherein the TNFAIP3-P2A-PD-L-1 construct comprises a nucleotide sequence consisting essentially of SEQ ID NO:54.

[0266] In another composition, composition 54, the present disclosure provides a composition according to any one of compositions 48 to 53, comprising a third exogenous polynucleotide encoding CD39 and a sixth exogenous polynucleotide encoding PD-L-1, wherein the third exogenous polynucleotide encoding CD39 is linked to the sixth exogenous polynucleotide encoding PD-L-1 by a polynucleotide encoding a P2A peptide, such that the third exogenous polynucleotide, the polynucleotide encoding the P2A peptide, and the sixth exogenous polynucleotide form a CD39-P2A-PD-L-1 construct.

[0267] In another composition, composition 55, the present disclosure provides a composition according to composition 54, wherein the CD39-P2A-PD-L-1 construct is operably linked to an exogenous promoter.

[0268] In another composition, composition 56, the present disclosure provides a composition according to composition 54 or 55, wherein the CD39-P2A-PD-L-1 construct comprises a nucleotide sequence consisting essentially of SEQ ID NO:53.

[0269] In another composition, composition 57, the present disclosure provides a composition according to any one of compositions 48 to 56, comprising a first exogenous polynucleotide encoding MANF, a second exogenous polynucleotide encoding TNFAIP3, and a sixth exogenous polynucleotide encoding PD-L-1, wherein the first exogenous polynucleotide is linked to the second exogenous polynucleotide by a first polynucleotide encoding a P2A peptide, and the second exogenous polynucleotide is linked to the sixth exogenous polynucleotide by a second polynucleotide encoding the P2A peptide, such that the first exogenous polynucleotide encoding MANF, the first polynucleotide encoding the P2A peptide, the second exogenous polynucleotide encoding TNFAIP3, the second polynucleotide encoding the P2A peptide, and the sixth exogenous polynucleotide encoding PD-L-1 form a MANF-P2A-TNFAIP3-P2A-PD-L-1 construct.

[0270] In another composition, composition 58, the present disclosure provides a composition according to composition 57, wherein MANF-P2A-TNFAIP3-P2A-PD-L-1 is operably linked to an exogenous promoter.

[0271] In another composition, composition 59, the present disclosure provides a composition according to composition 57 or 58, wherein the MANF-P2A-TNFAIP3-P2A-PD-L-1 construct comprises a nucleotide sequence consisting essentially of SEQ ID NO:52.

[0272] In another composition, composition 60, the present disclosure provides a composition according to any one of compositions 48 to 59, comprising a third exogenous polynucleotide encoding CD39, a fourth exogenous polynucleotide encoding CD73, and a sixth exogenous polynucleotide encoding PD-L-1, wherein the third exogenous polynucleotide encoding CD39 is linked to the fourth exogenous polynucleotide encoding CD73 by a first polynucleotide encoding a P2A peptide, and the fourth exogenous polynucleotide is linked to the sixth exogenous polynucleotide by a second polynucleotide encoding the P2A peptide, such that the third exogenous polynucleotide encoding CD39, the first polynucleotide encoding the P2A peptide, the fourth exogenous polynucleotide encoding CD73, the second polynucleotide encoding the P2A peptide, and the sixth exogenous polynucleotide encoding PD-L-1 form a CD39-P2A-CD73-P2A-PD-L-1 construct.

[0273] In another composition, composition 61, the present disclosure provides a composition according to composition 60, wherein the CD39-P2A-CD73-P2A-PD-L-1 construct is operably linked to an exogenous promoter.

[0274] In another composition, composition 62, the present disclosure provides a composition according to composition 60 or 61, wherein the CD39-P2A-CD73-P2A-PD-L-1 construct comprises a nucleotide sequence consisting essentially of SEQ ID NO:56.

[0275] In another composition, composition 63, the present disclosure provides a composition according to any one of compositions 25 to 62, wherein the genetically modified cells comprise a disrupted B2M gene, a disrupted TXNIP gene, a disrupted CIITA gene, and / or a disrupted TGFβ gene, and the cells have disrupted expression of B2M protein, TXNIP protein, CIITA protein and / or TGFβ protein.

[0276] In another composition, composition 64, the present disclosure provides a composition according to any one of compositions 25 to 63, wherein a first exogenous polynucleotide encoding MANF, a second exogenous polynucleotide encoding TNFAIP3, a third exogenous polynucleotide encoding CD39, and / or a fourth exogenous polynucleotide encoding CD73 are each independently inserted within or near the B2M gene, the TXNIP gene, the CIITA gene, and / or the TGFβ gene, thereby disrupting expression of the B2M protein, the TXNIP protein, the CIITA protein, and / or the TGFβ protein.

[0277] In another composition, composition 65, the present disclosure provides a composition according to any one of compositions 25 to 64, further comprising a fifth exogenous polynucleotide encoding an HLA class I histocompatibility antigen, alpha chain E (HLA-E), wherein the fifth exogenous polynucleotide encoding HLA-E is inserted within or near the B2M gene, the TXNIP gene, the CIITA gene, and / or the TGFβ gene, thereby disrupting expression of the B2M protein, the TXNIP protein, the CIITA protein, and / or the TGFβ protein, and the universal donor cells further express HLA-E.

[0278] In another composition, Composition 66, the present disclosure provides a composition according to any one of Compositions 25 to 65, further comprising a sixth exogenous polynucleotide encoding programmed death-ligand 1 (PD-L-1), wherein the sixth exogenous polynucleotide encoding PD-L-1 is inserted within or near the B2M gene, the TXNIP gene, the CIITA gene, and / or the TGFβ gene, thereby disrupting expression of the B2M protein, the TXNIP protein, the CIITA protein, and / or the TGFβ protein, and wherein the universal donor cells express PD-L-1.

[0279] In another composition, composition 67, the present disclosure provides a composition according to any one of compositions 64 to 66, wherein a first exogenous polynucleotide encoding MANF is inserted within or near a B2M gene, a TXNIP gene, or a CIITA gene, and / or a TGFβ gene.

[0280] In another composition, composition 68, the present disclosure provides a composition according to any one of compositions 64 to 67, wherein a first exogenous polynucleotide encoding MANF is inserted within or near the B2M gene and / or the TXNIP gene, thereby disrupting expression of the B2M protein and / or the TXNIP protein.

[0281] In another composition, composition 69, the present disclosure provides a composition according to compositions 64 to 68, wherein a second exogenous polynucleotide encoding TNFAIP3 is inserted within or near the B2M gene, the TXNIP gene, or the CIITA gene, and / or the TGFβ gene.

[0282] In another composition, composition 70, the present disclosure provides a composition according to compositions 64 to 69, wherein a second exogenous polynucleotide encoding TNFAIP3 is inserted within or near the B2M gene, thereby disrupting expression of the B2M protein.

[0283] In another composition, composition 71, the present disclosure provides a composition according to any one of compositions 64 to 70, wherein a third exogenous polynucleotide encoding CD39 is inserted within or near the B2M gene, the TXNIP gene, the CIITA gene, and / or the TGFβ gene.

[0284] In another composition, composition 72, the present disclosure provides a composition according to any one of compositions 64 to 71, wherein a third exogenous polynucleotide encoding CD39 is inserted within or near the CITTA gene and / or the B2M gene, thereby disrupting expression of the CITTA protein and / or the B2M protein.

[0285] In another composition, composition 73, the present disclosure provides a composition according to any one of compositions 64 to 72, wherein a fourth exogenous polynucleotide encoding CD73 is inserted within or near the B2M gene, the TXNIP gene, the CIITA gene, and / or the TGFβ gene.

[0286] In another composition, composition 74, the present disclosure provides a composition according to any one of compositions 64 to 73, wherein a fourth exogenous polynucleotide encoding CD73 is inserted within or near the B2M gene, thereby disrupting expression of the B2M protein.

[0287] In another composition, composition 75, the present disclosure provides a composition according to any one of compositions 64 to 74, comprising a MANF-P2A-HLA-E construct, wherein the MANF-P2A-HLA-E construct is inserted within or near the B2M gene, the TXNIP gene, the CIITA gene, or the TGFβ gene, thereby disrupting expression of the B2M protein, the TXNIP protein, the CIITA protein, and / or the TGFβ protein.

[0288] In another composition, composition 76, the present disclosure provides a composition according to any one of compositions 64 to 75, in which the MANF-P2A-HLA-E construct is inserted within or near the TXNIP gene, thereby disrupting expression of the TXNIP protein.

[0289] In another composition, composition 77, the present disclosure provides a composition according to any one of compositions 64 to 76, comprising a TNFAIP3-P2A-PD-L-1 construct, wherein the TNFAIP3-P2A-PD-L-1 construct is inserted within or near the B2M gene, the TXNIP gene, the CIITA gene, and / or the TGFβ gene, thereby disrupting expression of the B2M protein, the TXNIP protein, the CIITA protein, and / or the TGFβ protein.

[0290] In another composition, composition 78, the present disclosure provides a composition according to any one of compositions 64 to 77, wherein the TNFAIP3-P2A-PD-L-1 construct is inserted within or near the B2M gene, thereby disrupting expression of the B2M protein.

[0291] In another composition, composition 79, the present disclosure provides a composition according to any one of compositions 64 to 78, comprising a CD39-P2A-PD-L-1 construct, wherein the CD39-P2A-PD-L-1 construct is inserted within or near the B2M gene, the TXNIP gene, the CIITA gene, and / or the TGFβ gene, thereby disrupting expression of the B2M protein, the TXNIP protein, the CIITA protein, and / or the TGFβ protein.

[0292] In another composition, composition 80, the present disclosure provides a composition according to any one of compositions 64 to 79, wherein the CD39-P2A-PD-L-1 construct is inserted within or near the B2M gene, thereby disrupting expression of the B2M protein.

[0293] In another composition, composition 81, the present disclosure provides a composition according to any one of compositions 64 to 80, comprising a MANF-P2A-TNFAIP3-P2A-PD-L-1 construct, wherein the MANF-P2A-TNFAIP3-P2A-PD-L-1 construct is inserted within or near the B2M gene, the TXNIP gene, the CIITA gene, and / or the TGFβ gene, thereby disrupting expression of the B2M protein, the TXNIP protein, the CIITA protein, and / or the TGFβ protein.

[0294] In another composition, composition 82, the present disclosure provides a composition according to any one of compositions 64 to 81, wherein the MANF-P2A-TNFAIP3-P2A-PD-L-1 construct is inserted within or near the B2M gene, thereby disrupting expression of the B2M protein.

[0295] In another composition, composition 83, the present disclosure provides a composition according to any one of compositions 64 to 82, comprising a CD39-P2A-CD73 construct, wherein the CD39-P2A-CD73 construct is inserted within or near the B2M gene, the TXNIP gene, the CIITA gene, and / or the TGFβ gene, thereby disrupting expression of the B2M protein, the TXNIP protein, the CIITA protein, and / or the TGFβ protein.

[0296] In another composition, composition 84, the present disclosure provides a composition according to any one of compositions 64 to 83, wherein the CD39-P2A-CD73 construct is inserted within or near the B2M gene, thereby disrupting expression of the B2M protein.

[0297] In another composition, composition 85, the present disclosure provides a composition according to any one of compositions 64 to 84, comprising a CD39-P2A-CD73-P2A-PD-L-1 construct, wherein the CD39-P2A-CD73-P2A-PD-L-1 construct is inserted within or near the B2M gene, the TXNIP gene, the CIITA gene, and / or the TGFβ gene, thereby disrupting expression of the B2M protein, the TXNIP protein, the CIITA protein, and / or the TGFβ protein.

[0298] In another composition, composition 86, the present disclosure provides a composition according to any one of compositions 64 to 85, wherein the CD39-P2A-CD73-P2A-PD-L-1 construct is inserted within or near the B2M gene, thereby disrupting expression of the B2M protein.

[0299] In another composition, composition 87, the present disclosure provides a composition according to any one of compositions 25 to 86, wherein disrupting expression of B2M protein, TXNIP protein, CIITA protein, and / or TGFβ protein includes reducing or eliminating expression of B2M protein, TXNIP protein, CIITA protein, and / or TGFβ protein.

[0300] In another composition, composition 88, the present disclosure provides a composition according to any one of compositions 25 to 87, wherein the TGFβ protein is TGFβ-2.

[0301] In another composition, composition 89, the present disclosure provides a composition according to any one of compositions 25 to 88, wherein the cells are stem cells.

[0302] In another composition, composition 90, the present disclosure provides a composition according to composition 89, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0303] In another composition, composition 91, the present disclosure provides a composition according to any one of compositions 25 to 88, wherein the cell is a differentiated cell or a somatic cell.

[0304] In another composition, composition 92, the present disclosure provides a composition according to composition 91, wherein the cell is a lineage-restricted progenitor cell or a fully differentiated somatic cell.

[0305] In another composition, composition 93, the present disclosure provides a composition according to composition 92, wherein the lineage-restricted progenitor cell is a definitive endoderm cell, an undifferentiated intestinal cell, a posterior foregut cell, a pancreatic endoderm progenitor cell, a pancreatic endocrine progenitor cell, a pancreatic endocrine cell, or an immature beta cell, and the fully differentiated somatic cell is a pancreatic beta cell.

[0306] In another composition, composition 94, the present disclosure provides a composition comprising a plurality of genetically modified cells according to any one of claims 1-93.

[0307] In another composition, composition 95, the present disclosure provides a composition according to composition 94, wherein at least about 50% of the cells express MANF, HLA-E, TNFAIP3, PD-L-1, CD39 and / or CD73.

[0308] In another composition, composition 96, the present disclosure provides a composition comprising a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from a plurality of genetically modified cells of composition 94 or 95.

[0309] In another composition, composition 97, the present disclosure provides a composition according to composition 96, wherein the lineage-restricted progenitor cell is a definitive endoderm cell, an undifferentiated intestinal cell, a posterior foregut cell, a pancreatic endoderm precursor, a pancreatic endocrine precursor, or an immature beta cell, and the fully differentiated somatic cell is a pancreatic beta cell.

[0310] In another composition, composition 98, the present disclosure provides a composition according to composition 96 or 97, wherein at least about 50% of the cells express MANF, HLA-E, TNFAIP3, PD-L-1, CD39 and / or CD73.

[0311] In another composition, composition 99, the present disclosure provides a composition comprising a plurality of cells of composition 94 or 95 or a population of cells of any one of compositions 96-98.

[0312] In another composition, composition 100, the present disclosure provides a composition according to composition 99, further comprising at least one pharmaceutically acceptable excipient.

[0313] In another composition, composition 101, the present disclosure provides a composition according to composition 99 or 100 for use in treating a pancreatic disease or disorder.

[0314] In another composition, composition 102, the present disclosure provides a composition according to composition 101, wherein the pancreatic disease or disorder is type I diabetes, type II diabetes, or pancreatectomy.

[0315] In another composition, composition 103, the present disclosure provides a composition according to composition 101 or 102, wherein the human comprises a pancreatic disease or disorder.

[0316] In another method, method 104, the present disclosure provides a method for treating a subject in need thereof, comprising: (a) obtaining or obtaining a plurality of genetically modified cells of claim 94 or 95 after differentiation into pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, and / or pancreatic beta cells; and (b) administering the pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, and / or pancreatic beta cells to the subject.

[0317] In another method, method 105, the present disclosure provides a method for treating a subject in need thereof, comprising: (a) obtaining or obtaining a population of lineage-restricted progenitor cells or fully differentiated somatic cells according to any one of claims 96-98, wherein the lineage-restricted progenitor cells or fully differentiated somatic cells comprise pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells; and (b) administering the pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, and / or beta cells to the subject.

[0318] In another method, method 106, the present disclosure provides a method for treating a subject in need thereof, comprising: (a) obtaining or obtaining a plurality of genetically modified cells of claim 94 or 95, wherein the plurality of genetically modified cells comprises stem cells; (b) differentiating the genetically modified cells into pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells; and (c) administering the pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells to the subject.

[0319] In another method, Method 107, the present disclosure provides the method provided in any one of Methods 104 to 106, wherein the administering step includes implanting into the subject a device comprising pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, or mature beta cells.

[0320] In another method, method 108, the present disclosure provides the method provided in any one of methods 104 to 107, wherein the subject has, is suspected of having, or is at risk for a pancreatic disease or disorder.

[0321] In another method, method 109, the present disclosure provides the method provided in method 108, wherein the pancreatic disease or disorder is type I diabetes, type II diabetes, or pancreatectomy.

[0322] In another method, method 110, the present disclosure provides the method provided in any one of methods 104 to 109, wherein the subject is a human.

[0323] In another composition, composition 111, the present disclosure provides a composition comprising a genetically modified cell comprising (a) a first polynucleotide encoding midbrain astrocyte-derived neurotrophic factor (MANF) inserted within or near a gene encoding thioredoxin interacting protein (TXNIP), and (b) a second polynucleotide encoding tumor necrosis factor alpha-induced protein 3 (TNFAIP3) inserted within or near a gene encoding beta2-microglobulin (B2M), wherein the genetically modified cell expresses MANF and TNFAIP3 and has disrupted expression of TXNIP and B2M.

[0324] In another composition, composition 112, the present disclosure provides a composition according to composition 111, wherein disrupting expression of B2M and TXNIP comprises reducing or eliminating expression of B2M and / or TXNIP.

[0325] In another composition, composition 113, the present disclosure provides a composition according to composition 111 or 112, further comprising a third polynucleotide encoding an HLA class I histocompatibility antigen, alpha chain E (HLA-E), inserted within or near the TXNIP gene.

[0326] In another composition, composition 114, the present disclosure provides a composition according to composition 113, wherein the third polynucleotide encoding HLA-E comprises a polynucleotide encoding an HLA-E trimer, the HLA-E trimer comprising a B2M membrane protein fused to HLA-E without a signal peptide, an HLA-G-presenting peptide fused thereto, and a B2M signal peptide fused thereto.

[0327] In another composition, composition 115, the present disclosure provides a composition according to composition 113 or 114, wherein a first polynucleotide encoding MANF and a third polynucleotide encoding HLA-E are operably linked to an exogenous promoter.

[0328] In another composition, composition 116, the present disclosure provides a composition according to any one of compositions 113 to 115, wherein a first polynucleotide encoding MANF is linked to a third polynucleotide encoding HLA-E by a polynucleotide encoding a P2A peptide, such that the first polynucleotide, the polynucleotide encoding the P2A peptide, and the third polynucleotide form a MANF-P2A-HLA-E construct.

[0329] In another composition, composition 117, the present disclosure provides a composition according to composition 116, wherein the MANF-P2A-HLA-E construct comprises a polynucleotide sequence consisting essentially of SEQ ID NO:55.

[0330] In another composition, composition 118, the present disclosure provides a composition according to any one of compositions 111 to 117, further comprising a fourth polynucleotide encoding programmed death-ligand 1 (PD-L-1) inserted within or near the B2M gene.

[0331] In another composition, composition 119, the present disclosure provides a composition according to composition 118, wherein the second polynucleotide encoding TNFAIP3 and the fourth polynucleotide encoding PD-L-1 are operably linked to an exogenous promoter.

[0332] In another composition, composition 120, the present disclosure provides a composition according to composition 118 or 119, wherein a second polynucleotide encoding TNFAIP3 is linked to a fourth polynucleotide sequence encoding PD-L-1 and by a polynucleotide encoding a P2A peptide, such that the second polynucleotide, the polynucleotide encoding the P2A peptide, and the fourth polynucleotide form a TNFAIP3-P2A-PD-L-1 construct.

[0333] In another composition, composition 121, the present disclosure provides a composition according to composition 120, wherein the TNFAIP3-P2A-PD-L-1 construct comprises a nucleotide sequence consisting essentially of SEQ ID NO:54.

[0334] In another composition, composition 122, the present disclosure provides a composition according to any one of compositions 111 to 121, wherein the cells are stem cells.

[0335] In another composition, composition 123, the present disclosure provides a composition according to composition 112, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0336] In another composition, composition 124, the present disclosure provides a composition according to any one of compositions 111 to 121, wherein the cell is a differentiated cell or a somatic cell.

[0337] In another composition, composition 125, the present disclosure provides a composition according to composition 124, wherein the cell is a lineage-restricted progenitor cell or a fully differentiated somatic cell.

[0338] In another composition, composition 126, the present disclosure provides a composition according to composition 125, wherein the lineage-restricted progenitor cell is a definitive endoderm cell, an undifferentiated intestinal cell, a posterior foregut cell, a pancreatic endoderm progenitor cell, a pancreatic endocrine progenitor cell, a pancreatic endocrine cell, or an immature beta cell, and the fully differentiated somatic cell is a pancreatic beta cell.

[0339] In another composition, composition 127, the present disclosure provides a composition comprising a plurality of genetically modified cells of any one of claims 111-126.

[0340] In another composition, composition 128, the present disclosure provides a composition according to composition 127, wherein at least about 50% of the cells express MANF, HLA-E, TNFAIP3, and / or PD-L-1.

[0341] In another composition, composition 129, the present disclosure provides a composition comprising a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from a plurality of genetically modified cells of composition 127 or 128.

[0342] In another composition, composition 130, the present disclosure provides a composition according to composition 129, wherein the lineage-restricted progenitor cell is a definitive endoderm cell, an undifferentiated intestinal cell, a posterior foregut cell, a pancreatic endoderm precursor, a pancreatic endocrine precursor, or an immature beta cell, and the fully differentiated somatic cell is a pancreatic beta cell.

[0343] In another composition, composition 131, the present disclosure provides a composition according to composition 129 or 130, wherein at least about 50% of the cells express MANF, HLA-E, TNFAIP3, and / or PD-L-1.

[0344] In another composition, composition 132, the present disclosure provides a composition comprising a plurality of cells of composition 17 or 18 or a population of cells of any one of compositions 130 or 131.

[0345] In another composition, composition 133, the present disclosure provides a composition according to composition 132, further comprising at least one pharmaceutically acceptable excipient.

[0346] In another composition, composition 134, the present disclosure provides a composition according to composition 132 or 133 for use in treating a pancreatic disease or disorder.

[0347] In another composition, composition 135, the present disclosure provides a composition according to composition 134, wherein the human comprises a pancreatic disease or disorder.

[0348] In another composition, composition 136, the present disclosure provides a composition according to composition 134 or 135, wherein the pancreatic disease or disorder is type I diabetes, type II diabetes, or pancreatectomy.

[0349] In another method, method 137, the present disclosure provides a method for treating a subject in need thereof, comprising: (a) obtaining or obtaining a plurality of genetically modified cells described in any one of claims 111 to 139 after differentiation into pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, and / or pancreatic beta cells; and (b) administering the pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, and / or pancreatic beta cells to the subject.

[0350] In another method, method 138, the present disclosure provides a method for treating a subject in need thereof, comprising: (a) obtaining or obtaining a population of lineage-restricted progenitor cells or fully differentiated somatic cells according to claim 129, wherein the lineage-restricted progenitor cells or fully differentiated somatic cells comprise pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells; and (b) administering the pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, and / or beta cells to the subject.

[0351] In another method, method 139, the present disclosure provides a method for treating a subject in need thereof, the method comprising: (a) obtaining or obtaining a plurality of genetically modified cells of claim 127 or 128, wherein the plurality of genetically modified cells comprises stem cells; (b) differentiating the genetically modified cells into pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells; and (c) administering the pancreatic endoderm cells, pancreatic endocrine precursor cells, immature beta cells, and / or pancreatic beta cells to the subject.

[0352] In another method, method 140, the present disclosure provides the method provided in any one of methods 137 to 139, wherein the administering step includes implanting into the subject a device comprising pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, or mature beta cells.

[0353] In another method, method 141, the present disclosure provides a method as provided in any one of methods 137 to 140, wherein the subject has, is suspected of having, or is at risk for a pancreatic disease or disorder.

[0354] In another method, method 142, the present disclosure provides the method provided in method 141, wherein the pancreatic disease or disorder is type I diabetes, type II diabetes, or pancreatectomy.

[0355] In another method, method 143, the present disclosure provides the method provided in any one of methods 137 to 142, wherein the subject is a human.

[0356] In another method, method 144, the present disclosure provides an in vitro method for generating universal donor cells. 1. An in vitro method comprising: (a) a first RNA-guided nuclease and a first guide RNA (gRNA) that targets a target site in a beta-2 microglobulin (B2M) gene locus; (b) a first vector comprising nucleic acids, the nucleic acid comprising: (i) a nucleotide sequence encoding tumor necrosis factor alpha-inducible protein 3 (TNFAIP3) and a nucleotide sequence encoding programmed death-ligand 1 (PD-L-1); (ii) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the B2M gene locus; and (iii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the B2M gene locus, wherein (i) is flanked by (ii) and (iii), the first vector cleaving the B2M gene locus at the target site and inserting into the B2M gene locus a nucleic acid comprising the nucleotide sequences encoding TNFAIP3 and PD-L-1, thereby disrupting the B2M gene; and (c) a second RNA-guided nuclease and a thioredoxin-interacting protein (TXNIP and (d) a second vector comprising a nucleic acid, wherein the nucleic acid comprises: (i) a nucleotide sequence encoding midbrain astrocyte-derived neurotrophic factor (MANF) and a nucleotide sequence encoding HLA class I histocompatibility antigen, alpha chain E (HLA-E); (ii) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the TXNIP locus; and (iii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the TXNIP locus, wherein (i) is flanked by (ii) and (iii), wherein the second vector cleaves the TXNIP locus at the target site and inserts a nucleic acid comprising nucleotide sequences encoding MANF and HLA-E into the TXNIP locus, thereby disrupting the TXNIP gene, wherein the universal donor cells express TNFAIP3, PD-L-1, MANF, and HLA-E and have disrupted expression of B2M and TXNIP.

[0357] In another method, method 145, the present disclosure provides an in vitro method as provided in method 144, wherein disrupting expression of B2M and TXNIP comprises reducing or eliminating expression of B2M and / or TXNIP.

[0358] In another method, Method 146, the present disclosure provides the in vitro method provided in Methods 144 or 145, wherein the nucleotide sequence of (b)(i) comprises a nucleotide sequence encoding P2A linked to a nucleotide sequence encoding PD-L-1, which in turn comprises a nucleotide sequence encoding TNFAIP3 linked thereto.

[0359] In another method, method 147, the present disclosure provides the in vitro method provided in any one of methods 144 to 146, wherein the nucleotide sequence of (b)(i) comprises SEQ ID NO:54.

[0360] In another method, method 148, the present disclosure provides the in vitro method provided in any one of methods 144 to 147, wherein the nucleotide sequence of (b)(i) is operably linked to an exogenous promoter.

[0361] In another method, method 149, the present disclosure provides the in vitro method provided in method 148, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0362] In another method, method 150, the present disclosure provides the in vitro method provided in any one of methods 144 to 149, wherein the nucleotide sequence of (b)(ii) comprises or consists essentially of SEQ ID NO:15.

[0363] In another method, method 151, the present disclosure provides the in vitro method provided in any one of methods 144 to 150, wherein the nucleotide sequence of (b)(iii) comprises or consists essentially of SEQ ID NO:22.

[0364] In another method, method 152, the present disclosure provides the in vitro method provided in any one of methods 144 to 151, wherein the first RNA-guided nuclease and the first gRNA are present in a ratio of about 1:1 to about 1:10.

[0365] In another method, method 153, the present disclosure provides the in vitro method provided in any one of methods 144 to 152, wherein the first RNA-guided nuclease is a first Cas9 nuclease.

[0366] In another method, method 154, the present disclosure provides the in vitro method provided in method 153, wherein the first Cas9 nuclease is linked to at least one nuclear localization signal.

[0367] In another method, method 155, the present disclosure provides the in vitro method provided in any one of methods 144 to 154, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer, the HLA-E trimer comprising a B2M membrane protein fused to HLA-E without a signal peptide, an HLA-G presenting peptide fused thereto, and a B2M signal peptide fused thereto.

[0368] In another method, Method 156, the present disclosure provides the in vitro method provided in any one of Methods 144 to 155, wherein the nucleotide sequence of (d)(i) comprises a nucleotide sequence encoding P2A linked to a nucleotide sequence encoding HLA-E, linked thereto a nucleotide sequence encoding MANF.

[0369] In another method, method 157, the present disclosure provides the in vitro method provided in any one of methods 144 to 156, wherein the nucleotide sequence of (d)(i) comprises SEQ ID NO:55.

[0370] In another method, method 158, the disclosure provides the in vitro method provided in any one of methods 144 to 157, wherein the nucleotide sequence of (d)(i) is operably linked to an exogenous promoter.

[0371] In another method, method 159, the present disclosure provides the in vitro method provided in method 158, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0372] In another method, method 160, the present disclosure provides the in vitro method provided in any one of methods 144 to 159, wherein the nucleotide sequence of (d)(ii) consists essentially of SEQ ID NO:42.

[0373] In another method, Method 161, the present disclosure provides the in vitro method provided in any one of Methods 144 to 160, wherein the nucleotide sequence of (d)(iii) consists essentially of SEQ ID NO:44.

[0374] In another method, method 162, the present disclosure provides the in vitro method provided in any one of methods 144 to 161, wherein the second RNA-guided nuclease and the second gRNA are present in a ratio of about 1:1 to about 1:10.

[0375] In another method, method 163, the present disclosure provides the in vitro method provided in any one of methods 144 to 162, wherein the second RNA-guided nuclease is a second Cas9 nuclease.

[0376] In another method, method 164, the present disclosure provides the in vitro method provided in method 163, wherein the second Cas9 nuclease is linked to at least one nuclear localization signal.

[0377] In another method, method 165, the present disclosure provides an in vitro method as provided in any one of methods 144 to 164, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0378] In another method, method 166, the present disclosure provides the in vitro method provided in any one of methods 144 to 165, wherein the stem cells are human stem cells.

[0379] In another method, method 167, the present disclosure provides an in vitro method as provided in any one of methods 144 to 166, wherein the universal donor cells have increased immune evasion and / or post-transplant survival compared to comparable cells that have not been subjected to nucleic acid insertion and gene disruption.

[0380] In another method, method 168, the disclosure provides an in vitro method for generating universal donor cells, comprising: (a) a first nucleic acid comprising an RNA-guided nuclease and a guide RNA (gRNA) targeting a target site in a first target locus, and a nucleotide sequence encoding tumor necrosis factor alpha-inducible protein 3 (TNFAIP3), midbrain astrocyte-derived neurotrophic factor (MANF), cluster of differentiation 39 (CD39), and / or cluster of differentiation 73 (CD73), wherein the first nucleic acid cleaves the first target locus at the target site and inserts the first nucleic acid comprising the nucleotide sequence encoding TNFAIP3, MANF, CD39, and / or CD73 into the target locus, thereby disrupting the target gene; and / or (b) an RNA-guided nuclease and a guide RNA (gRNA) targeting a target site in a beta-2 microglobulin (B2M) locus, wherein the guide RNA cleaves the B2M locus at the target site, thereby disrupting the B2M gene; and / or (c) a guide RNA (gRNA) that targets an RNA-guided nuclease and a target site in the thioredoxin interacting protein (TXNIP) locus, where the guide RNA cleaves the TXNIP locus at the target site, thereby disrupting the TXNIP gene; and / or (d) a guide RNA (gRNA) that targets an RNA-guided nuclease and a target site in the class II transactivator (CIITA) locus, where the guide RNA cleaves the CIITA locus at the target site, thereby disrupting the CIITA gene; and / or (e) a guide RNA (gRNA) that targets an RNA-guided nuclease and a target site in the transforming growth factor beta (TGFβ) locus, where the guide RNA cleaves the TGFβ locus at the target site, thereby disrupting the TGFβ gene.

[0381] In another method, method 169, the present disclosure provides the in vitro method provided in method 168, wherein the target locus of (a) is selected from the beta 2 microglobulin (B2M) locus, the thioredoxin interacting protein (TXNIP) locus, the class II transactivator of thymocytes (CIITA) locus and / or the transforming growth factor beta (TGFβ) locus, and the universal donor cells have disruption of expression of B2M, TXNIP, CIITA and / or TGFβ.

[0382] In another method, method 170, the present disclosure provides an in vitro method as provided in method 168 or 169, wherein disrupting expression of B2M, TXNIP, CIITA and / or TGFβ comprises reducing or eliminating expression of B2M, TXNIP, CIITA and / or TGFβ.

[0383] In another method, Method 171, the present disclosure provides the in vitro method provided in Method 169 or 170, wherein the target locus of (a) is the B2M locus, and the nucleic acid further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the B2M locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the B2M locus, wherein nucleotide sequences encoding TNFAIP3, MANF, CD39 and / or CD73 are flanked by (i) and (ii), and the universal donor cell has disrupted expression of B2M.

[0384] In another method, method 172, the present disclosure provides the in vitro method provided in method 171, wherein the nucleotide sequence of (i) comprises or consists essentially of SEQ ID NO:15.

[0385] In another method, method 173, the present disclosure provides the in vitro method provided in any one of methods 171 or 172, wherein the nucleotide sequence of (ii) comprises or consists essentially of SEQ ID NO:22.

[0386] In another method, method 174, the present disclosure provides an in vitro method as provided in method 169, wherein the target locus of (a) is the TXNIP locus, and the nucleic acid further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the TXNIP locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the TXNIP locus, and nucleotide sequences encoding TNFAIP3, MANF, CD39 and / or CD73 are flanked by (i) and (ii), and the universal donor cell has disrupted expression of TXNIP.

[0387] In another method, method 175, the present disclosure provides the in vitro method provided in method 174, wherein the nucleotide sequence of (i) comprises or consists essentially of SEQ ID NO:42.

[0388] In another method, method 176, the present disclosure provides the in vitro method provided in method 174 or 175, wherein the nucleotide sequence of (ii) comprises or consists essentially of SEQ ID NO:44.

[0389] In another method, method 177, the present disclosure provides the in vitro method provided in method 169, wherein the target locus of (a) is the CIITA locus, and the nucleic acid further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the CIITA locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the CIITA locus, wherein nucleotide sequences encoding TNFAIP3, MANF, CD39 and / or CD73 are flanked by (i) and (ii), and the universal donor cell has disrupted expression of CIITA.

[0390] In another method, method 178, the present disclosure provides the in vitro method provided in method 177, wherein the nucleotide sequence of (i) comprises or consists essentially of SEQ ID NO:26.

[0391] In another method, method 179, the present disclosure provides the in vitro method provided in method 177 or 178, wherein the nucleotide sequence of (ii) comprises or consists essentially of SEQ ID NO:28.

[0392] In another method, Method 180, the present disclosure provides the in vitro method provided in Method 169, wherein the target locus of (a) is the TGFβ locus, and the nucleic acid further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the TGFβ locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the TGFβ locus, wherein nucleotide sequences encoding TNFAIP3, MANF, CD39 and / or CD73 are flanked by (i) and (ii), and the universal donor cell has disrupted expression of TGFβ.

[0393] In another method, Method 181, the present disclosure provides the in vitro method provided in any one of Methods 168 to 180, wherein the target site of (b) comprises a nucleotide sequence consisting essentially of any one of SEQ ID NOs: 1-13.

[0394] In another method, Method 182, the present disclosure provides the in vitro method provided in any one of Methods 168 to 181, wherein the target site of (c) comprises a nucleotide sequence consisting essentially of any one of SEQ ID NOs: 32-41.

[0395] In another method, Method 183, the present disclosure provides the in vitro method provided in any one of Methods 168 to 182, wherein the target site of (d) comprises a nucleotide sequence consisting essentially of any one of SEQ ID NOs: 25 and 48-51.

[0396] In another method, Method 184, the present disclosure provides the in vitro method provided in any one of Methods 168 to 183, wherein the target site of (e) comprises a nucleotide sequence consisting essentially of SEQ ID NO:57.

[0397] In another method, Method 185, the present disclosure provides the in vitro method as provided in any one of Methods 168 to 184, further comprising the step of: (f) delivering to a stem cell a nucleic acid comprising an RNA-guided nuclease and a guide RNA (gRNA) targeting a target site in the target locus, and a nucleotide sequence encoding tumor necrosis factor alpha-inducible protein 3 (TNFAIP3), midbrain astrocyte-derived neurotrophic factor (MANF), cluster of differentiation 39 (CD39), cluster of differentiation 73 (CD73), HLA class I histocompatibility antigen, alpha chain E (HLA-E), and / or programmed death-ligand 1 (PD-L-1), wherein the target locus is cleaved at the target site and the nucleic acid comprising the nucleotide sequence encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 is inserted into the target locus, thereby disrupting the target gene.

[0398] In another method, method 186, the present disclosure provides the in vitro method provided in method 185, wherein the target locus of (f) is selected from the beta 2 microglobulin (B2M) locus, the thioredoxin interacting protein (TXNIP) locus, the class II transactivator of HIV-1 (CIITA) locus, and / or the transforming growth factor beta (TGFβ) locus.

[0399] In another method, Method 187, the present disclosure provides the in vitro method provided in Method 186, wherein the target locus of (f) is the B2M locus, and the nucleic acid of (f) further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the B2M locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the B2M locus, wherein nucleotide sequences encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 are flanked between (i) and (ii).

[0400] In another method, method 188, the present disclosure provides the in vitro method provided in method 187, wherein the nucleotide sequence of (i) comprises or consists essentially of SEQ ID NO:15.

[0401] In another method, method 189, the present disclosure provides the in vitro method provided in any one of methods 187 or 188, wherein the nucleotide sequence of (ii) comprises or consists essentially of SEQ ID NO:22.

[0402] In another method, Method 190, the present disclosure provides an in vitro method as provided in Method 186, wherein the target locus of (f) is the TXNIP locus, and the nucleic acid of (f) further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the TXNIP locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the TXNIP locus, and wherein nucleotide sequences encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 are flanked between (i) and (ii).

[0403] In another method, method 191, the present disclosure provides the in vitro method provided in method 190, wherein the nucleotide sequence of (i) comprises or consists essentially of SEQ ID NO:42.

[0404] In another method, method 192, the present disclosure provides the in vitro method provided in method 190 or 191, wherein the nucleotide sequence of (ii) comprises or consists essentially of SEQ ID NO:44.

[0405] In another method, Method 193, the present disclosure provides the in vitro method provided in Method 186, wherein the target locus of (f) is the CIITA locus, and the nucleic acid of (f) further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the CIITA locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the CIITA locus, wherein nucleotide sequences encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 are flanked between (i) and (ii).

[0406] In another method, method 194, the present disclosure provides the in vitro method provided in method 193, wherein the nucleotide sequence of (i) comprises or consists essentially of SEQ ID NO:26.

[0407] In another method, method 195, the present disclosure provides the in vitro method provided in method 193 or 195, wherein the nucleotide sequence of (ii) comprises or consists essentially of SEQ ID NO:28.

[0408] In another method, Method 196, the present disclosure provides the in vitro method provided in Method 186, wherein the target locus of (f) is the TGFβ locus, and the nucleic acid of (f) further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the TGFβ locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the TGFβ locus, wherein nucleotide sequences encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 are flanked between (i) and (ii).

[0409] In another method, method 197, the present disclosure provides the in vitro method provided in any one of methods 185 to 196, wherein the target locus of (f) is the same as the target locus of (a).

[0410] In another method, method 198, the present disclosure provides the in vitro method provided in any one of methods 185 to 197, wherein the target locus of (f) is different from the target locus of (a).

[0411] In another method, Method 199, the present disclosure provides the in vitro method provided in any one of Methods 168 to 198, further comprising the step of: (g) delivering to a stem cell a nucleic acid comprising an RNA-guided nuclease and a guide RNA (gRNA) targeting a target site in a target locus, and a nucleotide sequence encoding tumor necrosis factor alpha-induced protein 3 (TNFAIP3), midbrain astrocyte-derived neurotrophic factor (MANF), cluster of differentiation 39 (CD39), cluster of differentiation 73 (CD73), HLA-E, and / or PD-L-1, wherein the target locus is cleaved at the target site and the nucleic acid comprising the nucleotide sequence encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 is inserted into the target locus, thereby disrupting the target gene.

[0412] In another method, method 200, the present disclosure provides an in vitro method as provided in method 199, wherein the target locus of (g) is selected from the beta 2 microglobulin (B2M) locus, the thioredoxin interacting protein (TXNIP) locus, the class II transactivator of HIV-1 (CIITA) locus, and / or the transforming growth factor beta (TGFβ) locus.

[0413] In another method, Method 201, the present disclosure provides the in vitro method provided in Method 200, wherein the target locus of (g) is the B2M locus, and the nucleic acid of (g) further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the B2M locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the B2M locus, wherein nucleotide sequences encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 are flanked between (i) and (ii).

[0414] In another method, method 202, the present disclosure provides the in vitro method provided in method 201, wherein the nucleotide sequence of (i) comprises or consists essentially of SEQ ID NO:15.

[0415] In another method, method 203, the disclosure provides the in vitro method provided in method 201 or 202, wherein the nucleotide sequence of (ii) comprises or consists essentially of SEQ ID NO:22.

[0416] In another method, Method 204, the present disclosure provides an in vitro method as provided in Method 200, wherein the target locus of (g) is the TXNIP locus, and the nucleic acid of (g) further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the TXNIP locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the TXNIP locus, and wherein nucleotide sequences encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 are flanked between (i) and (ii).

[0417] In another method, method 205, the present disclosure provides the in vitro method provided in method 204, wherein the nucleotide sequence of (i) comprises or consists essentially of SEQ ID NO:42.

[0418] In another method, method 206, the present disclosure provides the in vitro method provided in method 204 or 205, wherein the nucleotide sequence of (ii) comprises or consists essentially of SEQ ID NO:44.

[0419] In another method, Method 207, the present disclosure provides the in vitro method provided in Method 200, wherein the target locus of (g) is the CIITA locus, and the nucleic acid of (g) further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the CIITA locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the CIITA locus, wherein nucleotide sequences encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 are flanked between (i) and (ii).

[0420] In another method, method 208, the present disclosure provides the in vitro method provided in method 207, wherein the nucleotide sequence of (i) comprises or consists essentially of SEQ ID NO:26.

[0421] In another method, method 209, the present disclosure provides the in vitro method provided in method 207 or 208, wherein the nucleotide sequence of (ii) comprises or consists essentially of SEQ ID NO:28.

[0422] In another method, Method 210, the present disclosure provides the in vitro method provided in Method 200, wherein the target locus of (g) is the TGFβ locus, and the nucleic acid of (g) further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the TGFβ locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the TGFβ locus, wherein nucleotide sequences encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 are flanked between (i) and (ii).

[0423] In another method, method 211, the present disclosure provides the in vitro method provided in any one of methods 199 to 210, wherein the target locus of (g) is the same as the target locus of (a) and / or (f).

[0424] In another method, method 212, the present disclosure provides an in vitro method as provided in any one of methods 199 to 211, wherein the target locus of (g) is different from the target locus of (a) and / or (f).

[0425] In another method, Method 213, the present disclosure provides the in vitro method provided in any one of Methods 168 to 212, further comprising the step of: (h) delivering to a stem cell a nucleic acid comprising an RNA-guided nuclease and a guide RNA (gRNA) targeting a target site in a target locus, and a nucleotide sequence encoding tumor necrosis factor alpha-induced protein 3 (TNFAIP3), midbrain astrocyte-derived neurotrophic factor (MANF), cluster of differentiation 39 (CD39), cluster of differentiation 73 (CD73), HLA-E, and / or PD-L-1, wherein the target locus is cleaved at the target site and the nucleic acid comprising the nucleotide sequence encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 is inserted into the target locus, thereby disrupting the target gene.

[0426] In another method, method 214, the present disclosure provides the in vitro method provided in method 213, wherein the target locus of (h) is selected from the beta 2 microglobulin (B2M) locus, the thioredoxin interacting protein (TXNIP) locus, the class II transactivator of HIV-1 (CIITA) locus, and / or the transforming growth factor beta (TGFβ) locus.

[0427] In another method, Method 215, the present disclosure provides the in vitro method provided in Method 214, wherein the target locus of (h) is the B2M locus, and the nucleic acid of (h) further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the B2M locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the B2M locus, wherein nucleotide sequences encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 are flanked between (i) and (ii).

[0428] In another method, method 216, the present disclosure provides an in vitro method provided in method 215, wherein the nucleotide sequence of (i) comprises or consists essentially of SEQ ID NO:15.

[0429] In another method, method 217, the present disclosure provides the in vitro method provided in method 215 or 216, wherein the nucleotide sequence of (ii) comprises or consists essentially of SEQ ID NO:22.

[0430] In another method, Method 218, the present disclosure provides an in vitro method as provided in Method 214, wherein the target locus of (h) is the TXNIP locus, and the nucleic acid of (h) further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the TXNIP locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the TXNIP locus, and wherein nucleotide sequences encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 are flanked between (i) and (ii).

[0431] In another method, method 219, the present disclosure provides the in vitro method provided in method 218, wherein the nucleotide sequence of (i) comprises or consists essentially of SEQ ID NO:42.

[0432] In another method, method 220, the present disclosure provides the in vitro method provided in method 218 or 219, wherein the nucleotide sequence of (ii) comprises or consists essentially of SEQ ID NO:44.

[0433] In another method, Method 221, the present disclosure provides the in vitro method provided in Method 214, wherein the target locus of (h) is the CIITA locus, and the nucleic acid of (h) further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the CIITA locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the CIITA locus, wherein nucleotide sequences encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 are flanked between (i) and (ii).

[0434] In another method, method 222, the present disclosure provides the in vitro method provided in method 221, wherein the nucleotide sequence of (i) comprises or consists essentially of SEQ ID NO:26.

[0435] In another method, method 223, the present disclosure provides the in vitro method provided in method 221 or 222, wherein the nucleotide sequence of (ii) comprises or consists essentially of SEQ ID NO:28.

[0436] In another method, Method 224, the present disclosure provides the in vitro method provided in Method 214, wherein the target locus of (h) is the TGFβ locus, and the nucleic acid of (h) further comprises (i) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the TGFβ locus; and (ii) a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the TGFβ locus, wherein nucleotide sequences encoding TNFAIP3, MANF, CD39, CD73, HLA-E, and / or PD-L-1 are flanked between (i) and (ii).

[0437] In another method, Method 225, the present disclosure provides the in vitro method provided in any one of Methods 213 to 224, wherein the target locus of (h) is the same as the target locus of (a), (f) and / or (g).

[0438] In another method, method 226, the present disclosure provides an in vitro method as provided in any one of methods 213 to 225, wherein the target locus of (h) is different from the target locus of (a), (f) and / or (g).

[0439] In another method, method 227, the present disclosure provides the in vitro method provided in any one of methods 168 to 226, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g) and / or the nucleic acid of (h) comprises a nucleotide sequence encoding MANF, and the universal donor cell expresses MANF.

[0440] In another method, method 228, the present disclosure provides the in vitro method provided in method 227, wherein the nucleotide sequence encoding MANF consists essentially of SEQ ID NO:17.

[0441] In another method, Method 229, the present disclosure provides the in vitro method provided in any one of Methods 168 to 228, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g) and / or the nucleic acid of (h) comprises a nucleotide sequence encoding TNFAIP3, and the universal donor cell expresses TNFAIP3.

[0442] In another method, method 230, the present disclosure provides the in vitro method provided in method 229, wherein the nucleotide sequence encoding TNFAIP3 consists essentially of SEQ ID NO:19.

[0443] In another method, method 231, the present disclosure provides the in vitro method provided in any one of methods 168 to 230, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence encoding CD39, and the universal donor cells express CD39.

[0444] In another method, method 232, the present disclosure provides the in vitro method provided in method 231, wherein the nucleotide sequence encoding CD39 consists essentially of SEQ ID NO:27.

[0445] In another method, Method 233, the present disclosure provides the in vitro method provided in any one of Methods 168 to 232, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence encoding CD73, and the universal donor cells express CD73.

[0446] In another method, method 234, the present disclosure provides the in vitro method provided in method 233, wherein the nucleotide sequence encoding CD73 consists essentially of SEQ ID NO:46.

[0447] In another method, method 235, the present disclosure provides the in vitro method provided in any one of methods 168 to 234, wherein the nucleic acid of (a) further comprises a nucleotide sequence encoding an HLA class I histocompatibility antigen, alpha chain E (HLA-E), and the universal donor cells further express HLA-E.

[0448] In another method, method 236, the present disclosure provides the in vitro method provided in any one of methods 168 to 235, wherein the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence encoding an HLA class I histocompatibility antigen, alpha chain E (HLA-E), and the universal donor cells express HLA-E.

[0449] In another method, method 237, the present disclosure provides the in vitro method provided in methods 168 to 236, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer, the HLA-E trimer comprising a B2M membrane protein fused to HLA-E without a signal peptide, an HLA-G presenting peptide fused thereto, and a B2M signal peptide fused thereto.

[0450] In another method, method 238, the present disclosure provides an in vitro method as provided in any one of methods 235 to 237, wherein the nucleotide sequence encoding HLA-E consists essentially of SEQ ID NO:43.

[0451] In another method, method 239, the present disclosure provides the in vitro method provided in any one of methods 235 to 238, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g) and / or the nucleic acid of (h) comprises a nucleotide sequence encoding MANF and a nucleotide sequence encoding HLA-E, and the universal donor cells express MANF and HLA-E.

[0452] In another method, method 240, the present disclosure provides the in vitro method provided in method 239, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence encoding a P2A peptide linked to a nucleotide sequence encoding HLA-E, linked thereto a nucleotide sequence encoding MANF.

[0453] In another method, Method 241, the present disclosure provides an in vitro method as provided in Method 240, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g) and / or the nucleic acid of (h) comprise a nucleotide sequence consisting of SEQ ID NO:55.

[0454] In another method, method 242, the present disclosure provides the in vitro method provided in any one of methods 168 to 241, wherein the nucleic acid of (a) further comprises a nucleotide sequence encoding programmed death-ligand 1 (PD-L-1), and the universal donor cells further express PD-L-1.

[0455] In another method, Method 243, the present disclosure provides the in vitro method provided in any one of Methods 168 to 242, wherein the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence encoding PD-L-1, and the universal donor cells express PD-L-1.

[0456] In another method, Method 244, the present disclosure provides the in vitro method provided in Method 242 or 243, wherein the nucleotide sequence encoding PD-L-1 consists essentially of SEQ ID NO:20.

[0457] In another method, Method 245, the present disclosure provides the in vitro method provided in Methods 242 to 244, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence encoding TNFAIP3 and a nucleotide sequence encoding PD-L-1, and the universal donor cells express TNFAIP3 and PD-L-1.

[0458] In another method, Method 246, the present disclosure provides the in vitro method provided in any one of Methods 242 to 245, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence encoding a P2A peptide linked to a nucleotide sequence encoding PD-L-1, linked thereto a nucleotide sequence encoding TNFAIP3.

[0459] In another method, Method 247, the present disclosure provides the in vitro method provided in Method 246, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprise a nucleotide sequence consisting essentially of SEQ ID NO:54.

[0460] In another method, Method 248, the present disclosure provides the in vitro method provided in any one of Methods 242 to 247, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence for CD39 and a nucleotide sequence encoding PD-L-1, and the universal donor cells express CD39 and PD-L-1.

[0461] In another method, Method 249, the present disclosure provides the in vitro method provided in Method 248, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence encoding a P2A peptide linked to a nucleotide sequence encoding PD-L-1, linked thereto a nucleotide sequence for CD39.

[0462] In another method, Method 250, the present disclosure provides the in vitro method provided in Method 249, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence consisting essentially of SEQ ID NO:53.

[0463] In another method, Method 251, the present disclosure provides the in vitro method provided in any one of Methods 185 to 250, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence encoding MANF, a nucleotide sequence encoding TNFAIP3, and a nucleotide sequence encoding PD-L-1, and the universal donor cells express MANF, TNFAIP3, and PD-L-1.

[0464] In another method, Method 252, the present disclosure provides the in vitro method provided in Method 251, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence encoding MANF linked to a nucleotide sequence encoding TNFAIP3 by a first nucleotide sequence encoding a P2A peptide, and a nucleotide sequence encoding TNFAIP3 linked to a nucleotide sequence encoding PD-L-1 by a second nucleotide sequence encoding a P2A peptide.

[0465] In another method, Method 253, the present disclosure provides the in vitro method provided in Method 252, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprise a nucleotide sequence consisting essentially of SEQ ID NO:52.

[0466] In another method, Method 254, the present disclosure provides the in vitro method provided in any one of Methods 185 to 253, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence encoding CD39, a nucleotide sequence encoding CD73, and a nucleotide sequence encoding PD-L-1, and the universal donor cells express CD39, CD73, and PD-L-1.

[0467] In another method, Method 255, the present disclosure provides the in vitro method provided in Method 254, wherein the nucleotide sequence encoding CD39 is linked to the nucleotide sequence encoding CD73 by a first nucleotide sequence encoding a P2A peptide, and the nucleotide sequence encoding CD73 is linked to the nucleotide sequence encoding PD-L-1 by a second nucleotide sequence encoding a P2A peptide.

[0468] In another method, Method 256, the present disclosure provides an in vitro method as provided in Method 255, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprise a nucleotide sequence consisting essentially of SEQ ID NO:56.

[0469] In another method, Method 257, the present disclosure provides the in vitro method provided in any one of Methods 168 to 256, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprises a nucleotide sequence encoding CD39 and a nucleotide sequence encoding CD73, and the universal donor cells express CD39 and CD73.

[0470] In another method, method 258, the present disclosure provides the in vitro method provided in method 257, wherein a nucleotide sequence encoding CD39 is linked to a nucleotide sequence encoding CD73 by a nucleotide sequence encoding a P2A peptide.

[0471] In another method, Method 259, the present disclosure provides the in vitro method provided in Method 258, wherein the nucleic acid of (a), the nucleic acid of (f), the nucleic acid of (g), and / or the nucleic acid of (h) comprise a nucleotide sequence consisting essentially of SEQ ID NO:58.

[0472] In another method, Method 260, the present disclosure provides an in vitro method as provided in any one of Methods 168 to 259, wherein the nucleotide sequence of any of the nucleic acids of (a), (f), (g), and / or (h) is operably linked to an exogenous promoter.

[0473] In another method, method 261, the present disclosure provides the in vitro method provided in method 260, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0474] In another method, Method 262, the present disclosure provides the in vitro method provided in any one of Methods 168 to 261, wherein the RNA-guided nuclease of (a), (b), (c), (d), (e), (f), (g) and / or (h) and the gRNA are present in a ratio of about 1:1 to about 1:10.

[0475] In another method, Method 263, the present disclosure provides the in vitro method provided in any one of Methods 168 to 262, wherein the RNA-guided nuclease and gRNA of each of (a), (b), (c), (d), (e), (f), (g), and / or (h) are present in a ratio of about 1:1 to about 1:10.

[0476] In another method, Method 264, the present disclosure provides the in vitro method provided in any one of Methods 168 to 263, wherein the RNA-guided nuclease of each of (a), (b), (c), (d), (e), (f), (g), and / or (h) is a Cas9 nuclease.

[0477] In another method, method 265, the present disclosure provides the in vitro method provided in method 264, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0478] In another method, method 266, the present disclosure provides an in vitro method as provided in any one of methods 168 to 264, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0479] In another method, method 267, the present disclosure provides an in vitro method as provided in any one of methods 168 to 266, wherein the stem cells are human stem cells.

[0480] In another method, method 268, the present disclosure provides an in vitro method as provided in any one of methods 1 to 267, wherein the universal donor cells have increased immune evasion and / or post-transplant survival compared to comparable cells that have not been subjected to nucleic acid insertion and / or gene disruption.

[0481] In another method, method 269, the disclosure provides an in vitro method for generating a universal donor cell, comprising: (a) a first RNA-guided nuclease and a first guide RNA (gRNA) that targets a target site in the beta 2 microglobulin (B2M) locus; (b) a first vector comprising nucleic acids, the nucleic acid comprising: (i) a nucleotide sequence encoding tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and a nucleotide sequence encoding programmed death-ligand 1 (PD-L-1); (ii) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the B2M locus; and (iii) a nucleotide sequence encoding a genomic region located to the right of the target site in the B2M locus. and (iii) to deliver to stem cells a first vector comprising a nucleotide sequence having sequence homology to a genomic region to be inserted into the B2M locus, wherein (i) is flanked by (ii) and (iii), wherein the first vector cleaves the B2M locus at a target site and inserts a nucleic acid comprising a nucleotide sequence encoding TNFAIP3 and PD-L-1 into the B2M locus, thereby disrupting the B2M gene, wherein the universal donor cells express TNFAIP3 and PD-L-1 and have increased immune evasion and / or post-transplant survival compared to comparable cells without the nucleic acid insertion and gene disruption.

[0482] In another method, method 270, the present disclosure provides an in vitro method as provided in method 269, wherein disrupting the B2M gene comprises reducing or eliminating expression of B2M.

[0483] In another method, Method 271, the present disclosure provides the in vitro method provided in Methods 269 or 270, wherein the nucleotide sequence of (b)(i) comprises a nucleotide sequence encoding a P2A peptide linked to a nucleotide sequence encoding PD-L-1, linked thereto a nucleotide sequence encoding TNFAIP3.

[0484] In another method, method 272, the present disclosure provides the in vitro method provided in any one of methods 269 to 271, wherein the nucleotide sequence of (b)(i) comprises SEQ ID NO:54.

[0485] In another method, method 273, the disclosure provides the in vitro method provided in any one of methods 269 to 272, wherein the nucleotide sequence of (b)(i) is operably linked to an exogenous promoter.

[0486] In another method, method 274, the present disclosure provides the in vitro method provided in method 273, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0487] In another method, Method 275, the present disclosure provides the in vitro method provided in any one of Methods 269 to 274, wherein the nucleotide sequence of (b)(ii) comprises or consists essentially of SEQ ID NO:15.

[0488] In another method, Method 276, the present disclosure provides the in vitro method provided in any one of Methods 269 to 275, wherein the nucleotide sequence of (b)(iii) comprises or consists essentially of SEQ ID NO:22.

[0489] In another method, method 277, the present disclosure provides the in vitro method provided in any one of methods 269 to 276, wherein the first RNA-guided nuclease and the first gRNA are present in a ratio of about 1:1 to about 1:10.

[0490] In another method, method 278, the present disclosure provides the in vitro method provided in any one of methods 269 to 277, wherein the first RNA-guided nuclease is a first Cas9 nuclease.

[0491] In another method, method 279, the present disclosure provides the in vitro method provided in method 278, wherein the first Cas9 nuclease is linked to at least one nuclear localization signal.

[0492] In another method, method 280, the present disclosure provides a method for producing a genomic vector comprising: (c) a second gRNA that targets a target site in a second RNA-guided nuclease and thioredoxin interacting protein (TXNIP) locus; and (d) a second vector comprising nucleic acids, the nucleic acids comprising: (i) a nucleotide sequence encoding midbrain astrocyte-derived neurotrophic factor (MANF) and a nucleotide sequence encoding HLA class I histocompatibility antigen, alpha chain E (HLA-E); (ii) a nucleotide sequence having sequence homology to a genomic region located to the left of the target site in the TXNIP locus; and (iii) a second vector comprising a nucleic acid ... ) An in vitro method as provided in any one of methods 269 to 279, further comprising the step of delivering to stem cells a second vector comprising a nucleotide sequence having sequence homology to a genomic region located to the right of the target site in the TXNIP locus, wherein (i) is flanked by (ii) and (iii), thereby cleaving the TXNIP locus at the target site and inserting a nucleic acid comprising a nucleotide sequence encoding MANF and HLA-E into the TXNIP locus, thereby disrupting the TXNIP gene, wherein the universal donor cells further express MANF and HLA-E.

[0493] In another method, method 281, the present disclosure provides an in vitro method as provided in method 280, wherein disrupting the TXNIP gene comprises reducing or eliminating expression of TXNIP.

[0494] In another method, method 282, the present disclosure provides the in vitro method provided in method 280 or 281, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer, the HLA-E trimer comprising a B2M membrane protein fused to HLA-E without a signal peptide, an HLA-G presenting peptide fused thereto, and a B2M signal peptide fused thereto.

[0495] In another method, Method 283, the present disclosure provides the in vitro method provided in any one of Methods 280 to 282, wherein the nucleotide sequence of (d)(i) comprises a nucleotide sequence encoding a P2A peptide linked to a nucleotide sequence encoding HLA-E, linked thereto a nucleotide sequence encoding MANF.

[0496] In another method, method 284, the present disclosure provides the in vitro method provided in any one of methods 280 to 283, wherein the nucleotide sequence of (d)(i) comprises SEQ ID NO:55.

[0497] In another method, method 285, the present disclosure provides the in vitro method provided in any one of methods 280 to 284, wherein the nucleotide sequence of (d)(i) is operably linked to an exogenous promoter.

[0498] In another method, method 286, the present disclosure provides the in vitro method provided in method 285, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0499] In another method, Method 287, the present disclosure provides the in vitro method provided in any one of Methods 280 to 286, wherein the nucleotide sequence of (d)(ii) consists essentially of SEQ ID NO:42.

[0500] In another method, Method 288, the present disclosure provides the in vitro method provided in any one of Methods 280 to 287, wherein the nucleotide sequence of (d)(iii) consists essentially of SEQ ID NO:44.

[0501] In another method, method 289, the present disclosure provides the in vitro method provided in any one of methods 280 to 288, wherein the second RNA-guided nuclease and the second gRNA are present in a ratio of about 1:1 to about 1:10.

[0502] In another method, method 290, the present disclosure provides the in vitro method provided in any one of methods 280 to 289, wherein the second RNA-guided nuclease is a second Cas9 nuclease.

[0503] In another method, method 291, the present disclosure provides the in vitro method provided in method 290, wherein the second Cas9 nuclease is linked to at least one nuclear localization signal.

[0504] In another method, method 292, the present disclosure provides an in vitro method as provided in any one of methods 269 to 291, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0505] In another method, method 293, the present disclosure provides the in vitro method provided in any one of methods 269 to 292, wherein the stem cells are human stem cells.

[0506] In another composition, composition 294, the present disclosure provides a composition comprising genetically modified cells comprising a nucleotide sequence encoding midbrain astrocyte-derived neurotrophic factor (MANF), a nucleotide sequence encoding tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a nucleotide sequence encoding cluster of differentiation 73 (CD73), and / or a nucleotide sequence encoding cluster of differentiation 39 (CD39) inserted within or near a gene encoding beta2-microglobulin (B2M), thioredoxin interacting protein (TXNIP), or class II transactivator of mitochondrial T cells (CIITA), wherein the genetically modified cells express MANF, TNFAIP3, CD73, and / or CD39 and have disrupted expression of B2M, TXNIP, and / or CIITA.

[0507] In another composition, composition 295, the present disclosure provides a composition according to composition 294, wherein disrupting expression of B2M, TXNIP, and / or CIITA comprises reducing or eliminating expression of B2M, TXNIP, and / or CIITA.

[0508] In another composition, composition 296, the present disclosure provides a composition according to composition 294 or 295, further comprising a nucleotide sequence encoding programmed death-ligand 1 (PD-L-1) and / or a nucleotide sequence encoding HLA class I histocompatibility antigen, alpha chain E (HLA-E) inserted within or near the B2M, TXNIP, or CIITA gene.

[0509] In another composition, composition 297, the present disclosure provides a composition according to composition 296, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer, the HLA-E trimer comprising a B2M membrane protein fused to HLA-E without a signal peptide, an HLA-G presenting peptide fused thereto, and a B2M signal peptide fused thereto.

[0510] In another composition, composition 298, the present disclosure provides a composition according to composition 296 or 297, wherein the genetically modified cells comprise a nucleotide sequence encoding MANF and a nucleotide sequence encoding HLA-E inserted within or near the TXNIP gene.

[0511] In another composition, composition 299, the present disclosure provides a composition according to composition 298, wherein a nucleotide sequence encoding MANF and a nucleotide sequence encoding HLA-E are operably linked to an exogenous promoter.

[0512] In another composition, composition 300, the present disclosure provides a composition according to composition 298 or 299, wherein a nucleotide sequence encoding MANF is linked to a nucleotide sequence encoding HLA-E by a nucleotide sequence encoding a ribosomal skip.

[0513] In another composition, composition 301, the present disclosure provides a composition according to composition 300, wherein the ribosomal skip is a 2A sequence family member.

[0514] In another composition, composition 302, the present disclosure provides a composition according to any one of compositions 294 to 301, wherein the genetically modified cells comprise a nucleotide sequence encoding TNFAIP3 and a nucleotide sequence encoding PD-L-1 inserted within or near the B2M gene.

[0515] In another composition, composition 303, the present disclosure provides a composition according to composition 302, wherein a nucleotide sequence encoding TNFAIP3 and a nucleotide sequence encoding PD-L-1 are operably linked to an exogenous promoter.

[0516] In another composition, composition 304, the present disclosure provides a composition according to composition 302 or 303, wherein a nucleotide sequence encoding TNFAIP3 is linked to a nucleotide sequence encoding PD-L-1 by a nucleotide sequence encoding a ribosomal skip.

[0517] In another composition, composition 305, the disclosure provides a composition according to composition 304, wherein the ribosomal skip is a 2A sequence family member.

[0518] In another composition, composition 306, the present disclosure provides a composition according to composition 296 or 297, wherein the genetically modified cells comprise a nucleotide sequence encoding TNFAIP3, a nucleotide sequence encoding MANF, and a nucleotide sequence encoding PD-L-1 inserted within or near the B2M gene.

[0519] In another composition, composition 307, the present disclosure provides a composition according to composition 306, wherein a nucleotide sequence encoding TNFAIP3, a nucleotide sequence encoding MANF, and a nucleotide sequence encoding PD-L-1 are operably linked to an exogenous promoter.

[0520] In another composition, composition 308, the present disclosure provides a composition according to composition 306 or 307, wherein the nucleotide sequence encoding TNFAIP3 is linked to the nucleotide sequence encoding MANF by a nucleotide sequence encoding a ribosomal skip, and the nucleotide sequence encoding MANF is linked to the nucleotide sequence encoding PD-L-1 by a nucleotide sequence encoding a ribosomal skip.

[0521] In another composition, composition 309, the disclosure provides a composition according to composition 308, wherein the ribosomal skip is a 2A sequence family member.

[0522] In another composition, composition 310, the present disclosure provides a composition according to any one of compositions 294 to 309, wherein the genetically modified cells comprise a nucleotide sequence encoding CD39 inserted within or near the CIITA gene.

[0523] In another composition, composition 311, the present disclosure provides a composition according to composition 310, wherein the nucleotide sequence encoding CD39 is operably linked to an exogenous promoter.

[0524] In another composition, composition 312, the present disclosure provides a composition according to composition 296 or 297, wherein the genetically modified cells comprise a nucleotide sequence encoding CD39 and a nucleotide sequence encoding PD-L-1 inserted within or near the B2M gene.

[0525] In another composition, composition 313, the present disclosure provides a composition according to composition 312, wherein a nucleotide sequence encoding CD39 and a nucleotide sequence encoding PD-L-1 are operably linked to an exogenous promoter.

[0526] In another composition, composition 314, the present disclosure provides a composition according to composition 312 or 313, wherein a nucleotide sequence encoding CD39 is linked to a nucleotide sequence encoding PD-L-1 by a nucleotide sequence encoding a ribosomal skip.

[0527] In another composition, composition 315, the disclosure provides a composition according to composition 314, wherein the ribosomal skip is a 2A sequence family member.

[0528] In another composition, composition 316, the present disclosure provides a composition comprising a nucleotide sequence encoding PD-L-1 inserted within or near the B2M gene, a nucleotide sequence encoding HLA-E inserted within or near the TXNIP gene, and / or a nucleotide sequence encoding CD39 inserted within or near the CIITA gene or the B2M gene, wherein genetically modified cells express PD-L-1, HLA-E, and / or CD39 and have disrupted expression of B2M, TXNIP, and / or CIITA.

[0529] In another composition, composition 317, the present disclosure provides a composition according to composition 316, wherein disrupting expression of B2M, TXNIP, and / or CIITA includes reducing or eliminating expression of B2M, TXNIP, and / or CIITA.

[0530] In another composition, composition 318, the present disclosure provides a composition according to any one of compositions 294 to 317, wherein the cells are stem cells.

[0531] In another composition, composition 319, the present disclosure provides a composition according to composition 318, wherein the stem cells are embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0532] In another composition, composition 320, the present disclosure provides a composition according to any one of compositions 294 to 319, wherein the cell is a differentiated cell or a somatic cell.

[0533] In another composition, composition 321, the present disclosure provides a composition according to composition 320, in which the cells are differentiated into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0534] In another composition, composition 322, the present disclosure provides a composition according to composition 321, wherein the lineage-restricted progenitor cell is a definitive endoderm cell, an undifferentiated intestinal cell, a posterior foregut cell, a pancreatic endoderm precursor, a pancreatic endocrine precursor, or a pancreatic endocrine cell, and the fully differentiated somatic cell is an immature beta cell or a mature beta cell.

[0535] In another composition, composition 323, the present disclosure provides a composition comprising a plurality of genetically modified cells of any one of claims 294-322.

[0536] In another composition, composition 324, the present disclosure provides a composition according to composition 323, wherein at least about 50% of the cells express MANF, HLA-E, TNFAIP3, PD-L-1, and / or CD39.

[0537] In another composition, composition 325, the present disclosure provides a composition comprising a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from a plurality of genetically modified cells of composition 323 or 324.

[0538] In another composition, composition 326, the present disclosure provides a composition according to composition 325, wherein the lineage-restricted progenitor cell is a definitive endoderm cell, an undifferentiated intestinal cell, a posterior foregut cell, a pancreatic endoderm precursor, a pancreatic endocrine precursor, or a pancreatic endocrine cell, and the fully differentiated somatic cell is an immature beta cell or a mature beta cell.

[0539] In another composition, composition 327, the present disclosure provides a composition according to composition 325 or 326, wherein at least about 50% of the cells express MANF, HLA-E, TNFAIP3, PD-L-1, and / or CD39.

[0540] In another composition, composition 328, the disclosure provides a composition comprising a plurality of cells of composition 323 or 324 or a population of cells of any one of compositions 325 through 327.

[0541] In another composition, composition 329, the present disclosure provides a composition according to composition 328, further comprising at least one pharmaceutically acceptable excipient.

[0542] In another composition, composition 330, the present disclosure provides a composition according to composition 328 or 329 for use in treating a subject in need thereof.

[0543] In another composition, composition 331, the present disclosure provides a composition according to composition 330, wherein the subject has, is suspected of having, or is at risk for a disease or disorder.

[0544] In another composition, composition 332, the present disclosure provides a composition according to composition 330 or 331, wherein the disease or disorder is a genetic disease, such as type I diabetes.

[0545] In another composition, composition 333, the present disclosure provides a composition according to composition 332, wherein the disease or disorder is type II diabetes or pancreatectomy.

[0546] In another composition, composition 334, the present disclosure provides a composition according to any one of compositions 328 to 333, wherein the subject is a human.

[0547] In another method, method 335, the present disclosure provides a method for treating a pancreatic disease or disorder in a subject in need thereof, comprising: (a) obtaining or obtaining a plurality of genetically modified cells described in any one of claims 294 to 322 following differentiation into pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, or mature beta cells; and (b) administering the pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, or mature beta cells to the subject.

[0548] In another method, method 336, the present disclosure provides the method provided in method 335, wherein the administering step includes implanting into the subject a device comprising pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, or mature beta cells.

[0549] In another method, method 337, the present disclosure provides the method provided in method 335 or 336, wherein the pancreatic disease or disorder is type I diabetes, type II diabetes, or pancreatectomy.

[0550] In another method, method 338, the present disclosure provides the method provided in any one of methods 335 to 337, wherein the subject is a human.

[0551] In another method, method 339, the disclosure provides an in vitro method for generating a universal donor cell, comprising: (a) a ribonucleoprotein (RNP) complex comprising an RNA-guided nuclease and a guide RNA (gRNA) that targets a target site in the beta 2 microglobulin (B2M) locus; (b) a vector comprising nucleic acids, the nucl...

Claims

1. A genetically modified cell comprising: (a) a disrupted gene for thioredoxin interacting protein (TXNIP) and a first insertion into the disrupted TXNIP gene of a first exogenous polynucleotide encoding midbrain astrocyte-derived neurotrophic factor (MANF); and (b) a disrupted beta 2-microglobulin (B2M) gene and a second insertion into the disrupted B2M gene of a second exogenous polynucleotide encoding tumor necrosis factor alpha-induced protein 3 (TNFAIP3); A genetically modified cell, wherein the genetically modified cell expresses MANF and TNFAIP3 and has disrupted expression of B2M protein and TXNIP protein.

2. The genetically modified cell of claim 1, wherein the first exogenous polynucleotide comprises SEQ ID NO:17 and / or the second exogenous polynucleotide comprises SEQ ID NO:

19.

3. said first exogenous polynucleotide encoding MANF is linked to an exogenous polynucleotide encoding HLA-E by a polynucleotide encoding a P2A peptide, whereby said first exogenous polynucleotide encoding MANF, the polynucleotide encoding a P2A peptide and said exogenous polynucleotide encoding HLA-E form a MANF-P2A-HLA-E construct; the exogenous polynucleotide encoding HLA-E comprises a polynucleotide encoding an HLA-E trimer, the HLA-E trimer comprising a B2M membrane protein fused to HLA-E without a signal peptide, an HLA-G presenting peptide fused thereto, and a B2M signal peptide fused thereto; 3. The genetically modified cell of claim 1 or 2, optionally wherein the MANF-P2A-HLA-E construct is operably linked to an exogenous promoter.

4. The genetically modified cell of claim 3, wherein the MANF-P2A-HLA-E construct comprises the polynucleotide sequence of SEQ ID NO:

55.

5. said second exogenous polynucleotide encoding TNFAIP3 is linked to a second exogenous polynucleotide encoding PD-L1 by a polynucleotide encoding a P2A peptide, whereby said second exogenous polynucleotide, the polynucleotide encoding a P2A peptide and said exogenous polynucleotide sequence encoding PD-L1 form a TNFAIP3-P2A-PD-L1 construct; 5. The genetically modified cell of any one of claims 1-4, optionally wherein the TNFAIP3-P2A-PD-L1 construct is operably linked to an exogenous promoter.

6. 6. The genetically modified cell of claim 5, wherein the TNFAIP3-P2A-PD-L1 construct comprises the nucleotide sequence of SEQ ID NO:

54.

7. 7. The genetically modified cell of any one of claims 1 to 6, wherein disrupting expression of the B2M protein and / or the TXNIP protein comprises reducing or eliminating expression of the B2M protein and / or the TXNIP protein. (a) the cell is a stem cell, and optionally the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell; or (b) the cell is a differentiated cell, optionally the differentiated cell is a somatic cell.

9. The cell is a lineage-restricted progenitor cell or a fully differentiated somatic cell, 9. Optionally, the lineage-restricted progenitor cell is a definitive endoderm cell, an undifferentiated intestinal cell, a posterior foregut cell, a pancreatic endoderm progenitor cell, a pancreatic endocrine progenitor cell, a pancreatic endocrine cell, or an immature beta cell, and the fully differentiated somatic cell is a pancreatic beta cell.

10. 10. The plurality of genetically modified cells of any one of claims 1-9, wherein at least about 50% of the cells express MANF, HLA-E, TNFAIP3, and / or PD-L1.

11. A population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from a plurality of genetically modified cells of claim 10, comprising: Optionally, said lineage-restricted progenitor cells, definitive endoderm cells, undifferentiated intestinal cells, posterior foregut cells, pancreatic endoderm precursors, pancreatic endocrine precursors, or immature beta cells, and said fully differentiated somatic cells are pancreatic beta cells, and optionally, at least about 50% of said cells express MANF, HLA-E, TNFAIP3, and / or PD-L1.

12. 12. A composition comprising a plurality of genetically modified cells of claim 10 or a population of cells of claim 11, optionally further comprising at least one pharma- ceutically acceptable excipient.

13. The composition described in claim 12 for treating a pancreatic disease or disorder in a subject, wherein the pancreatic disease or disorder is type I diabetes, type II diabetes or pancreatic resection.

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