universal donor cells

Abstract

This document provides genetically modified cells compatible with multiple subjects, such as universal donor cells; and methods for generating said genetically modified cells. These universal donor cells contain at least one genetic modification within or near at least one gene encoding a survival factor, wherein the genetic modification includes the insertion of a polynucleotide encoding a tolerogenic factor. These universal donor cells may further contain 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 regulatory factors of the MHC-I or MHC-II complex, wherein said genetic modification includes the insertion of a polynucleotide encoding a second tolerogenic factor.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 896,473, filed September 5, 2019, and U.S. Provisional Application No. 62 / 979,771, filed February 21, 2020, the disclosures of which are hereby incorporated in their entirety by reference.

[0003] By referencing and incorporating into the sequence list

[0004] This application contains a sequence list submitted via EFS-Web in ASCII format, and its entirety is hereby incorporated by reference. The ASCII copy created on September 2, 2020, is named CT124-PCT-100867-666508-Sequence-Listing_ST25.txt and is approximately 53,000 bytes in size. Technical Field

[0005] This invention relates to the field of gene editing, and in some embodiments, to genetic modifications for the purpose of producing cells compatible with multiple subjects (e.g., universal donor cells). Background Technology

[0006] Several approaches have been proposed to overcome allogeneic rejection of transplanted or implanted cells, including HLA matching, blocking pathways that trigger T cell activation with antibodies, using mixtures of immunosuppressive drugs, and autologous cell therapy. Another strategy to suppress graft rejection involves minimizing allogeneic differences between transplanted or implanted cells and recipients. Human leukocyte antigens (HLA), expressed on the cell surface and encoded by genes located in the human major histocompatibility complex on chromosome 6, are major mediators of immune rejection. A single mismatch of a single HLA gene between donor and recipient can elicit a robust immune response (Fleischhauer K. et al., “Bone marrow-allograft rejection by T lymphocytes recognizing a single amino acid difference in HLA-B44,” N Engl J Med., 1990, 323:1818-1822). HLA genes are classified into MHC class I (MHC-I) and MHC class II (MHC-II). MHC-I genes (HLA-A, HLA-B, and HLA-C) are expressed in almost all tissue and cell types, presenting peptides treated with “non-self” antigens to CD8+ T cells, thereby promoting their activation into cytolytic CD8+ T cells. Transplantation or implantation of cells expressing “non-self” MHC-I molecules elicits a robust cellular immune response against these cells, ultimately leading to cell death through activated cytolytic CD8+ T cells. MHC-I proteins are closely associated with β-2-microglobulin (B2M) in the endoplasmic reticulum, which is crucial for the formation of functional MHC-I molecules on the cell surface.

[0007] In contrast to the widespread cellular expression of MHC-I genes, the expression of MHC-II genes is limited to antigen-presenting cells, such as dendritic cells, macrophages, and B cells. HLA antigen genes are among the most polymorphic genes observed in the human genome (Rubinstein P., “HLA matching for bone marrow transplantation—how much is enough?” N Engl J Med., 2001, 345:1842-1844). The generation of “universal donor” cells compatible with any HLA genotype offers an alternative strategy to address the associated economic costs of current approaches to immune rejection and immune evasion.

[0008] To generate such universal donor cell lines, a previous approach involved functionally disrupting the expression of MHC-I and MHC-II genes. This could be achieved, for example, by genetically disrupting two genetic alleles encoding the MHC-I light chain B2M. The resulting B2M-null cell lines and their derivatives were expected to exhibit significantly reduced surface MHC-I, thereby exhibiting reduced immunogenicity against allogeneic CD8+ T cells. A transcription activator-like effector nuclease (TALEN) targeting approach has been used to generate B2M-deficient hESC lines by deleting certain nucleotides in exon 2 of the B2M gene (Lu, P. et al., “Generating hypoimmunogenic human embryonic stem cells by the disruption of beta 2-microglobulin,” StemCell Rev. 2013, 9:806-813). Although B2M-targeting hESC lines appear to be HLA-I-deficient on the surface, they have been found to still contain mRNAs specific to B2M and MHC-I. B2M and MHC-I mRNAs are expressed at levels comparable to those in non-targeting hESCs (constitutive and IFN-γ-induced). Therefore, there is concern that these TALEN B2M-targeting hESC lines may express residual cell-surface MHC-I sufficient to induce immune rejection, as has been observed in B2M2 / 2 mouse cells that 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 off-target cleavage events in TALEN B2M-targeted hESC lines were not examined, the occurrence of non-specific cleavage during TALEN use remains a significant concern, posing substantial safety risks for its clinical application (Grau, J. et al., “TALENoffer: genome-wide TALEN off-target prediction,” Bioinformatics, 2013, 29:2931-2932; Guilinger JP et al., “Broadspecificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity,” Nat Methods, 2014, 11:429-435). In addition, another report describes iPS cells that evade allogeneic recognition by knocking out the first B2M allele and knocking in the HLA-E gene at the second B2M allele, resulting in the surface expression of HLA-E dimers or trimers in the absence of surface expression of HLA-A, HLA-B, or HLA-C (Gornalusse, GG et al., “HLA-E-expressing pluripotentstem cells escape allogeneic responses and lysis by NK cells,” Nature Biotechnology, 2017, 35, 765-773).

[0009] One potential limitation of the above strategies is that MHC class I negative cells are susceptible to lysis by natural killer (NK) cells because HLA molecules act as major ligand inhibitors of natural killer (NK) cells. It has been shown that host NK cells can eliminate transplanted or engrafted B2M- / - donor cells, and a similar phenomenon has occurred in vitro in the case of MHC class I negative human leukemia cell lines (Bix, M. et al., “Rejection of class IMHC-deficient hematopoietic 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 previous methods to generate universal donor cells capable of evading immune responses, and to generate cells that can survive post-implantation. As described herein, post-implantation cell survival can be mediated by host pathways independent of allogeneic rejection (e.g., hypoxia, reactive oxygen species, nutrient deprivation, and oxidative stress). Additionally, as described herein, the genetic introduction of survival factors (genes and / or proteins) can contribute to cell survival after implantation. As described herein, universal donor cell lines can combine the properties that address both allogeneic rejection and post-implantation survival. Summary of the Invention

[0010] In some aspects, this disclosure covers methods for generating universal donor cells. The method includes delivering to a cell (a) a site-directed nuclease targeting a site within or near a gene encoding a survival factor, and (b) a nucleic acid containing a nucleotide sequence encoding a tolerance factor, the flanking nucleotide sequence being (i) a nucleotide sequence homologous to a region to the left of the target site in (a) and (ii) a nucleotide sequence homologous to a region to the right of the target site in (a), wherein the site-directed nuclease cleaves the target site in (a), and the nucleic acid in (b) is inserted at a site that partially overlaps, completely overlaps with, or is contained within the site in (a), thereby generating universal donor cells, wherein the universal donor cells have increased cell survival compared to cells in which the nucleic acid in (b) is not inserted.

[0011] In some embodiments, the survival factor is TXNIP, ZNF143, FOXO1, JNK, or MANF, and the tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47. In a specific embodiment, the survival factor is TXNIP and the tolerogenic factor is HLA-E. In embodiments where the site-directed nuclease is a CRISPR system comprising a CRISPR nuclease and a guide RNA (gRNA), the CRISPR nuclease is a type II Cas9 nuclease or a type V Cfp1 nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal. In some embodiments, the gRNA targets the following polynucleotide sequences: selected from SEQ ID NO: 15-24 or 45-54, and (i) consisting essentially of the nucleotide sequence of SEQ ID NO: 25, and (ii) consisting essentially of the nucleotide sequence of SEQ ID NO: 32.

[0012] In some embodiments, the method further includes delivering to the cell (c) a site-directed nuclease targeting a site within or near a gene encoding one or more of the MHC-I or MHC-II human leukocyte antigens or components or transcriptional regulatory factors of the MHC-I or MHC-II complex, and (d) a nucleic acid containing a nucleotide sequence encoding a tolerogenic factor, the flanking nucleotide sequence being (iii) a nucleotide sequence homologous to a region to the left of the target site in (c) and (iv) a nucleotide sequence homologous to a region to the right of the target site in (c), wherein the tolerogenic factor in (d) is different from the tolerogenic factor in (b), wherein the site-directed nuclease cleaves the target site in (c), and the nucleic acid in (d) is inserted at a site that partially overlaps, completely overlaps with, or is contained within the site in (c), wherein the universal donor cell has increased immune evasion and / or cell survival compared to a cell in which the nucleic acid in (d) is not inserted.

[0013] In some embodiments, the gene encoding one or more MHC-I or MHC-II human leukocyte antigens, or components or transcriptional regulators of the MHC-I or MHC-II complex, is an MHC-I gene selected from HLA-A, HLA-B, or HLA-C; an MHC-II gene selected from HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR; or a gene selected from B2M, NLRC5, CIITA, RFX5, RFXAP, or RFXANK; and the tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47. In a specific embodiment, the gene encoding one or more MHC-I or MHC-II human leukocyte antigens, or components or transcriptional regulators of the MHC-I or MHC-II complex, is B2M, and the tolerogenic factor is PD-L1. In embodiments where the site-directed nuclease is a CRISPR system comprising a CRISPR nuclease and gRNA, the CRISPR nuclease is a type II Cas9 nuclease or a type V Cfp1 nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal. In some embodiments, the gRNA targets the following polynucleotide sequences: selected from SEQ ID NO:1-3 or 35-44, and (iii) consisting essentially of the nucleotide sequence of SEQ ID NO:7, and (iv) consisting essentially of the nucleotide sequence of SEQ ID NO:13.

[0014] In some embodiments, the nucleotide sequences encoding the tolerogenic factors of (b) and (d) are operatively linked to an exogenous promoter. The exogenous promoter may be selected from constitutive promoters, inducible promoters, time-specific promoters, tissue-specific promoters, or cell-type-specific promoters. In some embodiments, the exogenous promoter is a CMV, EFi, PGK, CAG, or UBC promoter. In a specific embodiment, the exogenous promoter is a CAG promoter.

[0015] This disclosure also covers universal donor cells generated by the methods disclosed herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a pluripotent stem cell (PSC), embryonic stem cell (ESC), adult stem cell (ASC), induced pluripotent stem cell (iPSC), or hematopoietic stem cell and progenitor cell (HSPC) (also known as hematopoietic stem cell (HSC)). In some embodiments, the cell is a differentiated cell. In some embodiments, the cell is a somatic cell.

[0016] Typically, the universal donor cells disclosed herein can differentiate into lineage-restricted progenitor cells or fully differentiated somatic cells. In some embodiments, these lineage-restricted progenitor cells are pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells, and these fully differentiated somatic cells are endocrine secretory cells, such as pancreatic β cells, epithelial cells, endoderm cells, macrophages, hepatocytes, adipocytes, kidney cells, blood cells, or immune system cells. In some embodiments, these fully differentiated somatic cells are cardiomyocytes.

[0017] Another aspect of this disclosure provides a method for treating a subject in need, wherein the method includes obtaining, or having already obtained, universal donor cells as disclosed herein differentiated into lineage-restricted progenitor cells or fully differentiated somatic cells, and administering these lineage-restricted progenitor cells or fully differentiated somatic cells to the subject. A method for obtaining cells for administration to a subject in need is also provided, comprising obtaining, or having already obtained, universal donor cells as disclosed herein, and maintaining these universal donor cells for a time and under conditions sufficient to differentiate these cells into lineage-restricted progenitor cells or fully differentiated somatic cells. In some embodiments, the subject is a person who has a disease, is suspected of having a disease, or is at risk of having a disease. In some embodiments, the disease is a genetically heritable disease.

[0018] Another aspect of the invention covers gRNAs that target polynucleotide sequences selected from SEQ ID NO:15-24 or 45-54.

[0019] While this disclosure is susceptible to various modifications and alternatives, specific embodiments thereof are shown by way of example in the accompanying drawings and will be described in detail herein. However, it should be understood that the accompanying drawings and specific embodiments provided herein are not intended to limit this disclosure to the particular embodiments disclosed, but rather, on the contrary, are intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of this disclosure as defined by the appended claims.

[0020] Other features and advantages of this disclosure will become clear from the following detailed description of embodiments of the invention, with reference to the accompanying drawings. Attached Figure Description

[0021] Figure 1 TIDE analysis of B2M gRNA cleavage in CyT49 cells is shown. B2M-1, B2M-2, and B2M-3 gRNAs were tested.

[0022] Figure 2A-2B This shows the effect in WT CyT49 cells ( Figure 2A ) and B2M KO CyT49 cells ( Figure 2BFlow cytometry assessment of B2M expression with and without IFN-γ.

[0023] Figure 3 The plasmid map of the B2M-CAGGS-PD-L1 donor vector for HDR is shown.

[0024] Figure 4 Flow cytometry analysis of pluripotency of B2M KO / PD-L1 KI CyT49 stem cells is shown. Derived clones were >99% double positive for OCT4 and SOX2 (two transcription factors crucial for pluripotency). IgG was used as a negative control.

[0025] Figures 5A-5B It shows WT CyT49 ( Figure 5A ) and B2M KO / PD-L1 KI( Figure 5B Flow cytometry analysis of derived stem cell clones. WT cells responded to IFNγ-upregulated B2M expression. The B2M KO / PD-L1 KI clone fully expressed PD-L1 and did not express B2M with or without IFNγ treatment. NT-1 = untreated. INTG-1 = cells treated with 50 ng / mL IFNγ for 48 hours.

[0026] Figure 6 Flow cytometry of FOXA2 and SOX17 in stage 1 (determined endoderm) cells differentiated from wild-type CyT49, PD-L1 KI / B2M KO, or B2M KO CyT49 cells is shown.

[0027] Figure 7 The quantitative percentages of FOXA2 and SOX17 expression in stage 1 (determined endoderm) cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells are shown.

[0028] Figure 8 The quantitative percentages of CHGA, PDX1, and NKX6.1 expression are shown in stage 4 (PEC) cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells.

[0029] Figure 9 The heterogeneous cell population at stage 4 (PEC) is shown.

[0030] Figure 10 The selected gene expression at different stages of differentiation is shown in cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells.

[0031] Figure 11A-11FThe selected gene expression at different stages of differentiation is shown in cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells. Figure 11A B2M expression in wild-type cells is shown. Figure 11B B2M expression in B2M KO cells is shown. Figure 11C B2M expression in PD-L1 KI / B2M KO cells is shown. Figure 11D PD-L1 expression in wild-type cells is shown. Figure 11E PD-L1 expression in B2M KO cells is shown. Figure 11F PD-L1 expression in PD-L1KI / B2M KO cells is shown.

[0032] Figure 12A-12F MHC class I and II expression at the PEC stage are shown in cells differentiated from wild-type, PD-L1 KI / B2M KO, or B2M KO cells. Figure 12A MHC class I expression is shown in wild-type cells. Figure 12B MHC class I expression is shown in B2M KO cells. Figure 12C MHC class I expression is shown in PD-L1 KI / B2M KO cells. Figure 12D The expression of MHC class II PD-L1 in wild-type cells is shown. Figure 12E MHC class II expression is shown in B2M KO cells. Figure 12F MHC class II expression was shown in PD-L1 KI / B2M KO cells.

[0033] Figure 13 TIDE analysis of TXNIP gRNA cleavage in TC1133 hiPSC is shown. Guide T5 appears to be optimal for cleavage at exon 1.

[0034] Figure 14 The plasmid map of the TXNIP-CAGGS-HLA-E donor vector for HDR is shown.

[0035] Figure 15 Flow cytometry analysis of the pluripotency of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI CyT49 stem cells is shown. The derived clones were >99% double-positive for OCT4 and SOX2 (two transcription factors crucial for pluripotency). These clones also did not express B2M. These clones did not express MHC-I.

[0036] Figure 16Flow cytometry analysis of the pluripotency of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI CyT49 stem cells is shown. The derived clones expressed PD-L1 and HLA-E after differentiation to stage 6 (immature β cells). IgG was used as a negative control.

[0037] Figure 17 The quantitative percentages of CHGA, PDX1, and NKX6.1 expression are shown in stage 4 (PEC) cells differentiated from wild-type, B2M KO, PD-L1 KI / B2M KO (V1A), or TXNIP KO / HLA-E KI (V1B) hESCs.

[0038] Figures 18A-18B TXNIP KO cells were shown. Figure 18A ) or TXNIP KO / HLA-E KI(V1B)( Figure 18B The gene expression selected by cells during the differentiation process.

[0039] Figures 19A-19B Flow cytometry analysis of T cell activation using CFSE proliferation assay is shown. Human primary CD3+ T cells were co-incubated with PECs derived from WT, B2M KO, B2M KO / PD-L1 KI, or B2M KO / PD-L1 KI+TXNIP KO / HLA-EKI CyT49 clones. Figure 19A ). Figure 19B T cell activation in various cell types was summarized. One-way ANOVA was performed with the "CFSE-T alone" set as a control (α = 0.05, using Dunnett's multiple comparison test). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. ns = not significant.

[0040] Figure 20 The selected gene expression in cells differentiated from TXNIP KO cells at different stages of the differentiation process is shown.

[0041] Figure 21 Flow cytometry assessment of PDX1 and NKX6.1 expression in PEC cells differentiated from TXNIP KO cells is shown.

[0042] Figure 22Morphology of various B2M KO / PD-L1KI and TXNIP KO / HLA-E KI clones ("S6-V1B-H9", "S6-V1B-3B11", "S6-V1B-1G7" and "S6-V1B-3C2") after differentiation to stage 6 is shown compared with wild-type ("WT") and non-cut guide control ("NCG#1") cells.

[0043] Figures 23A-23F The selected gene expression of clones after differentiation to stage 6 is shown. Figure 23A The selected gene expression at different stages of differentiation is shown in cells differentiated from exemplary B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones. Figure 23B-23F The figures show the INS values ​​in wild-type cells differentiated to stage 6 (“S6-Cyt49 WT”), uncut guide control (“S6-NCG#1”) cells, and various B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones (“S6-V1B-H9”, “S6-V1B-3B11”, “S6-V1B-1G7”, and “S6-V1B-3C2”). Figure 23B ), NKX6.1 Figure 23C ), GCG Figure 23D ), SST Figure 23E ) and GCK Figure 23F Gene expression of ) was measured, with undifferentiated B2M KO / PD-L1 KI and TXNIPKO / HLA-E KI clones (“ES-V1B-H9”) and wild-type islets (“islets”) serving as controls.

[0044] Figures 24A-24B The expression of INS and GCG in stage 6 cells differentiated from B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones is shown. Figure 24A ) and INS and NKX6.1 expressions ( Figure 24B Flow cytometry evaluation.

[0045] Figures 25A-25B The image shows INS expression in stage 6 cells differentiated from wild-type cells (“S6-WT”), uncut guide control cells (“S6-NCG#1”), and two B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones (“S6-V1B003” and “V1B-H9”). Figure 25A ) and NKX6.1 expression ( Figure 25B The percentage of ).

[0046] Figure 26AFlow cytometry assessment of PDX1 and NKX6.1 expression in stage 4 cells differentiated from clone 1 (B2M KO / PD-L1 KI and TXNIP KO / HLA-EKI) cells with different seeding densities is shown.

[0047] Figure 26B Flow cytometry assessment of PD-L1 and HLA-E expression in stage 4 cells differentiated from clone 1 (B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI) cells is shown.

[0048] Figures 27A-27C Characteristic analysis of seed clones differentiated to the PEC stage is shown. Figure 27A It shows morphology, Figure 27B The selected gene expression at different differentiation timelines is shown, and Figure 27C This shows the expression of CHGA in the differentiated population. - / NKX6.1 + / PDX1 + The percentage of cells.

[0049] Figure 28 The selected gene expression at different stages of differentiation is shown in cells differentiated from the TXNIP KO / HLA-E KI clone. Detailed Implementation

[0050] I. Definition

[0051] Deletion: As used herein, the term "deletion," which may be used interchangeably with the terms "genetic deletion" or "knockout," generally refers to the following genetic modification in which a site or region of genomic DNA is removed by any molecular biological method, such as those described herein, for example by delivering an endonuclease and at least one gRNA to the site of genomic DNA. Any number of nucleotides may be deleted. In some embodiments, deletion involves the removal of at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, at least twenty, or at least 25 nucleotides. In some embodiments, deletion involves the removal of 10–50, 25–75, 50–100, 50–200, or more than 100 nucleotides. In some embodiments, deletion involves the removal of an entire target gene, such as the B2M gene. In some embodiments, deletion involves the removal of a portion of a target gene, such as all or a portion of the promoter and / or coding sequence of the B2M gene. In some embodiments, deletion involves the removal of transcriptional regulators of a target gene, such as the promoter region. In some embodiments, deletion involves the removal of all or a portion of a coding region such that a product normally expressed by the coding region is no longer expressed, expressed in a truncated form, or expressed at a reduced level. In some embodiments, the deletion results in a reduction in gene expression relative to unmodified cells.

[0052] Endonuclease: As used herein, the term "endonuclease" generally refers to an enzyme that cleaves the phosphodiester bond within a polynucleotide. In some embodiments, an endonuclease specifically cleaves the phosphodiester bond within a DNA polynucleotide. In some embodiments, the endonuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a homing endonuclease (HE), a broad-spectrum nuclease, a MegaTAL, or a CRISPR-associated endonuclease. In some embodiments, the endonuclease is an RNA-guided endonuclease. In some aspects, the RNA-guided endonuclease is a CRISPR nuclease, such as type II CRISPR Cas9 endonuclease or 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, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm 6. Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonucleases, or their homologs, recombinant molecules thereof, their codon-optimized forms or modified forms, or combinations thereof. In some embodiments, the endonuclease may introduce one or more single-strand breaks (SSBs) and / or one or more double-strand breaks (DSBs).

[0053] Genetic modification: As used herein, the term "genetic modification" generally refers to a genomic DNA site that has been genetically edited or manipulated using any molecular biology method, such as those described herein, for example by delivering an endonuclease and at least one gRNA to the site of genomic DNA. Exemplary genetic modifications include insertions, deletions, duplications, inversions, and translocations, and combinations thereof. In some embodiments, the genetic modification is a deletion. In some embodiments, the genetic modification is an insertion. In other embodiments, the genetic modification is an insertion-deletion mutation (or indel) that causes a shift in the reading frame of the target gene, resulting in an altered gene product or no gene product.

[0054] Guide RNA (gRNA): As used herein, the term "guide RNA" or "gRNA" generally refers to a short ribonucleic acid that can interact with, for example, bind to, endonucleases and bind to or hybridize to a target genomic site or region. In some embodiments, gRNA is a single-molecule guide RNA (sgRNA). In some embodiments, gRNA may include a spacer extension region. In some embodiments, gRNA may include a tracrRNA extension region. In some embodiments, gRNA is single-stranded. In some embodiments, gRNA contains naturally occurring nucleotides. In some embodiments, gRNA is chemically modified gRNA. In some embodiments, chemically modified gRNA is gRNA containing at least one nucleotide with a chemical modification (e.g., 2′-O-methyl sugar modification). In some embodiments, chemically modified gRNA contains a modified nucleic acid backbone. In some embodiments, chemically modified gRNA contains 2′-O-methyl-phosphothioester residues. In some embodiments, gRNA may be pre-complexed with a DNA endonuclease.

[0055] Insertion: As used herein, the term "insertion," which may be used interchangeably with the terms "genetic insertion" or "knock-in," generally refers to the following genetic modification in which a polynucleotide is introduced or added to a site or region of genomic DNA by any molecular biology method, such as those described herein, for example by delivering an endonuclease and at least one gRNA to the site of genomic DNA. In some embodiments, the insertion may occur within or near a site of genomic DNA that is already a previously genetically modified site (e.g., a deletion or insertion-deletion mutation). In some embodiments, the insertion occurs at a site of genomic DNA that partially overlaps, completely overlaps with, or is contained within a previously genetically modified site (e.g., a deletion or insertion-deletion mutation). In some embodiments, the insertion occurs at a safe harbor locus. In some embodiments, the insertion involves the introduction of a polynucleotide encoding a target protein. In some embodiments, the insertion involves the introduction of a polynucleotide encoding a tolerance 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 foreign promoter, such as a constitutive promoter (e.g., a CAG promoter). In some embodiments, the insertion involves the introduction of a polynucleotide encoding a non-coding gene. Typically, the polynucleotide to be inserted is flanked by a sequence (e.g., a homology arm) that has significant sequence homology with the genomic DNA at or near the insertion site.

[0056] 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, such as humans); and whose function is to present peptides of non-self or foreign antigens (e.g., proteins) from within the cell (i.e., the cytosol) to cytotoxic T cells (e.g., CD8+ T cells) in order to stimulate an immune response. In some embodiments, the MHC-I biomolecule is an MHC-I gene or an MHC-I protein. The complexation of MHC-I protein with β-2 microglobulin (B2M) is required for the cell surface expression of all MHC-I proteins. In some embodiments, reduced expression of MHC-I human leukocyte antigen (HLA) relative to unmodified cells involves a reduction (or decrease) in MHC-I gene expression. In some embodiments, reduced expression of MHC-I human leukocyte antigen (HLA) relative to unmodified cells involves a reduction (or decrease) in the cell surface expression of MHC-I proteins. In some embodiments, the MHC-I biomolecule is HLA-A (NCBI gene ID: 3105), HLA-B (NCBI gene ID: 3106), HLA-C (NCBI gene ID: 3107), or B2M (NCBI gene ID: 567).

[0057] Major Histocompatibility Complex II (MHC-II): As used herein, the term "major histocompatibility complex II" or "MHC-II" generally refers to a class of biomolecules that are commonly found on the cell surface of antigen-presenting cells in vertebrates (including mammals, such as humans); and whose function is to present peptides of non-self or foreign antigens (e.g., proteins) to cytotoxic T cells (e.g., CD8+ T cells) from outside the cell (extracellular) in order to stimulate an immune response. In some embodiments, antigen-presenting cells are dendritic cells, macrophages, or B cells. In some embodiments, the MHC-II biomolecule is an MHC-II gene or an MHC-II protein. In some embodiments, reduced expression of MHC-II human leukocyte antigen (HLA) relative to unmodified cells involves a reduction (or decrease) in MHC-II gene expression. In some embodiments, reduced expression of MHC-II human leukocyte antigen (HLA) relative to unmodified cells involves a reduction (or decrease) in the expression of MHC-II protein on the cell surface. In some embodiments, the MHC-II biomolecule is HLA-DPA (NCBI gene ID: 3113), HLA-DPB (NCBI gene ID: 3115), HLA-DMA (NCBI gene ID: 3108), HLA-DMB (NCBI gene ID: 3109), HLA-DOA (NCBI gene ID: 3111), HLA-DOB (NCBI gene ID: 3112), HLA-DQA (NCBI gene ID: 3117), HLA-DQB (NCBI gene ID: 3119), HLA-DRA (NCBI gene ID: 3122), or HLA-DRB (NCBI gene ID: 3123).

[0058] Polynucleotide: As used herein, the term "polynucleotide," which may be used interchangeably with the term "nucleic acid," generally refers to a biomolecule containing two or more nucleotides. In some embodiments, a polynucleotide contains at least two, at least five, at least ten, at least twenty, at least 30, at least 40, at least 50, at least 100, at least 200, at least 250, at least 500, or any number of nucleotides. For example, a polynucleotide may include 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 within the genome of an unmodified cell or a universal donor cell. In some embodiments, the polynucleotide is an exogenous polynucleotide not integrated into the genomic DNA. In some embodiments, the polynucleotide is an exogenous polynucleotide integrated into the 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.

[0059] Safe harbor loci: As used herein, the term “safe harbor locus” generally refers to any location, site, or region of genomic DNA that can accommodate genetic insertions into said location, site, or region without adverse effects on the cell. In some embodiments, a safe harbor locus is an intragenic or extragenic region. In some embodiments, a safe harbor locus is a region of genomic DNA that is normally transcribedly silenced. In some embodiments, a safe harbor locus is AAVS1 (PPP1 R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1, TF, or TTR locus. In some embodiments, safe harbor loci are described in Sadelain, M. et al., “Safe harbors for the integration of new DNA in the human genome,” Nature Reviews Cancer, 2012, Vol. 12, pp. 51–58.

[0060] Safety Switch: As used herein, the term "safety switch" generally refers to a biomolecule that causes cells to undergo apoptosis. In some embodiments, the safety switch is a protein or gene. In some embodiments, the safety switch is a suicide gene. In some embodiments, the safety switch (e.g., herpes simplex virus thymidine kinase (HSV-tk)) causes cells to undergo apoptosis via a metabolizing prodrug (e.g., ganciclovir). In some embodiments, overexpression of the safety switch alone causes cells to undergo apoptosis. In some embodiments, the safety switch is a p53-based molecule, HSV-tk, or inducible caspase-9.

[0061] 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 a disease or disorder, is suspected of having a disease or disorder, or is at risk of having a disease or disorder. In some embodiments, the subject has one or more symptoms of a disease or disorder.

[0062] Survival Factor: As used herein, the term "survival factor" generally refers to a protein (e.g., expressed by a polynucleotide as described herein) that, when increased or decreased in cells, enables cells (e.g., universal donor cells) to survive with a higher survival rate than unmodified cells after transplantation or engraftment into a host subject. In some embodiments, the survival factor is a human survival factor. In some embodiments, the survival factor is a member of a key pathway involved in cell survival. In some embodiments, the key pathway involved in cell survival is 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 universal donor cells to survive for a longer period of time after engraftment than unmodified cells, for example, at least 1.05 times, at least 1.1 times, at least 1.25 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, or at least 50 times longer. In some embodiments, the survival factor is ZNF143 (NCBI gene ID: 7702), TXNIP (NCBI gene ID: 10628), FOXO1 (NCBI gene ID: 2308), JNK (NCBI gene ID: 5599), or MANF (NCBI gene ID: 7873). In some embodiments, the survival factor is inserted into cells (e.g., universal donor cells). In some embodiments, the survival factor is deleted from cells (e.g., universal donor cells). In some embodiments, the insertion of a polynucleotide encoding MANF enables cells (e.g., universal donor cells) to survive with a higher survival rate than unmodified cells after transplantation or engraftment into a host subject. In some embodiments, deletions or insertion-deletion mutations within or near the ZNF143, TXNIP, FOXO1, or JNK genes enable cells (e.g., universal donor cells) to survive with a higher survival rate than unmodified cells after transplantation or engraftment into a host subject.

[0063] Tolerogenic factors: As used herein, the term "tolerogenic factor" generally refers to a protein (e.g., expressed by a polynucleotide as described herein) that, when increased or decreased in cells, enables cells (e.g., universal donor cells) to suppress or evade immune rejection at a higher rate than unmodified cells 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 cells (e.g., universal donor cells) to suppress or evade immune rejection at a rate at least 1.05-fold, at least 1.1-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold higher than unmodified cells after engraftment. In some embodiments, the tolerogenic factor is HLA-E (NCBI gene ID: 3133), HLA-G (NCBI gene ID: 3135), CTLA-4 (NCBI gene ID: 1493), CD47 (NCBI gene ID: 961), or PD-L1 (NCBI gene ID: 29126). In some embodiments, the tolerogenic factor is inserted into cells (e.g., universal donor cells). In some embodiments, the tolerogenic factor is deleted from cells (e.g., universal donor cells). In some embodiments, the insertion of polynucleotides encoding HLA-E, HLA-G, CTLA-4, CD47, and / or PD-L1 enables the cells (e.g., universal donor cells) to suppress or evade immune rejection after transplantation or engraftment into a host subject.

[0064] MHC-I or MHC-II transcriptional regulators: As used herein, the term "MHC-I or MHC-II transcriptional regulator" generally refers to a biomolecule that regulates (e.g., increases or decreases) the 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, MHC-I or MHC-II transcriptional regulators increase or decrease the cell surface expression of at least one MHC-I or MHC-II protein. In some embodiments, MHC-I or MHC-II transcriptional regulators increase or decrease the expression of at least one MHC-I or MHC-II gene. In some embodiments, the transcriptional regulator is CIITA (NCBI gene ID: 4261) or NLRC5 (NCBI gene ID: 84166). In some embodiments, the loss or reduction of CIITA or NLRC5 expression reduces the expression of at least one MHC-I or MHC-II gene.

[0065] Universal donor cells: As used herein, the term "universal donor cell" 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 as described herein are universal donor cells. In some embodiments, universal donor cells have increased immune evasion and / or cell survival compared to unmodified cells. In some embodiments, universal donor cells have increased cell survival compared to unmodified cells. In some embodiments, universal donor cells may be stem cells. In some embodiments, universal donor cells may be embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), or hematopoietic stem cells or progenitor cells (HSPCs) (also referred to as hematopoietic stem cells (HSCs)). In some embodiments, universal donor cells may be differentiated cells. In some embodiments, universal donor cells may be somatic cells (e.g., immune system cells). In some embodiments, universal donor cells are administered to a subject. In some embodiments, universal donor cells are administered to a subject who has a disease, is suspected of having a disease, or is at risk of having 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, lineage-restricted progenitor cells are pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells. In some embodiments, fully differentiated somatic cells are endocrine cells such as pancreatic β cells, epithelial cells, endoderm cells, macrophages, hepatocytes, adipocytes, kidney cells, blood cells, or immune system cells. In some embodiments, these fully differentiated somatic cells are cardiomyocytes.

[0066] Unmodified cells: As used herein, the term "unmodified cell" refers to a cell that has not undergone genetic modification involving polynucleotides or genes encoding transcriptional regulatory factors, survival factors, and / or tolerogenic factors of MHC-I, MHC-II, MHC-II, or MHC-II. In some embodiments, unmodified cells may be stem cells. In some embodiments, unmodified cells may be embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), or hematopoietic stem cells or progenitor cells (HSPCs) (also referred to as hematopoietic stem cells (HSCs)). In some embodiments, unmodified cells may be differentiated cells. In some embodiments, unmodified cells may be selected from somatic cells (e.g., immune system cells, such as T cells, such as CD8+ T cells). When a universal donor cell is compared "relative to an unmodified cell," the universal donor cell and the unmodified cell are of the same cell type or have a common parental cell line, for example, comparing a universal donor iPSC relative to an unmodified iPSC.

[0067] Within or near a gene: As used herein, the term "within or near a gene" refers to a site or region of genomic DNA that is a component of an intron or exon of the gene or located proximal to the gene. In some embodiments, the site of genomic DNA is considered to be within the gene if it contains at least a portion of an intron or exon of the gene. In some embodiments, the site of genomic DNA 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, the site of genomic DNA near a gene may be a promoter region or repressor region that regulates the expression of the gene. In some embodiments, the site of genomic DNA near a gene may be on the same chromosome as the gene. In some embodiments, the site or region of genomic DNA is considered to be near the gene if it is within or closer to 50 Kb, 40 Kb, 30 Kb, 20 Kb, 10 Kb, 5 Kb, or 1 Kb of the 5' or 3' end of the gene (e.g., at the 5' or 3' end of the coding region of the gene).

[0068] II. Genome Editing Methods

[0069] Genome editing generally refers to the process of modifying the nucleotide sequence of a genome in a precise or predetermined manner. In some embodiments, genome editing methods as described herein (e.g., CRISPR-endonuclease systems) can be used to genetically modify cells as described herein, for example, to generate universal donor cells. In some embodiments, genome editing methods as described herein (e.g., CRISPR-endonuclease systems) can be used to genetically modify cells as described herein, for example, to introduce at least one genetic modification within or near at least one gene that reduces the expression of one or more MHC-I and / or MHC-II human leukocyte antigens or other components of the MHC-I or MHC-II complex relative to unmodified cells; to introduce at least one genetic modification that increases the expression of at least one polynucleotide encoding a tolerance factor relative 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 relative to unmodified cells.

[0070] Examples of genome editing methods described in this article include those that use site-directed nucleases to cut deoxyribonucleic acid (DNA) at precise target sites within the genome, thereby creating single-stranded or double-stranded DNA breaks at specific locations within the genome. Such breaks can and regularly are repaired by natural endogenous cellular processes such as homologous targeted 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 main DNA repair processes consist of a series of alternative pathways. NHEJ directly joins DNA ends resulting from double-strand breaks, sometimes losing or adding nucleotide sequences, which can disrupt or enhance gene expression. HDR utilizes homologous or donor sequences as templates to insert a defined DNA sequence at the break point. Homologous sequences can be found in the endogenous genome, such as sister chromatids. Alternatively, the donor sequence can be a foreign polynucleotide, such as a plasmid, single-stranded oligonucleotide, double-stranded oligonucleotide, duplex oligonucleotide, or virus, which has a region highly homologous to the locus cleaved by the nuclease (e.g., left and right homologous arms), but may also contain additional sequences or sequence variations (including deletions that can be incorporated into the cleaved target locus). A third repair mechanism can be microhomology-mediated end joining (MMEJ), also known as "alternative NHEJ," which has similar genetic outcomes to NHEJ because small deletions and insertions can occur at the cleavage site. MMEJ can utilize homologous sequences flanking a few base pairs at 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, the likely repair outcome can be predicted based on the analysis of potential microhomology at the DNA break site.

[0071] Each of these genome editing mechanisms can be used to produce the desired genetic modification. One step in the genome editing process can be to generate one or two DNA breaks, either double-strand breaks or two single-strand breaks, at a target locus near the intended mutation site. As described and illustrated in this paper, this can be achieved using endonucleases.

[0072] CRISPR endonuclease system

[0073] The CRISPR-endonuclease system is a naturally occurring defense mechanism in prokaryotes, which has been repurposed as an RNA-guided DNA-targeting platform for gene editing. CRISPR systems include types I, II, III, IV, V, and VI. In some respects, the CRISPR system is the type II CRISPR / Cas9 system. In others, it is the type V CRISPR / Cprf system. The CRISPR system relies on a DNA endonuclease (e.g., Cas9) and two non-coding RNAs (crisprRNA (crRNA) and trans-activating RNA (tracrRNA)) to target DNA cleavage.

[0074] crRNA drives the sequence recognition and specificity of the CRISPR-endonuclease complex through Watson-Crick base pairing, typically involving approximately 20 nucleotides (nt) of the target DNA sequence. Altering the 5' 20 nt sequence of the crRNA allows the CRISPR-endonuclease complex to target a specific locus. If the target sequence is followed by a specific short DNA motif (the sequence is NGG) (called the prototype spacer adjacent motif (PAM)), the CRISPR-endonuclease complex binds only to DNA sequences containing the first 20 nt sequence that matches the single guide RNA (sgRNA).

[0075] TracrRNA hybridizes to the 3' end of crRNA to form an RNA double-stranded structure, which binds to an endonuclease to form a catalytically active CRISPR-endonuclease complex, which can then cleave target DNA.

[0076] Once the CRISPR-endonuclease complex binds to DNA at the target site, the two independent nuclease domains within the endonuclease each cleave one of the DNA strands three bases upstream of the PAM site, leaving a double-strand break (DSB), where the two strands of DNA terminate with a base pair (blunt end).

[0077] In some embodiments, the endonuclease is Cas9 (CRISPR-associated protein 9). In some embodiments, the Cas9 endonuclease is derived from *Streptococcus pyogenes*, but other Cas9 homologs can be used, such as *Staphylococcus aureus* Cas9, *Neisseria meningitidis* Cas9, *Streptococcus thermophilus* CRISPR 1 Cas9, *Streptococcus 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, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease. In some embodiments, a wild-type variant may be used. In some embodiments, modified forms of the aforementioned endonucleases (e.g., their homologs, recombinations of their naturally occurring molecules, codon optimizations, or modified forms thereof) may be used.

[0078] CRISPR nucleases can be linked to at least one nuclear localization signal (NLS). The at least one NLS can be located at or within 50 amino acids from the amino terminus of the CRISPR nuclease, and / or at least one NLS can be located at or within 50 amino acids from the carboxyl terminus of the CRISPR nuclease.

[0079] Exemplary CRISPR / Cas peptides include the Cas9 peptide disclosed 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 the Cas gene, the CRISPR / Cas gene nomenclature system has undergone extensive rewriting. Fonfara et al. also provided PAM sequences of Cas9 peptides from various species.

[0080] Zinc finger nucleases

[0081] Zinc finger nucleases (ZFNs) are modular proteins consisting of engineered zinc finger DNA-binding domains linked to the catalytic domain of the type II endonuclease FokI. Since FokI functions only as a dimer, a pair of ZFNs must be engineered to bind to homologous target "half-sites" on opposite DNA strands, with precise spacing between them enabling the formation of catalytically active FokI dimers. Following the dimerization of the non-sequence-specific FokI domains, double-strand breaks occur between the ZFN half-sites, serving as the initiation step for genome editing.

[0082] Each ZFN's DNA-binding domain typically consists of 3-6 zinc fingers with an abundant Cys2-His2 architecture. Each finger primarily recognizes a nucleotide triplet on one strand of the target DNA sequence, although cross-strand interactions with a fourth nucleotide can also be important. Changes in the amino acids of the fingers at key DNA contact sites alter the sequence specificity of a given finger. Thus, a four-fingered zinc finger protein will selectively recognize a 12 bp target sequence, which is a synthesis of the triplet preference contributed by each finger, although the triplet preference may be influenced to varying degrees by adjacent fingers. An important aspect of ZFNs is that they can be easily retargeted to almost any genomic address by modifying only a single finger. In most applications of ZFNs, 4-6 fingered proteins are used, each recognizing 12-18 bp. Therefore, a pair of ZFNs will typically recognize a combined target sequence of 24-36 bp (excluding the typical 5-7 bp spacer between halves). Binding sites can be further separated by larger spacers (including 15-17 bp). Assuming repetitive sequences or genetic homologs are excluded during the design process, a target sequence of that length is likely unique in the human genome. However, ZFN protein-DNA interactions are not absolute in their specificity, so off-target binding and cleavage events do occur, either as heterodimers between two ZFNs or as homodimers of one or the other. The latter possibility is effectively eliminated by engineering the dimerization interface of the FokI domain to produce “positive” and “negative” variants (also known as obligate heterodimer variants, which can only dimerize with each other and not with themselves). Facilitating obligate heterodimers prevents the formation of homodimers. This significantly improves the specificity of ZFNs and any other nucleases employing these FokI variants.

[0083] Various ZFN-based systems have been described in this field, with modifications reported regularly, and numerous references describe the rules and parameters used to guide ZFN design; 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.

[0084] Transcription activator-like effector nucleases (TALENs)

[0085] TALEN represents another form of modular nuclease, in which, like ZFN, an engineered DNA-binding domain is linked to a FokI nuclease domain, and a pair of TALENs work in tandem to achieve targeted DNA cleavage. The main difference from ZFN lies in the nature of the DNA-binding domain and the associated target DNA sequence recognition characteristics. The TALEN DNA-binding domain is derived from TALE proteins, which were initially described in species of the plant bacterial pathogen *Xanthomonas* sp. TALE consists of a tandem array of 33–35 amino acid repeat sequences, each repeat recognizing a single base pair in the target DNA sequence. This repeat is typically 20 bp long, resulting in a total target sequence length of 40 bp. The nucleotide specificity of each repeat sequence is determined by the variable double residue repeat (RVD), which includes two amino acids only at positions 12 and 13. Guanine, adenine, cytosine, and thymine bases are primarily recognized by four RVDs: Asn-Asn, Asn-Ile, His-Asp, and Asn-Gly, respectively. This constitutes a much simpler recognition code than the zinc finger, thus offering an advantage over the zinc finger in nuclease design. However, like ZFN, the protein-DNA interaction of TALEN is not absolute in its specificity, and TALEN also benefits from using a specific heterodimeric variant of the FokI domain to reduce off-target activity.

[0086] Additional variants have been developed that inactivate the FokI domain in its catalytic function. If half of the TALEN or ZFN pair contains the inactivated FokI domain, only single-stranded DNA cleavage (creating a nick) occurs at the target site, without DSB. The results are comparable to using CRISPR / Cas9 or CRISPR / Cpf1 “nickase” mutants (where one of the Cas9 cleavage domains is inactivated). DNA nicks can be used to drive genome editing via HDR, but at a lower efficiency than DSB. Unlike DSB, the main benefit is that off-target nicks are repaired rapidly and accurately, whereas DSB is susceptible to NHEJ-mediated false repair.

[0087] Various TALEN-based systems have been described in this field, and their modifications are reported regularly; 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. Several groups have described the use of TALEN based on the "Golden Gate" platform or cloning protocol; 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.

[0088] homing endonuclease

[0089] Homing endonucleases (HEs) are sequence-specific endonucleases with long recognition sequences (14–44 base pairs) that typically cleave DNA at a unique site within the genome with high specificity. There are at least six known families of HEs classified by their structure, including GIY-YIG, His-Cis boxes, HNH, PD-(D / E)xK, and Vsr-like enzymes, derived from a wide range of hosts, including eukaryotes, protozoa, bacteria, archaea, cyanobacteria, and bacteriophages. Like ZFNs and TALENs, HEs can be used to generate site-specific cleavage sites (DSBs) at target loci as a starting step in genome editing. Additionally, some natural and engineered HEs cleave only single-stranded DNA, thus functioning as site-specific cleavage enzymes. The large target sequences of HEs and the specificity they provide make them attractive candidates for generating site-specific DSBs.

[0090] Several HE-based systems have been described in this field, and their modifications are reported regularly; see reviews such as those in the following literature: 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.

[0091] MegaTAL / Tev-mTALEN / MegaTev

[0092] As another example of hybrid nucleases, the MegaTAL and Tev-mTALEN platforms utilize the fusion of the TALE DNA-binding domain and the catalytically active HE, taking advantage of both the tunable DNA binding and specificity of TALE and the cleavage sequence specificity of HE; see, for example, 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.

[0093] In another variant, the MegaTev architecture is a fusion of a large-scale nuclease (Mega) with a nuclease domain derived from the GIY-YIG homing endonuclease I-TevI ​​(Tev). These two active sites are approximately 30 bp apart on the DNA substrate, generating two DSBs with incompatible sticky ends; see, for example, Wolfs et al., Nucleic Acids Res., 2014, 42, 8816-29. It is foreseeable that other combinations of existing nuclease-based methods will be developed and used to achieve the targeted genome modifications described herein.

[0094] dCas9-FokI or dCpf1-Fok1 and other nucleases

[0095] Combining the structural and functional characteristics of the aforementioned nuclease platforms offers an alternative approach to genome editing that may overcome some inherent limitations. For example, CRISPR genome editing systems typically use a single Cas9 endonuclease to generate DSBs. Target specificity is driven by a 20 or 24-nucleotide sequence in the guide RNA that performs a Watson-Crick base pairing with the target DNA (in the case of Cas9 from Streptococcus pyogenes, this is supplemented by two additional bases from the adjacent NAG or NGG PAM sequence). This sequence is long enough to be unique in the human genome; however, the specificity of the RNA / DNA interaction is not absolute and can sometimes tolerate significant contamination, especially at the 5' half of the target sequence, which effectively reduces the number of bases driving specificity. One solution is to completely inactivate the catalytic function of Cas9 or Cpf1 (retaining only RNA-guided DNA binding function) and fuse the FokI domain with 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. Since FokI must dimerize to become catalytically active, two guide RNAs are needed to tether the two FokI fusions to a close proximity to form a dimer and cleave DNA. This essentially doubles the number of bases at the combined target site, thereby increasing the tightness of targeting by CRISPR-based systems.

[0096] As another example, the fusion of the TALE DNA-binding domain with catalytically active HE (such as I-TevI) leverages the tunable DNA binding and specificity of TALE, as well as the cleavage sequence specificity of I-TevI, which is expected to further reduce off-target cleavage.

[0097] RNA-guided endonuclease

[0098] The RNA-guided endonuclease system used herein may contain an amino acid sequence that has 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 with a wild-type exemplary endonuclease (e.g., Cas9 from Streptococcus pyogenes, US 2014 / 0068797 Sequence ID No. 8 or Sepranauskas et al., Nucleic Acids Res, 39(21):9275-9282(2011)). The endonuclease may contain at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type endonuclease (e.g., Cas9 from Streptococcus pyogenes, as above) in 10 consecutive amino acids. The endonuclease may contain at most: at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with the wild-type endonuclease (e.g., Cas9 from Streptococcus pyogenes, as above) in 10 consecutive amino acids. An endonuclease may at least contain at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with a wild-type endonuclease (e.g., Cas9 from Streptococcus pyogenes, as above) on 10 consecutive amino acids in the HNH nuclease domain of the endonuclease. An endonuclease may at most contain at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with a wild-type endonuclease (e.g., Cas9 from Streptococcus pyogenes, as above) on 10 consecutive amino acids in the HNH nuclease domain of the endonuclease. An endonuclease may at least contain at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with a wild-type endonuclease (e.g., Cas9 from Streptococcus pyogenes, as above) on ...).

[0099] Endonucleases may include modified forms of wild-type exemplary endonucleases. Modified forms of wild-type exemplary endonucleases may contain mutations that reduce the nucleic acid cleavage activity of the endonuclease. Modified forms of wild-type exemplary endonucleases 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 Streptococcus pyogenes, ibid.). Modified forms of endonucleases may not have significant nucleic acid cleavage activity. When an endonuclease is a modified form without significant nucleic acid cleavage activity, it is referred to herein as "enzyme-inactivated".

[0100] The envisioned mutations can include substitution, addition, and deletion, or any combination thereof. A mutation may convert a mutated amino acid to alanine. A mutation may convert a 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). A mutation may convert a mutated amino acid to a non-natural amino acid (e.g., selenomethionine). A mutation may convert a mutated amino acid to an amino acid mimic (e.g., a phosphomimic). Mutations can be conserved. For example, a mutation may convert a mutated amino acid to an amino acid similar in size, shape, charge, polarity, conformation, and / or rotational isomer to the mutated amino acid (e.g., cysteine / serine mutation, lysine / asparagine mutation, histidine / phenylalanine mutation). Mutations can cause reading frame shifts and / or premature stop codon generation. Mutations can cause changes in the regulatory regions of genes or loci that affect the expression of one or more genes.

[0101] Guide RNA

[0102] This disclosure provides guide RNAs (gRNAs) that can direct the activity of an associated endonuclease to a specific target site within a polynucleotide. The guide RNA may comprise at least one spacer sequence that hybridizes to the target nucleic acid sequence, and a CRISPR repeat sequence. In type II CRISPR systems, the gRNA also comprises a second RNA sequence called tracrRNA. In type II CRISPR guide RNAs (gRNAs), the CRISPR repeat sequence and the tracrRNA sequence hybridize to form a double strand. In type V CRISPR systems, the gRNA comprises crRNA that forms the double strand. In some embodiments, the gRNA can bind to an endonuclease, causing the gRNA and the endonuclease to form a complex. The gRNA can provide target specificity to the complex due to its association with the endonuclease. Therefore, targeting a genomic nucleic acid can direct the activity of the endonuclease.

[0103] Exemplary guide RNAs include spacer sequences comprising 15-200 nucleotides, wherein 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 may 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.

[0104] gRNA can be a bimolecular guide RNA. gRNA can also be a single-molecule guide RNA.

[0105] A bimolecular guide RNA can consist of two RNA strands. The first strand contains an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence in the 5' to 3' direction. The second strand can contain a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0106] The single-molecule guide RNA (sgRNA) may contain, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single-molecule guide adapter, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension sequence may contain elements that contribute additional function (e.g., stability) to the guide RNA. The single-molecule guide adapter can link the minimal CRISPR repeat sequence and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension may contain one or more hairpins.

[0107] In some embodiments, the sgRNA includes a 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA includes a spacer sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA includes a spacer sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA includes a spacer sequence of variable length having 17-30 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA includes a spacer extension sequence of length exceeding 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 nucleotides. In some embodiments, the sgRNA comprises a spacer extension sequence of less than 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides in length.

[0108] In some embodiments, the sgRNA comprises a spacer extension sequence containing another portion (e.g., a stability control sequence, an endonuclease-binding sequence, or a ribozyme). This portion can reduce or increase the stability of the target nucleic acid. This portion can be a transcription termination segment (i.e., a transcription termination sequence). This portion can function in eukaryotic cells. This portion can function in prokaryotic cells. This portion can function in both eukaryotic and prokaryotic cells. Non-limiting examples of suitable portions include: 5' caps (e.g., 7-methylguanylate caps (m7 G)), riboswitch sequences (e.g., sequences that allow proteins and protein complexes to regulate stability and / or accessibility), sequences that form dsRNA duplexes (i.e., hairpins), sequences that target RNA to subcellular locations (e.g., the nucleus, mitochondria, chloroplasts, etc.), modifications or sequences that provide tracking (e.g., direct conjugation to fluorescent molecules, conjugation to portions that promote fluorescence detection, sequences that allow fluorescence detection, etc.), and / or modifications or sequences that provide binding sites for proteins (e.g., proteins that act on DNA, including transcription activators, transcription repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.).

[0109] In some embodiments, the sgRNA includes a spacer sequence that hybridizes with a sequence in the target polynucleotide. The spacer of the gRNA can interact with the target polynucleotide in a sequence-specific manner via hybridization (i.e., base pairing). The nucleotide sequence of the spacer can vary depending on the sequence of the target nucleic acid.

[0110] In CRISPR-endonuclease systems, spacer sequences can be designed to hybridize with the target polynucleotide at the 5' end of the PAM of the endonuclease used in the system. The spacer can be a perfect match to the target sequence or may have a mismatch. Each endonuclease (e.g., Cas9 nuclease) has a specific PAM sequence that allows the endonuclease to recognize the target DNA. For example, Streptococcus pyogenes Cas9 recognizes a PAM containing the sequence 5'-NRG-3', where R contains either A or G, N is any nucleotide, and N is immediately adjacent to the 3' end of the target nucleic acid sequence targeted by the spacer sequence.

[0111] The target polynucleotide sequence can contain 20 nucleotides. The target polynucleotide can contain fewer than 20 nucleotides. The target polynucleotide can contain more than 20 nucleotides. The target polynucleotide can contain at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 or more nucleotides. The target polynucleotide sequence can contain up to 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 or more nucleotides. The target polynucleotide sequence can contain the 20 bases at the 5' end of the first nucleotide immediately adjacent to the PAM.

[0112] The spacer sequence for hybridization with 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 10 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 instances, the spacer sequence may contain 20 nucleotides. In some instances, the spacer may contain 19 nucleotides. In some instances, the spacer may contain 18 nucleotides. In some instances, the spacer may contain 22 nucleotides.

[0113] In some instances, the percentage 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 instances, the percentage complementarity between the spacer sequence and the target nucleic acid is at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 97%, at most about 98%, at most about 99%, or 100%. In some instances, the percentage complementarity between the spacer sequence and the target nucleic acid is 100% over six consecutive 5' nucleotides of the target sequence on the complementary strand of the target nucleic acid. The percentage complementarity between the spacer sequence and the target nucleic acid can be at least 60% over approximately 20 consecutive nucleotides. The lengths of the spacer sequence and the target nucleic acid can differ by 1 to 6 nucleotides, which can be considered as one or more protrusions.

[0114] The tracrRNA sequence may contain nucleotides that hybridize with the minimum CRISPR repeat sequence in the cell. The minimum tracrRNA sequence and the minimum CRISPR repeat sequence may form a double helix, i.e., a double-stranded structure with base pairing. The minimum tracrRNA sequence and the minimum CRISPR repeat sequence may together bind to an RNA-guided endonuclease. At least a portion of the minimum tracrRNA sequence may hybridize with the minimum CRISPR repeat sequence. The minimum tracrRNA sequence may have complementarity with the minimum CRISPR repeat sequence of 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%.

[0115] The minimum tracerRNA sequence can have a length ranging from about 7 nucleotides to about 100 nucleotides. For example, the length of the minimum tracerRNA 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. The minimum tracerRNA sequence can also be about 9 nucleotides long. The minimum tracerRNA sequence can also be about 12 nucleotides long. The smallest tracrRNA can consist of tracrRNA nt 23-48 as described by Jinek et al. (ibid.).

[0116] The minimal tracrRNA sequence may share at least about 60% identity with a reference minimal tracrRNA (e.g., wild-type tracrRNA from Streptococcus pyogenes) sequence over a segment of at least 6, 7, or 8 consecutive nucleotides. For example, the minimal tracrRNA sequence may share at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% identity with a reference minimal tracrRNA sequence over a segment of at least 6, 7, or 8 consecutive nucleotides.

[0117] The double helix between the smallest CRISPR RNA and the smallest tracrRNA contains a double helix. The double helix between the smallest CRISPR RNA and the smallest tracrRNA can contain at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. The double helix between the smallest CRISPR RNA and the smallest tracrRNA can contain at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.

[0118] A bichain may contain mismatches (i.e., the two chains of the bichain are not 100% complementary). A bichain may contain at least about 1, 2, 3, 4, or 5 OR mismatches. A bichain may contain at most about 1, 2, 3, 4, or 5 OR mismatches. A bichain may contain no more than 2 mismatches.

[0119] In some embodiments, the tracrRNA may be a 3' tracrRNA. In some embodiments, the 3' tracrRNA sequence may include a sequence having 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% sequence identity with a reference tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes).

[0120] In some embodiments, the gRNA may include a tracrRNA extension sequence. The tracrRNA extension sequence may have a length from about 1 nucleotide to about 400 nucleotides. The tracrRNA extension sequence may have a length greater than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 nucleotides. The tracrRNA extension sequence may have a length from about 20 to about 5000 or more nucleotides. The tracrRNA extension sequence may have a length less than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides. The length of the tracrRNA extension sequence may contain less than 10 nucleotides. The length of the tracrRNA extension sequence may be 10-30 nucleotides. The length of the tracrRNA extension sequence may be 30-70 nucleotides.

[0121] The tracrRNA extension sequence may include a functional portion (e.g., a stability control sequence, a ribozyme, or an endonuclease-binding sequence). The functional portion may include a transcription termination region (i.e., a transcription termination sequence). The functional portion may have a total length ranging from about 10 nucleotides (nt) to about 100 nucleotides, from about 10 nt to about 20 nt, from about 20 nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90 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.

[0122] In some embodiments, the sgRNA may comprise a linker sequence of length from about 3 nucleotides to about 100 nucleotides. In Jinek et al. (ibid.), for example, a simple 4-nucleotide "tetracyclic" (-GAAA-) was used (Jinek et al., Science, 2012, 337(6096):816-821). Illustrative linkers have lengths from about 3 nucleotides (nt) to about 90 nt, from about 3 nt to about 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60 nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, from about 3 nt to about 30 nt, from about 3 nt to about 20 nt, and from 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 for a single-molecule guiding nucleic acid can be between 4 and 40 nucleotides. The linker can be at least about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides. The linker can be at most about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.

[0123] The adapter can contain any of a variety of sequences, but in some instances, the adapter will not contain sequences of broad regions homologous to other parts of the guide RNA that could otherwise cause intramolecular binding to other functional regions of the guide. In Jinek et al. (ibid.), a simple 4-nucleotide sequence -GAAA- was used (Jinek et al., Science, 2012, 337(6096):816-821), but many other sequences, including longer ones, can also be used.

[0124] The adapter sequence may contain functional parts. For example, the adapter sequence may contain one or more features, including aptamers, ribozymes, protein-protein interaction hairpins, protein-binding sites, CRISPR arrays, introns, or exons. The adapter sequence may contain at least about one, two, three, four, or five or more functional parts. In some instances, the adapter sequence may contain at most about one, two, three, four, or five or more functional parts.

[0125] In some embodiments, the sgRNA does not contain uracil at the 3' end of the sgRNA sequence. In some embodiments, the sgRNA contains 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.

[0126] sgRNA can be chemically modified. In some embodiments, the chemically modified gRNA is a gRNA containing at least one nucleotide with a chemical modification (e.g., 2′-O-methyl sugar modification). In some embodiments, the chemically modified gRNA contains a modified nucleic acid backbone. In some embodiments, the chemically modified gRNA contains 2′-O-methyl-phosphate thioester residues. In some embodiments, chemical modifications enhance stability, reduce the likelihood or extent of innate immune responses, and / or enhance other properties as described in the art.

[0127] In some embodiments, the modified gRNA may contain a modified backbone, such as thiophosphate, phosphate triester, morpholino, methylphosphonate, short-chain alkyl or cycloalkyl interglucose bond, or short-chain heteroatom or heterocyclic interglucose bond.

[0128] Morpholin-based compounds are described in the following literature: Braasch and David Corey, Biochemistry, 2002, 41(14):4503-4510; Genesis, 2001, Vol. 30, No. 3; Heasman, Dev. Biol., 2002, 243:209-214; Nasevicius et al., Nat. Genet., 2000, 26:216-220; Lacera et al., Proc. Natl. Acad. Sci., 2000, 97:9591-9596; and U.S. Patent No. 5,034,506, issued on July 23, 1991.

[0129] Cyclohexenyl nucleic acid oligonucleotide mimics are described in Wang et al., J. Am. Chem. Soc. [Journal of the American Chemical Society], 2000, 122: 8595-8602.

[0130] In some embodiments, the modified gRNA may include one or more substituted sugar moieties at the 2' position, such as one of the following: OH, SH, SCH3, F, OCN, OCH3, OCH3 O(CH2)n CH3, O(CH2)n NH2, or O(CH2)n CH3, where n is from 1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkylaryl, or aryl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocyclic alkyl; heterocyclic alkylaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving group; reporter group; intercalating agent; 2'-O-(2-methoxyethyl); 2'-methoxy(2'-O-CH3); 2'-propoxy(2'-OCH2) CH2CH3); and 2'-fluorine (2'-F). Similar modifications can also be made at other positions on the gRNA, particularly at the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of the 5' terminal nucleotide. In some instances, both the sugar and the nucleotide internucleotide bond (i.e., the backbone) can be replaced by novel groups.

[0131] Guide RNA may also include additional or alternative nucleobase modifications or substitutions (often simply referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include those rarely or transiently found in native nucleic acids, such as hypoxanthine, 6-methyladenine, 5-methylpyrimidine, especially 5-methylcytosine (also known as 5-methyl-2'-deoxycytosine and commonly referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentiobiose HMC, as well as synthetic nucleobases, such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine or other heterosubstituted alkyladenine, 2-thiouracil, 2-thiothymidine, 5-bromouracil, 5-hydroxymethyluracil, 8-nitroguanine, 7-denitroguanine, 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. It may also include “universal” bases known in the art, such as inosine. 5-Me-C substitution has been shown to improve the stability of nucleic acid duplexes by 0.6 °C–1.2 °C. (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278) is an example of base substitution.

[0132] Modified nucleobases may 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, and cytosine. Pyrimidines and thymines, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogen, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-nitroguanine and 8-nitroadenine, 7-denitroguanine and 7-denitroadenine, and 3-denitroguanine and 3-denitroadenine.

[0133] A complex of nucleic acids and endonucleases targeting the genome

[0134] gRNA interacts with endonucleases (e.g., RNA-guided nucleases such as Cas9) to form a complex. The gRNA then guides the endonuclease to the target polynucleotide.

[0135] The endonuclease and gRNA can be administered separately to cells or a subject. In some embodiments, the endonuclease can be pre-complexed with one or more guide RNAs, or one or more crRNAs and tracrRNAs. The pre-complex can then be administered to cells or a subject. This pre-complex is called a ribonucleoprotein particle (RNP). The endonuclease in the RNP can be, for example, a Cas9 endonuclease or a Cpf1 endonuclease. The endonuclease can have one or more nuclear localization signals (NLS) attached to its N-terminus, C-terminus, or both. For example, a Cas9 endonuclease can have two NLSs attached, 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 the genomic target nucleic acid to the endonuclease in the RNP can range from about 1:1 to about 10:1. For example, the molar ratio of sgRNA to Cas9 endonuclease in the RNP can be 3:1.

[0136] Nucleic acids encoding system components

[0137] This disclosure provides nucleic acids comprising the nucleotide sequences of any nucleic acid or protein molecules necessary for various aspects of the methods described herein, including nucleic acids encoding the target genome of this disclosure, endonucleases of this disclosure, and / or methods of this disclosure. The encoding nucleic acid may be RNA, DNA, or a combination thereof.

[0138] Nucleic acids encoding the target genome disclosed herein, the endonucleases disclosed herein, and / or any nucleic acid or protein molecule necessary for various aspects of the methods disclosed herein may constitute a vector (e.g., a recombinant expression vector).

[0139] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid it is linked to. One type of vector is the "plasmid," which is a circular double-stranded DNA loop that can link another nucleic acid segment. Another type of vector is a viral vector, in which the additional nucleic acid segment can be linked to the viral genome. Some vectors are capable of autonomous replication in the host cells they are introduced into (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attachable mammalian vectors) are integrated into the host cell's genome after introduction and thus replicate along with the host genome.

[0140] In some instances, vectors can direct the expression of nucleic acids that are operatively linked to them. Such vectors are referred to herein as “recombinant expression vectors,” or more simply “expression vectors,” which have equivalent functions.

[0141] The term "operably linked" means that the target nucleotide sequence is linked to one or more regulatory sequences in a manner that allows the expression of that 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 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 nucleotide sequences in many types of host cells and those that direct nucleotide sequence expression only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will recognize that the design of expression vectors can depend on factors such as the selection of target cells and the desired expression level.

[0142] The expression vectors considered include, but are not limited to, viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviruses (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and other recombinant vectors. Other vectors considered for use in eukaryotic target cells include, but are not limited to, vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Other vectors may be used, provided they are compatible with the host cells.

[0143] In some instances, the vector may contain one or more transcriptional and / or translational control elements. Depending on the host / vector system used, any of a number of suitable transcriptional and translational control elements can be used in the expression vector, including constitutive and inducible promoters, transcriptional enhancer elements, transcription terminators, etc. The vector may be a self-inactivating vector that inactivates viral sequences or components or other elements of the CRISPR machine.

[0144] Non-restricted examples of suitable eukaryotic promoters (i.e., promoters that are 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-1α promoter (EF1α), chicken β-actin promoter (CBA), ubiquitin C promoter (UBC), hybrid constructs of CMV enhancers containing fusions with chicken β-actin promoter (CAG), promoters containing chicken β-actin gene (CAG or CAGGS), hybrid constructs of CMV enhancers containing fusions with first exon and first intron, mouse stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I promoter.

[0145] Promoters can be inducible promoters (e.g., heat shock promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, estrogen receptor-regulated promoters, etc.). Promoters can be constitutive promoters (e.g., CMV promoters, UBC promoters, CAG promoters). In some cases, promoters can be spatially restricted and / or temporally restricted promoters (e.g., tissue-specific promoters, cell type-specific promoters, etc.).

[0146] The introduction of the complexes, peptides, and nucleic acids disclosed herein into cells can occur through the following mechanisms: viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipid transfection, electroporation, nuclear transfection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-glucan-mediated transfection, liposome-mediated transfection, gene gun technology, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery.

[0147] III. Strategies for evading immune responses and increasing survival rates

[0148] This document describes strategies that enable genetically modified cells (i.e., universal donor cells) to increase their survival or viability and / or evade immune responses after implantation in a subject. In some embodiments, these strategies enable universal donor cells to survive and / or evade immune responses with a higher success rate compared to unmodified cells. In some embodiments, the genetically modified cells include the introduction of at least one genetic modification within or near at least one gene encoding a survival factor, wherein the genetic modification includes the insertion of a polynucleotide encoding a tolerogenic factor. These universal donor cells may further include 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 regulatory factors of the MHC-I or MHC-II complex, wherein the genetic modification includes the insertion of a polynucleotide encoding a second tolerogenic factor.

[0149] In some embodiments, the genetically modified cells include the introduction of at least one genetic modification that reduces the expression of one or more MHC-1 and MHC-2 human leukocyte antigens relative to unmodified cells within or near at least one gene; at least one genetic modification that increases the expression of at least one polynucleotide encoding a tolerance-inducing factor relative to unmodified cells; and at least one genetic modification that alters the expression of at least one gene encoding a survival factor relative to unmodified cells. In other embodiments, the genetically modified cells include at least one deletion or insertion-deletion mutation that alters the expression of one or more MHC-1 and MHC-2 human leukocyte antigens relative to unmodified cells within or near at least one gene; and at least one insertion of a polynucleotide encoding at least one tolerance-inducing factor at a site that partially overlaps, completely overlaps with, or is contained within a gene deletion site that alters the expression of one or more MHC-1 and MHC-2 HLA. In yet another embodiment, the genetically modified cells include at least one genetic modification that alters the expression of at least one gene encoding a survival factor relative to unmodified cells.

[0150] The genes encoding the major histocompatibility complex (MHC) are located on human chromosome 6p21. The proteins encoded by MHC genes are a series of surface proteins crucial for donor compatibility during cell transplantation. MHC genes are classified into MHC class I (MHC-I) and MHC class II (MHC-II). MHC-I genes (HLA-A, HLA-B, and HLA-C) are expressed in almost all tissue cell types, presenting peptides treated with “non-self” antigens to CD8+ T cells, thereby promoting their activation into cytolytic CD8+ T cells. Transplanted or implanted cells expressing “non-self” MHC-I molecules elicit a robust cellular immune response against these cells, ultimately leading to cell death through activated cytolytic CD8+ T cells. MHC-I proteins are closely associated with β-2-microglobulin (B2M) in the endoplasmic reticulum, which is essential for the formation of functional MHC-I molecules on the cell surface. In addition, there are three atypical MHC-Ib molecules (HLA-E, HLA-F, and HLA-G) with immunomodulatory functions. MHC-II biomolecules include HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR. Due to their major functions in the immune response, MHC-I and MHC-II biomolecules contribute to immune rejection following cell implantation into non-host cells (e.g., cell implantation for regenerative medicine purposes).

[0151] MHC-I cell surface molecules consist of the heavy chain (HLA-A, HLA-B, or HLA-C) encoded by MHC and the invariant subunit β-2-microglobulin (B2M). Therefore, reducing the intracellular concentration of B2M is an effective way to reduce the cell surface expression of MHC-I cell surface molecules.

[0152] In some embodiments, the cell contains genomic modifications of one or more MHC-I or MHC-II genes. In some embodiments, the cell contains genomic modifications of one or more polynucleotide sequences that regulate the expression of MHC-I and / or MHC-II. In some embodiments, the genetic modifications disclosed herein are performed using any gene editing method (including, but not limited to, those described herein).

[0153] In some embodiments, reducing the expression of one or more MHC-I and MHC-II human leukocyte antigens relative to unmodified cells is achieved by directly targeting, for example, a genetic deletion and / or insertion of at least one base pair in the MHC-I and / or MHC-II genes. In some embodiments, reducing the expression of one or more MHC-I and MHC-II human leukocyte antigens relative to unmodified cells is achieved by targeting the CIITA gene, for example, by a genetic 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 by targeting at least one transcriptional regulator of MHC-I or MHC-II, for example, by a genetic deletion. In some embodiments, the transcriptional regulator of MHC-I or MHC-II is the NLRC5 or CIITA gene. In some embodiments, the transcriptional regulator of MHC-I or MHC-II is the RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, IRF-1, and / or TAP1 genes.

[0154] In some embodiments, the cell's genome has been modified to lack all or part of the HLA-A, HLA-B, and / or HLA-C genes. In some embodiments, the cell's genome has been modified to lack all or part of the promoter regions of the HLA-A, HLA-B, and / or HLA-C genes. In some embodiments, the cell's genome has been modified to lack all or part of the genes encoding MHC-I or MHC-II transcriptional regulatory factors. In some embodiments, the cell's genome has been modified to lack all or part of the promoter regions of the genes encoding MHC-I or MHC-II transcriptional regulatory factors.

[0155] In some embodiments, the cell genome has been modified to reduce the expression of β-2-microglobulin (B2M). B2M is a non-polymorphic gene encoding a common protein subunit required for the surface expression of all polymorphic MHC class I heavy chains. HLA-I proteins are closely associated with B2M in the endoplasmic reticulum, which is essential for the formation of functionally expressed HLA-I molecules on the cell surface. In some embodiments, gRNA targets a site within the B2M gene containing the 5'-GCTACTCTCTCTTTCTGGCC-3' sequence (SEQ ID NO:1). In some embodiments, gRNA targets a site within the B2M gene containing the 5'-GGCCGAGATGTCTCGCTCCG-3' sequence (SEQ ID NO:2). In some embodiments, gRNA targets a site within the B2M gene containing the 5'-CGCGAGCACAGCTAAGGCCA-3' sequence (SEQ ID NO:3). In alternative embodiments, the gRNA targets a site within the B2M gene containing any of the following sequences: 5'-TATAAGTGGAGGCGTCGCGC-3' (SEQ ID NO:35), 5'-GAGTAGCGCGAGCACAGCTA-3' (SEQ ID NO:36), 5'-ACTGGACGCGTCGCGCTGGC-3' (SEQ ID NO:37), 5'-AAGTGGAGGCGTCGCGCTGG-3' (SEQ ID NO:38), 5-GGCCACGGAGCGAGACATCT-3' (SEQ ID NO:39), 5'-GCCCGAATGCTGTCAGCTTC-3' (SEQ ID NO:40), 5'-CTCGCGCTACTCTCTCTTTC-3' (SEQ ID NO:41), 5'-TCCTGAAGCTGACAGCATTC-3' (SEQ ID NO:42), 5'-TTCCTGAAGCTGACAGCATT-3' (SEQ ID NO:35). SEQ ID NO:43) or 5'-ACTCTCTCTTTCTGGCCTGG-3' (SEQ ID NO:44). In some embodiments, the gRNA comprises a polynucleotide sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44.The gRNA / CRISPR nuclease complex targets and cleaves target sites in the B2M locus. Repairing double-strand breaks via NHEJ can result in the deletion of at least one nucleotide and / or the insertion of at least one nucleotide, thereby disrupting or eliminating B2M expression. Alternatively, the B2M locus can be targeted by at least two CRISPR systems, each containing a different gRNA, such that cleavage at two sites within the B2M locus results in the deletion of the sequence between the two cleavage sites, thereby eliminating B2M expression.

[0156] In some embodiments, the cell genome has been modified to reduce the expression of thioredoxin-interacting protein (TXNIP). In some embodiments, gRNA targets a site within the TXNIP gene containing the 5'-GAAGCGTGTCTTCATAGCGC-3' sequence (SEQ ID NO: 15). In some embodiments, gRNA targets a site within the TXNIP gene containing the 5'-TTACTCGTGTCAAAGCCGTT-3' sequence (SEQ ID NO: 16). In some embodiments, gRNA targets a site within the TXNIP gene containing the 5'-TGTCAAAGCCGTTAGGATCC-3' sequence (SEQ ID NO: 17). In some embodiments, gRNA targets a site within the TXNIP gene containing the 5'-GCCGTTAGGATCCTGGCTTG-3' sequence (SEQ ID NO: 18). In some embodiments, gRNA targets a site within the TXNIP gene containing the 5'-GCGGAGTGGCTAAAGTGCTT-3' sequence (SEQ ID NO: 19). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-TCCGCAAGCCAGGATCCTAA-3' sequence (SEQ ID NO: 20). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-GTTCGGCTTTGAGCTTCCTC-3' sequence (SEQ ID NO: 21). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-GAGATGGTGATCATGAGACC-3' sequence (SEQ ID NO: 22). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-TTGTACTCATATTTGTTTCC-3' sequence (SEQ ID NO: 23). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-AACAAATATGAGTACAAGTT-3' sequence (SEQ ID NO: 24). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-GAAGCGTGTCTTCATAGCGCAGG-3' sequence (SEQ ID NO: 45). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-TTACTCGTGTCAAAGCCGTTAGG-3' sequence (SEQ ID NO:46). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-TGTCAAAGCCGTTAGGATCCTGG-3' sequence (SEQ ID NO:47).In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-GCCGTTAGGATCCTGGCTTGCGG-3' sequence (SEQ ID NO:48). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-GCGGAGTGGCTAAAGTGCTTTGG-3' sequence (SEQ ID NO:49). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-TCCGCAAGCCAGGATCCTAACGG-3' sequence (SEQ ID NO:50). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-GTTCGGCTTTGAGCTTCCTCAGG-3' sequence (SEQ ID NO:51). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-GAGATGGTGATCATGAGACCTGG-3' sequence (SEQ ID NO:52). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-TTGTACTCATATTTGTTTCCAGG-3' sequence (SEQ ID NO: 53). In some embodiments, the gRNA targets a site within the TXNIP gene containing the 5'-AACAAATATGAGTACAAGTTCGG-3' sequence (SEQ ID NO: 54). In some embodiments, the gRNA targets a target site within the TXNIP gene containing any one of SEQ ID NO: 15-24 or 45-54. In some embodiments, the gRNA targets a polynucleotide sequence containing any one of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. The gRNA / CRISPR nuclease complex targets and cleaves the target site in the TXNIP gene locus. Repairing double-strand breaks via NHEJ may result in the deletion of at least one nucleotide and / or the insertion of at least one nucleotide, thereby disrupting or eliminating TXNIP expression. Alternatively, inserting a polynucleotide encoding a foreign gene into the TXNIP locus can disrupt or eliminate TXNIP expression.

[0157] In some embodiments, the cell genome has been modified to reduce the expression of class II transactivator (CIITA). CIITA is a member of the LR or nucleotide-binding domain (NBD) leucine-rich repeat (LRR) protein family and regulates MHC-II transcription by associating with MHC enhancers. CIITA expression is induced in B cells and dendritic cells according to developmental stage and can be induced by IFN-γ in most cell types.

[0158] In some embodiments, the cell genome has been modified to reduce the expression of the NLR family CARD domain 5 (NLRC5). NLRC5 is a key regulator of MHC-I-mediated immune responses, and similar to CIITA, NLRC5 is highly inducible by IFN-γ and can translocate into the nucleus. NLRC5 activates the promoters of MHC-I genes and induces transcription of MHC-I and related genes involved in MHC-I antigen presentation.

[0159] In some embodiments, tolerogenic factors may be inserted into or re-inserted into genetically modified cells to generate immune-exempt universal donor cells. In some embodiments, the universal donor cells disclosed herein have been further modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, but are not limited to, one or more of HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI inhibitors, and IL-35. In some embodiments, genetic modification (e.g., insertion) of at least one polynucleotide encoding at least one tolerogenic factor enables the universal donor cells to suppress or evade immune rejection at a rate at least 1.05-fold, at least 1.1-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold higher than unmodified cells after implantation. In some embodiments, the insertion of polynucleotides encoding HLA-E, HLA-G, CTLA-4, CD47, and / or PD-L1 enables universal donor cells to suppress or evade immune rejection after transplantation or engraftment into a host subject.

[0160] Polynucleotides encoding tolerance factors typically contain left and right homologous arms flanking the sequence encoding the tolerance factor. These homologous arms exhibit significant sequence homology with the genomic DNA at or near the target insertion site. For example, the left homologous arm may be a nucleotide sequence homologous to a region to the left or upstream of the target or cleavage site, and the right homologous arm may be a nucleotide sequence homologous to a region to the right or downstream of the target or cleavage site. The proximal end of each homologous arm may be homologous to the genomic DNA sequence adjacent to the cleavage site. Alternatively, the proximal end of each homologous arm may be homologous to a genomic DNA sequence located up to about 10, 20, 30, 40, 50, 60, or 70 nucleotides away from the cleavage site. This allows the polynucleotide encoding the tolerance factor to be inserted into the target locus within about 10, 20, 30, 40, 50, 60, or 70 base pairs of the cleavage site, and may omit additional genomic DNA adjacent to the cleavage site (and not homologous to the homologous arms). The length of a homologous arm can range from about 50 nucleotides to several thousand nucleotides. In some embodiments, the length of a homologous arm can range from about 500 nucleotides to about 1000 nucleotides. The significant 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%.

[0161] In some embodiments, the homologous arm is used in conjunction with a B2M guide (e.g., a gRNA comprising the nucleotide sequences of SEQ ID NO: 1-3, 35-44). In some embodiments, the homologous arm is designed to be used with any B2M guide that will eliminate the start site of the B2M gene. In some embodiments, the B2M homologous arm may comprise, or consist of, a polynucleotide sequence of SEQ ID NO: 7 or 13 or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity with the polynucleotide sequence of SEQ ID NO: 7 or 13. In some embodiments, the left B2M homologous arm may comprise, or consist of, a polynucleotide sequence of SEQ ID NO: 7 or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity with the polynucleotide sequence of SEQ ID NO: 7. In some embodiments, the right B2M homologous arm may comprise, or consist of, a polynucleotide sequence of SEQ ID NO: 13 or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity with the polynucleotide sequence of SEQ ID NO: 13.

[0162] In some embodiments, the homology arm is used in conjunction with a TXNIP guide (e.g., a gRNA comprising the nucleotide sequences of SEQ ID NO: 15-24). In some embodiments, the homology arm is designed for use with any TXNIP guide (e.g., a gRNA comprising the nucleotide sequences of SEQ ID NO: 15-20) targeting exon 1 of TXNIP. In some embodiments, the TXNIP homology arm may comprise, or consist of, a polynucleotide sequence of SEQ ID NO: 25 or 32, or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity with the polynucleotide sequence of SEQ ID NO: 25 or 32, or substantially thereof. In some embodiments, the left TXNIP homology arm may comprise, or consist of, a polynucleotide sequence of SEQ ID NO: 25, or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity with the polynucleotide sequence of SEQ ID NO: 25, or substantially thereof. In some embodiments, the right TXNIP homologous arm may comprise, or consist substantially of, a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity with the polynucleotide sequence of SEQ ID NO:32 or the polynucleotide sequence of SEQ ID NO:32.

[0163] The at least one polynucleotide encoding at least one tolerogenic factor can be operatively 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, EFi, PGK, CAG, or UBC promoter.

[0164] In some embodiments, the at least one polynucleotide encoding at least one tolerogenic factor is inserted into a safe harbor locus (e.g., the AAVS 1 locus). In some embodiments, the at least one polynucleotide encoding at least one tolerogenic factor is inserted into a site or region of genomic DNA that partially overlaps, completely overlaps with, or is contained within (i.e., within or near) the MHC-I gene, MHC-II gene, or a transcriptional regulator of MHC-I or MHC-II.

[0165] In some embodiments, a polynucleotide encoding PD-L1 is inserted at a site within or near the B2M gene locus. In some embodiments, the polynucleotide encoding PD-L1 is inserted at a site within or near the B2M gene locus simultaneously with or after the deletion of all or part of the B2M gene or promoter. The polynucleotide encoding PD-L1 is operatively linked to a foreign promoter. The foreign promoter may be a CMV promoter. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:11.

[0166] In some embodiments, a polynucleotide encoding HLA-E is inserted at a site within or near the B2M gene locus. In some embodiments, the polynucleotide encoding HLA-E is inserted at a site within or near the B2M gene locus simultaneously with or after the deletion of all or part of the B2M gene or promoter. The polynucleotide encoding HLA-E is operatively linked to a foreign promoter. The foreign promoter may be a CMV promoter. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 26, 27, 28, 29, 30 and / or 30. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 55.

[0167] In some embodiments, a polynucleotide encoding HLA-G is inserted at or near the HLA-A, HLA-B, or HLA-C locus. In some embodiments, the polynucleotide encoding HLA-G is inserted at or near the HLA-A, HLA-B, or HLA-C locus simultaneously with or after the deletion of the HLA-A, HLA-B, or HLA-C gene or promoter.

[0168] In some embodiments, a polynucleotide encoding CD47 is inserted at or near the CIITA locus. In some embodiments, the polynucleotide encoding CD47 is inserted at or near the CIITA locus simultaneously with or after the deletion of the CIITA gene or promoter.

[0169] In some embodiments, while inserting a polynucleotide encoding CD47 at or near the CIITA locus, a polynucleotide encoding HLA-G is inserted at or near the HLA-A, HLA-B, or HLA-C locus.

[0170] In some embodiments, at least one polynucleotide encoding at least one tolerogenic factor may be delivered to cells as part of a vector. For example, the vector may be a plasmid vector. In various embodiments, the amount of plasmid vector delivered to cells may be from about 0.5 μg to about 10 μg (per about 10 μg of the target cell). 6The amount of plasmid may be in the range of approximately 1 μg to approximately 8 μg, approximately 2 μg to approximately 6 μg, or approximately 3 μg to approximately 5 μg, depending on the specific embodiment. In a particular embodiment, the amount of plasmid delivered to the cells may be approximately 4 μg.

[0171] In some embodiments, the cells contain increased or decreased expression of one or more survival factors. In some embodiments, the cells contain the insertion of one or more polynucleotide sequences encoding a survival factor. In some embodiments, the cells contain the deletion of one or more survival factors. In some embodiments, the genetic modifications disclosed herein are performed using any gene editing method (including, but not limited to, those described herein). In some embodiments, the cells contain increased or decreased expression of at least one survival factor relative to unmodified cells. In some embodiments, the survival factor is a member or key pathway involved in cell survival, such as hypoxia, reactive oxygen species, nutrient deprivation, and / or oxidative stress. In some embodiments, the genetic modification of at least one survival factor enables universal donor cells to survive for a longer period of time after implantation than unmodified cells, for example, at least 1.05 times, at least 1.1 times, at least 1.25 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, or at least 50 times longer. In some embodiments, the survival factor is ZNF143, TXNIP, FOXO1, JNK, or MANF.

[0172] In some embodiments, the cells contain an insertion of a polynucleotide encoding MANF, which enables universal donor cells to survive with a higher survival rate than unmodified cells after transplantation or engraftment into a host subject. In some embodiments, the polynucleotide encoding MANF is inserted into a safe harbor locus. In some embodiments, the polynucleotide encoding MANF is inserted into a gene belonging to MHC-I, MHC-II, or a transcriptional regulatory factor of MHC-I or MHC-II.

[0173] In some embodiments, the cell genome has been modified to delete all or part of the ZNF143, TXNIP, FOXO1, and / or JNK genes. In some embodiments, the cell genome has been modified to delete all or part of the promoter regions of the ZNF143, TXNIP, FOXO1, and / or JNK genes.

[0174] In some embodiments, more than one survival factor is genetically modified within the cell.

[0175] In some embodiments, cells lacking MHC-II expression and having moderate MHC-I expression are genetically modified to lack surface expression of either MHC-I or MHC-II. In another embodiment, cells lacking surface expression of MHC-I / II are further edited to express PD-L1, for example by inserting a polynucleotide encoding PD-L1. In yet another embodiment, cells lacking surface expression of MHC-I / II are further edited to express PD-L1, for example by inserting a polynucleotide encoding PD-L1, and are also genetically modified to increase or decrease the expression of at least one gene encoding a survival factor relative to unmodified cells.

[0176] In some embodiments, these cells further comprise increased or decreased expression (e.g., through genetic modification) of one or more additional genes that are not necessarily involved in post-implantation immune evasion or cell survival. In some embodiments, these cells further comprise increased expression of one or more safety switch proteins relative to unmodified cells. In some embodiments, these 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, for example, into a safe harbor locus. In some other embodiments, the genetically modified one or more additional genes encode one or more of the following: safety switch proteins; targeting patterns; receptors; signal transduction molecules; transcription factors; pharmacologically active proteins or peptides; drug target candidates; and proteins integrated with the construct that promote its implantation, transport, homing, viability, self-renewal, persistence, and / or survival.

[0177] One aspect of the invention provides a method for generating genome-engineered universal donor cells, wherein the universal donor cells contain at least one targeted genome modification at one or more selected sites in the genome, the method comprising genetically engineering a cell type as described herein by: introducing one or more constructs into the cells to achieve targeted modification at the selected sites; introducing one or more double-strand breaks at these selected sites into the cells using one or more endonucleases capable of recognizing the selected sites; and culturing these edited cells to allow endogenous DNA repair to produce targeted insertions or deletions at these selected sites; thereby obtaining genome-modified universal donor cells. Genome-modified universal donor cells may undergo successive rounds of genome modification, such that multiple sites are targeted and modified. Genome-modified cells are cultured, characterized, selected, and amplified using techniques well known in the art. Universal donor cells generated by this method will contain at least one functional targeted genome modification, and wherein the genome-modified cells (if they are stem cells) are then capable of differentiating into progenitor cells or fully differentiated cells.

[0178] In some other embodiments, the genome-engineered universal donor cell contains the introduction or increased expression of at least one of HLA-E, HLA-G, CD47, or PD-L1. In some embodiments, the genome-engineered universal donor cell is HLA class I and / or class II deficient. In some embodiments, the genome-engineered universal donor cell contains null or low B2M. In some embodiments, the genome-engineered universal donor cell contains an integrated or non-integrated exogenous polynucleotide encoding one or more of HLA-E, HLA-G, and PD-L1 proteins. In some embodiments, the introduced expression is an increased expression of a non-expressed or low-expressed gene contained in the cell. In some embodiments, the non-integrated exogenous polynucleotide is introduced using Sendai virus, AAV, episomes, or plasmids. In some embodiments, the universal donor cell is B2M null and has the introduced expression of one or more of HLA-E, HLA-G, and PD-L1, and increased or decreased expression of at least one safety switch protein. In another embodiment, the universal donor cell is HLA-A, HLA-B, and HLA-C nullified and has introduced expression of one or more of HLA-E, HLA-G, PD-L1, and at least one safety switch protein. In some embodiments, the universal donor cell is B2M nullified and has introduced expression of one or more of HLA-E, HLA-G, PD-L1, and increased or decreased expression of at least one survival factor (e.g., MANF). Methods for producing any of the genetically modified cells described herein are contemplated using at least any of the gene-editing methods described herein.

[0179] IV. Cell Type

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

[0181] Cells described herein (e.g., universal donor stem cells) can be differentiated into relevant cell types to assess HLA expression and evaluate the immunogenicity of universal stem cell lines. Typically, differentiation involves maintaining the target cells under conditions and for a sufficient period to differentiate them 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, β cells (e.g., pancreatic β cells), pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, hematopoietic progenitor cells, or neural progenitor cells (NPCs). In some embodiments, universal donor cells can differentiate into well-defined endoderm cells, gastrulation cells, hindbrain cells, pancreatic endoderm cells (PECs), pancreatic endocrine cells, immature β cells, or mature β cells.

[0182] Stem cells can both proliferate and produce more progenitor cells, which in turn have the ability to produce a large number of mother cells, which in turn can produce differentiated or potentially differentiated daughter cells. The daughter cells themselves can be induced to proliferate and produce offspring, which subsequently differentiate into one or more mature cell types while retaining one or more cells with the developmental potential of their parent. Therefore, the term "stem cell" refers to a cell that, under certain conditions, has the ability or potential to differentiate into a more specialized or differentiated phenotype, and in some cases, retains the ability to proliferate even without substantial differentiation. In one aspect, the term progenitor cell or stem cell refers to the mother cell in a broad sense, whose offspring typically specialize in different directions through differentiation that occurs in the gradual diversification of embryonic cells and tissues (e.g., by acquiring completely independent characteristics). Cell differentiation is a complex process that typically occurs through the division of many cells. Differentiated cells can originate from pluripotent cells, which themselves are also derived from pluripotent cells, etc. While each of these pluripotent cells can be considered a stem cell, the range of cell types that each pluripotent cell can produce can vary considerably. Some differentiated cells also have the ability to produce cells with greater developmental potential. This ability can be natural or artificially induced after treatment with various factors. In many biological instances, stem cells can also be "pluripotent" because they can produce offspring of more than one different cell type, but this is not necessary for "steminess".

[0183] "Differentiated cells" are cells that have progressed further in the developmental pathway than the cells in comparison. Thus, stem cells can differentiate into lineage-restricted precursor cells (e.g., myocyte progenitor cells), which in turn can differentiate into other types of precursor cells that further differentiate along the pathway (e.g., myocyte precursors), and then into terminally differentiated cells (e.g., myocytes) that play a specific role in certain tissue types and may or may not retain the ability to proliferate further. In some embodiments, the differentiated cells may be pancreatic β cells.

[0184] Embryonic stem cells

[0185] The cells described in this article may be embryonic stem cells (ESCs). ESCs are derived from embryonic cells of mammalian embryos and are capable of differentiating into any cell type and proliferating rapidly. ESCs are believed to also have a normal karyotype, maintain high-end granzyme activity, and exhibit significant long-term proliferative potential, making them excellent candidates for use as universal donor cells.

[0186] Adult stem cells

[0187] The cells described herein may refer to adult stem cells (ASCs). ASCs are undifferentiated cells that can be found in mammals (e.g., humans). ASCs are defined by their ability to self-renew (e.g., to maintain their undifferentiated state through several rounds of cell replication and passage) and their ability to differentiate into several different cell types (e.g., glial cells). Adult stem cells are a broad class of stem cells that may 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.

[0188] Induced pluripotent stem cells

[0189] The cells described in this article can be induced pluripotent stem cells (iPSCs). iPSCs can be generated directly from adult human cells by introducing genes encoding key transcription factors involved in pluripotency (e.g., OCT4, SOX2, cMYC, and KLF4). iPSCs can be derived from the same subject to be administered subsequent progenitor cells. That is, somatic cells can be obtained from a subject, reprogrammed into induced pluripotent stem cells, and then differentiated into progenitor cells (e.g., autologous cells) to be administered to the subject. However, in the case of autologous cells, there remains a risk of poor post-implantation immune response and viability.

[0190] Artificial hematopoietic stem cells and progenitor cells

[0191] The cells described in this article can be human hematopoietic stem cells and progenitor cells (hHSPCs). This stem cell lineage produces all types of blood cells, including erythroid cells (erythrocytes or red blood cells (RBCs)), bone marrow cells (monocytes and macrophages, neutrophils, basophils, eosinophils, megakaryocytes / platelets, and dendritic cells), and lymphoid cells (T cells, B cells, NK cells). Blood cells are produced by the proliferation and differentiation of a very small population of pluripotent hematopoietic stem cells (HSCs), which also have the ability to replenish themselves through self-renewal. During differentiation, the progeny of HSCs undergo various intermediate maturation stages before reaching full maturity, producing pluripotent progenitor cells and lineage-directed progenitor cells. Bone marrow (BM) is the primary site of blood cell production in the human body, and under normal conditions, only a small number of hematopoietic stem cells and progenitor cells (HSPCs) are 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 cells and progenitor cells into circulation.

[0192] Cell differentiation into other cell types

[0193] Another step in the method disclosed herein may include differentiating cells into fractionated cells. The differentiation step may be performed according to any method known in the art. For example, human iPSCs may be differentiated into fixed endoderm using various treatments including activin and B27 supplementation (Life Technologies). The fixed endoderm may be further differentiated into hepatocytes using treatments including: FGF4, HGF, BMP2, BMP4, tumor suppressor M, dexamethasone, etc. (Duan et al., Stem Cells, 2010; 28:674-686; Ma et al., Stem Cells Translational Medicine, 2013; 2:409-419). In another embodiment, the differentiation step may be performed according to Sawitza et al., Sci Rep. 2015; 5:13320. The differentiated cells may be any somatic cells of mammals (e.g., humans). In some embodiments, somatic cells may be exocrine epithelial cells (e.g., salivary gland mucus cells, prostate cells), hormone-secreting cells (e.g., anterior pituitary cells, intestinal cells, pancreatic islet cells), keratinizing epithelial cells (e.g., epidermal keratinocytes), moist stratification barrier epithelial cells, sensory transduction cells (e.g., photoreceptors), autonomic neurons, sensory organ and peripheral neuronal support cells (e.g., Schwann cells), central nervous system neurons, glial cells (e.g., astrocytes, oligodendrocytes), lens cells, adipocytes, kidney cells, barrier function cells (e.g., ductal cells), extracellular matrix cells, contractile cells (e.g., skeletal muscle cells, cardiomyocytes, smooth muscle cells), blood cells (e.g., erythrocytes), immune system cells (e.g., megakaryocytes, microglia, neutrophils, mast cells, T cells, B cells, natural killer cells), germ cells (e.g., sperm cells), trophic cells, or interstitial cells.

[0194] Typically, the universal donor cell population disclosed herein maintains expression of one or more inserted nucleotide sequences over time. For example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the universal donor cells express one or more tolerogenic factors. Furthermore, lineage-restricted or fully differentiated cell populations derived from the universal donor cells disclosed herein maintain expression of one or more inserted nucleotide sequences over time. For example, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the lineage-restricted or fully differentiated cells express one or more tolerogenic factors.

[0195] V. Preparation and Application

[0196] Formulation and delivery of gene editing

[0197] Guide RNA, polynucleotides (e.g., polynucleotides encoding tolerance factors or polynucleotides encoding endonucleases), and endonucleases as described herein can be formulated and delivered to cells in any manner known in the art.

[0198] Guide RNA and / or polynucleotides are formulated with pharmaceutically acceptable excipients (such as carriers, solvents, stabilizers, adjuvants, diluents, etc.) depending on the specific route of administration and dosage form. Guide RNA and / or polynucleotide compositions can be formulated to achieve physiologically compatible pH, and the range is from about 3 to about 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 may contain a therapeutically effective amount of at least one compound as described herein, and one or more pharmaceutically acceptable excipients. Optionally, the composition may contain a combination of compounds described herein, or may include a second active ingredient (e.g., but not limited to, an antimicrobial agent or antimicrobial agent) that can be used to treat or prevent bacterial growth, or may include a combination of reagents disclosed herein.

[0199] Suitable excipients include, for example, carrier molecules, which include large, slowly metabolized macromolecules (such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, and inactivated viral particles). Other exemplary excipients may include antioxidants (e.g., but not limited to ascorbic acid), chelating agents (e.g., but not limited to EDTA), carbohydrates (e.g., but not limited to dextrin, hydroxyalkyl cellulose, and hydroxyalkyl methyl cellulose), stearic acid, liquids (e.g., but not limited to oils, water, saline, glycerol, and ethanol), wetting agents or emulsifiers, pH buffers, etc.

[0200] Guide RNA polynucleotides (RNA or DNA) and / or one or more endonuclease polynucleotides (RNA or DNA) can be delivered using viral or non-viral delivery media known in the art. Alternatively, one or more endonuclease peptides can be delivered using viral or non-viral delivery media known in the art, such as electroporation or lipid nanoparticles. In another alternative aspect, DNA endonucleases can be delivered as one or more peptides alone or pre-complexed with one or more guide RNAs, or one or more crRNAs with tracrRNA.

[0201] Polynucleotides can be delivered via non-viral delivery media, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small RNA conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes. Some exemplary non-viral delivery media are described in Peer and Lieberman, Gene Therapy, 2011, 18:1127-1133 (which emphasizes that non-viral delivery media used for siRNA can also be used to deliver other polynucleotides).

[0202] For the polynucleotides disclosed herein, the formulation may be selected from any of those taught, for example, in international application PCT / US 2012 / 069610.

[0203] Lipid nanoparticles (LNPs) can deliver polynucleotides, such as guide RNA, sgRNA, and mRNA encoding endonucleases, to cells or subjects.

[0204] LNP refers to any particle with a diameter less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, the size range of nanoparticles can be from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

[0205] LNPs can be made from cationic, anionic, or neutral lipids. Neutral lipids (such as fused phospholipids DOPE or membrane component cholesterol) can be included in LNPs as "helper lipids" to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include reduced efficacy due to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses.

[0206] LNPs can also be composed of hydrophobic lipids, hydrophilic lipids, or both.

[0207] Any lipid or combination of lipids known in the art can be used to generate LNPs. Examples of lipids used to generate 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.

[0208] Lipids can be combined in any molar ratio to produce LNPs. Additionally, one or more polynucleotides can be combined with one or more lipids in a wide range of molar ratios to produce LNPs.

[0209] Recombinant adeno-associated virus (AAV) vectors can be used for delivery. Techniques for generating rAAV particles (whereby the AAV genome to be packaged (which includes the polynucleotides to be delivered, rep and cap genes, and helper viral functions)) are standard in the art. The generation of rAAV typically requires the presence of the following components within a single cell (referred to herein as the packaging cell): the rAAV genome, the AAV rep and cap genes separate from (i.e. not present in) the rAAV genome, and the helper viral functions. The AAV rep and cap genes can be derived from any AAV serotype (from which recombinant viruses can be derived), and can be derived from different AAV serotypes than the rAAV genomic ITR, including but not limited to the AAV serotypes described herein. The generation of pseudotyped rAAV is disclosed, for example, in International Patent Application Publication No. WO 01 / 83692.

[0210] Preparation and application of cells (e.g., universal donor cells)

[0211] Genetically modified cells (e.g., universal donor cells) as described herein can be formulated and administered to subjects in any manner known in the art.

[0212] The terms “application,” “introduction,” “implanting,” “engrafting,” and “transplantation” are used interchangeably in the context of placing cells (e.g., progenitor cells) into a subject (by a method or approach that results in the introduced cells being at least partially localized at a desired site). Cells (e.g., progenitor cells) or their differentiated progeny can be applied by any suitable approach that results in delivery to a desired site in the subject, where at least a portion of the implanted cells or cell components remain viable. Following application to the subject, the viability of the cells can range from short periods of time (e.g., twenty-four hours), days, to long periods of time, or even the lifespan of the subject (i.e., long-term implantation).

[0213] Genetically modified cells (e.g., universal donor cells) as described herein can remain viable after being administered to a subject for a longer period of time than unmodified cells.

[0214] In some embodiments, the composition comprising cells as described herein may be administered via a suitable route, which may include intravenous administration, such as as a bolus or by continuous infusion over a period of time. In some embodiments, intravenous administration may be performed via intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, or intrathecal routes. In some embodiments, the composition may be in solid, aqueous, or liquid form. In some embodiments, the aqueous or liquid form may be atomized or lyophilized. In some embodiments, the atomized or lyophilized form may be reconstituted with an aqueous or liquid solution.

[0215] Cellular compositions may also be emulsified or presented as liposome compositions, provided that the emulsification process does not adversely affect cell viability. Cells and any other active ingredients may be mixed with pharmaceutically acceptable and compatible excipients in amounts suitable for the therapeutic methods described herein.

[0216] Additional reagents contained in the cell composition may include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino group of a polypeptide) that are formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups may also be derived from inorganic bases (such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide) and organic bases (such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.).

[0217] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials other than the active ingredient and water, or contain buffers (such as sodium phosphate at physiological pH, physiological saline, or both, such as phosphate-buffered saline). Furthermore, aqueous carriers may contain more than one buffer salt, as well as salts (such as sodium chloride and potassium chloride), dextran, polyethylene glycol, and other solutes. In addition to and excluding water, liquid compositions may also contain a liquid phase. Examples of such additional liquid phases are glycerol, vegetable oils (such as cottonseed oil), and water-oil emulsions. The amount of an active compound used in a cellular composition to effectively treat a specific disorder or condition can depend on the nature of the disorder or condition and can be determined using standard clinical techniques.

[0218] In some embodiments, the cell-containing composition may be administered to a subject (e.g., a human subject) who has a disease, is suspected of having a disease, or is at risk of having a disease. In some embodiments, the composition may be administered to a subject who does not have a disease, is not suspected of having a disease, or is not at risk of having a disease. In some embodiments, the subject is a healthy person. In some embodiments, the subject (e.g., a human subject) has a genetically heritable disease, is suspected of having a genetically heritable disease, or is at risk of having a genetically heritable disease. In some embodiments, the subject is experiencing or is at risk of developing symptoms indicative of a disease. In some embodiments, the disease is diabetes, such as type 1 or type 2 diabetes.

[0219] VI. Specific compositions and methods disclosed herein

[0220] Therefore, this disclosure specifically relates to the following non-limiting compositions and methods.

[0221] In the first composition, namely composition 1, this disclosure provides a composition comprising a universal donor cell containing a nucleotide sequence encoding a first tolerogenic factor inserted within or near a gene encoding a survival factor, wherein the universal donor cell expresses the tolerogenic factor and has disrupted expression of the survival factor, and the universal donor cell has increased immune evasion and / or cell survival compared to control cells.

[0222] In another composition, namely composition 2, this disclosure provides a composition as provided in composition 1, wherein the control cell is a wild-type cell or a cell that does not contain the inserted nucleotide sequence.

[0223] In another composition, namely composition 3, this disclosure provides a composition as provided in composition 1 or 2, wherein the expression of the disrupted survival factor includes reduced or eliminated expression.

[0224] In another composition, namely composition 4, this disclosure provides a composition as provided in any one of compositions 1 to 3, wherein the first tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

[0225] In another composition, namely composition 5, this disclosure provides a composition as provided in any one of compositions 1 to 4, wherein the survival factor is TXNIP, ZNF143, FOXO1, JNK or MANF.

[0226] In another composition, namely composition 6, this disclosure provides a composition as provided in any one of claims 1 to 5, wherein the first tolerability-inducing factor is HLA-E and the survival factor is TXNIP.

[0227] In another composition, namely composition 7, this disclosure provides a composition as provided in composition 5 or 6, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide, the HLA-G presenting peptide being fused to a B2M membrane protein, the B2M membrane protein being fused to HLA-E without its signal peptide.

[0228] In another composition, namely composition 8, this disclosure provides a composition as provided in composition 7, wherein the sequence encoding the HLA-E trimer is substantially composed of SEQ ID NO:55.

[0229] In another composition, namely composition 9, this disclosure provides a composition as provided in any one of compositions 1 to 8, wherein the nucleotide sequence encoding the first tolerogenic factor is operatively linked to an exogenous promoter.

[0230] In another composition, namely composition 10, this disclosure provides a composition as provided in composition 9, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0231] In another composition, namely composition 11, this disclosure provides a composition as provided in any one of claims 1 to 10, the composition further comprising an inserted nucleotide sequence encoding a second tolerogenic factor within or near a gene encoding a component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or the MHC-I or MHC-II complex, wherein the universal donor cell expresses the tolerogenic factor and has disrupted expression of the MHC-I or MHC-II human leukocyte antigen or the component or transcriptional regulator of the MHC-I or MHC-II complex.

[0232] In another composition, namely composition 12, this disclosure provides a composition as provided in composition 11, wherein the expression of the disrupted MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of the MHC-I or MHC-II complex includes reduced or eliminated expression.

[0233] In another composition, namely composition 13, this disclosure provides a composition as provided in composition 11 or 12, wherein the second tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

[0234] In another composition, namely composition 14, this disclosure provides a composition as provided in any one of compositions 11 to 13, wherein the MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of the MHC-I or MHC-II complex is HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, B2M, NLRC5, CIITA, RFX5, RFXAP, or RFXANK.

[0235] In another composition, namely composition 15, this disclosure provides a composition as provided in any one of compositions 11 to 14, wherein the second tolerogenic factor is PD-L1 and the MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of the MHC-I or MHC-II complex is B2M.

[0236] In another composition, namely composition 16, this disclosure provides a composition as provided in composition 15, wherein the nucleotide sequence encoding PD-L1 consists substantially of SEQ ID NO:11.

[0237] In another composition, namely composition 17, this disclosure provides a composition as provided in any one of compositions 11 to 16, wherein the nucleotide sequence encoding the second tolerogenic factor is operatively linked to an exogenous promoter.

[0238] In another composition, namely composition 18, this disclosure provides a composition as provided in composition 17, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0239] In another composition, namely composition 19, this disclosure provides a composition as provided in any one of compositions 11 to 18, wherein the first tolerogenic factor is HLA-E, the survival factor is TXNIP, the second tolerogenic factor is PD-L1, and the MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of the MHC-I or MHC-II complex is B2M.

[0240] In another composition, namely composition 20, this disclosure provides a composition as provided in any one of compositions 1 to 19, wherein the cell is a stem cell.

[0241] In another composition, namely composition 21, this disclosure provides a composition as provided in composition 20, wherein the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.

[0242] In another composition, namely composition 22, this disclosure provides a composition as provided in any one of compositions 1 to 19, wherein the cell is a differentiated cell or a somatic cell.

[0243] In another composition, namely composition 23, this disclosure provides a composition as provided in any one of compositions 1 to 19, wherein the cell is capable of differentiating into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0244] In another composition, namely composition 24, this disclosure provides a composition as provided in composition 23, wherein these lineage-restricted progenitor cells are pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells.

[0245] In another composition, namely composition 25, this disclosure provides a composition as provided in composition 23, wherein these fully differentiated somatic cells are pancreatic β cells, epithelial cells, endoderm cells, macrophages, hepatocytes, adipocytes, kidney cells, blood cells, cardiomyocytes, or immune system cells.

[0246] In another composition, namely composition 26, this disclosure provides a composition as provided in any one of compositions 1 to 25, wherein the composition comprises a variety of universal donor cells.

[0247] In another composition, namely composition 27, this disclosure provides a composition as provided in composition 26, wherein the composition comprises a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from the plurality of universal donor cells.

[0248] In another composition, namely composition 28, this disclosure provides a composition as provided in composition 27, wherein the lineage-restricted progenitor cells are pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells, and the fully differentiated somatic cells are pancreatic β cells, epithelial cells, endodermal cells, macrophages, hepatocytes, adipocytes, kidney cells, blood cells, cardiomyocytes, or immune system cells.

[0249] In another composition, namely composition 29, this disclosure provides a composition as provided in composition 6 or 19, wherein the composition comprises a variety of universal donor cells.

[0250] In another composition, namely composition 30, this disclosure provides a composition as provided in composition 29, wherein the composition comprises a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from the plurality of universal donor cells.

[0251] In another composition, namely composition 31, this disclosure provides a composition as provided in composition 30, wherein the lineage-restricted progenitor cells are well-defined endoderm cells, gastrulation cells, hindbrain cells, pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, immature β cells or mature β cells, and the fully differentiated somatic cells are pancreatic β cells.

[0252] In another composition, namely composition 32, this disclosure provides a composition as provided in composition 26 or 29, wherein at least about 50%, at least about 70%, or at least about 90% of these cells express the first tolerogenic factor, the second tolerogenic factor, or the first tolerogenic factor and the second tolerogenic factor.

[0253] In another composition, namely composition 33, this disclosure provides a composition as provided in any one of compositions 27, 28, 30 or 31, wherein at least about 50%, at least about 70% or at least about 90% of these cells express the first tolerogenic factor, the second tolerogenic factor, or the first tolerogenic factor and the second tolerogenic factor.

[0254] In another composition, namely composition 34, this disclosure provides a composition comprising a plurality of cells of composition 26 or a cell population of composition 27 or 28.

[0255] In another composition, namely composition 35, this disclosure provides a composition as provided in composition 34 for treating a subject in need.

[0256] In another composition, namely composition 36, this disclosure provides a composition as provided in composition 35, wherein the subject has a disease, is suspected of having a disease, or is at risk of having a disease.

[0257] In another composition, namely composition 37, this disclosure provides a composition as provided in composition 36, wherein the disease is a genetically heritable disease.

[0258] In another composition, namely composition 38, this disclosure provides a composition comprising a plurality of cells of composition 29 or a cell population of composition 30 or 31.

[0259] In another composition, namely composition 39, this disclosure provides a composition as provided in composition 38 for treating diabetes in subjects in need.

[0260] In another composition, namely composition 40, this disclosure provides a composition as provided in composition 39, wherein the subject has type 1 or type 2 diabetes.

[0261] In another composition, namely composition 41, this disclosure provides a composition as provided in any one of compositions 35 to 40, wherein the subject is a human.

[0262] In the first method, namely method 1, this disclosure provides a method for obtaining cells for administration to a subject in need, the method comprising: (a) obtaining or having obtained a plurality of universal donor cells of any one of compositions 26, 29 or 32, and (b) maintaining the plurality of universal donor cells for a time and under conditions sufficient to differentiate these cells into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0263] In another method, namely method 2, this disclosure provides a method for treating a subject in need, the method comprising: (a) obtaining or having obtained, a plurality of universal donor cells of any one of compositions 26, 29 or 32 differentiated into lineage-restricted progenitor cells or fully differentiated somatic cells; and (b) administering such lineage-restricted progenitor cells or fully differentiated somatic cells to the subject.

[0264] In another method, namely method 3, this disclosure provides a method as provided in method 2, wherein administration includes implanting a device containing these lineage-restricted progenitor cells or fully differentiated somatic cells into the subject.

[0265] In another method, namely method 4, this disclosure provides a method as provided in any one of methods 1 to 3, wherein the lineage-restricted progenitor cells are pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells, and the fully differentiated somatic cells are pancreatic β cells, epithelial cells, endodermal cells, macrophages, hepatocytes, adipocytes, kidney cells, blood cells, cardiomyocytes, or immune system cells.

[0266] In another method, namely method 5, this disclosure provides a method as provided in any one of methods 1 to 4, wherein the subject has a disease, is suspected of having a disease, or is at risk of having a disease.

[0267] In another approach, namely approach 6, this disclosure provides a method as provided in approach 5, wherein the disease is a genetically heritable disease.

[0268] In another method, namely method 7, this disclosure provides a method as provided in any one of methods 1 to 6, wherein the subject is a human being.

[0269] In another method, namely method 8, this disclosure provides a method for treating diabetes in a subject in need, the method comprising: (a) obtaining or having obtained a variety of universal donor cells of composition 29 or 32 differentiated into pancreatic endoderm cells, pancreatic endocrine cells, immature β cells, mature β cells or pancreatic β cells; and (b) administering such pancreatic endoderm cells, pancreatic endocrine cells, immature β cells, mature β cells or pancreatic β cells to the subject.

[0270] In another method, namely method 9, this disclosure provides a method as provided in method 8, wherein administration includes implanting a device comprising these pancreatic endodermal cells, pancreatic endocrine cells, immature β cells, mature β cells or pancreatic β cells into the subject.

[0271] In another method, namely method 10, this disclosure provides a method as provided in method 8 or 9, wherein the subject has type 1 diabetes or type 2 diabetes.

[0272] In another method, namely method 11, this disclosure provides a method as provided in any one of methods 8 to 10, wherein the subject is a human being.

[0273] In another composition, namely composition 41, this disclosure provides a composition comprising universal donor cells containing a nucleotide sequence encoding HLA class I histocompatibility antigen α chain E (HLA-E) inserted within or near a gene encoding thioredoxin-interacting protein (TXNIP), wherein the universal donor cells express HLA-E and have disrupted TXNIP expression, and the universal donor cells have increased immune evasion and / or cell survival compared to a control.

[0274] In another composition, namely composition 42, this disclosure provides a composition as provided in composition 41, wherein the control cells are wild-type cells or cells that do not contain the inserted nucleotide sequence.

[0275] In another composition, namely composition 43, this disclosure provides a composition as provided in composition 41, wherein the disrupted TXNIP expression includes reduced or eliminated expression.

[0276] In another composition, namely composition 44, this disclosure provides a composition as provided in composition 41, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide, the HLA-G presenting peptide being fused to a B2M membrane protein, the B2M membrane protein being fused to HLA-E without its signal peptide.

[0277] In another composition, namely composition 45, this disclosure provides a composition as provided in composition 44, wherein the sequence encoding the HLA-E trimer consists substantially of SEQ ID NO:55.

[0278] In another composition, namely composition 46, this disclosure provides a composition as provided in composition 41, wherein the nucleotide sequence encoding HLA-E is operatively linked to an exogenous promoter.

[0279] In another composition, namely composition 47, this disclosure provides a composition as provided in composition 41, wherein the exogenous promoter is a CAG promoter.

[0280] In another composition, namely composition 48, this disclosure provides a composition as provided in composition 41, wherein the cell is a stem cell.

[0281] In another composition, namely composition 49, this disclosure provides a composition as provided in composition 48, wherein the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.

[0282] In another composition, namely composition 50, this disclosure provides a composition as provided in composition 41, wherein the cell is a differentiated cell or a somatic cell.

[0283] In another composition, namely composition 51, this disclosure provides a composition as provided in composition 41, wherein the cell is capable of differentiating into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0284] In another composition, namely composition 52, this disclosure provides a composition as provided in composition 51, wherein the lineage-restricted progenitor cells are well-defined endoderm cells, gastrulation cells, hindbrain cells, pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, immature β cells or mature β cells, and the fully differentiated somatic cells are pancreatic β cells.

[0285] In another composition, namely composition 53, this disclosure provides a composition comprising a variety of universal donor cells as provided in composition 41.

[0286] In another composition, namely composition 54, this disclosure provides a composition as provided in composition 53, wherein at least about 50% of these cells express HLA-E.

[0287] In another composition, namely composition 55, this disclosure provides a composition as provided in composition 53, wherein at least about 70% of these cells express HLA-E.

[0288] In another composition, namely composition 56, this disclosure provides a composition as provided in composition 53, wherein at least about 90% of these cells express HLA-E.

[0289] In another composition, namely composition 57, this disclosure provides a composition comprising a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from a variety of universal donor cells of composition 53.

[0290] In another composition, namely composition 58, this disclosure provides a composition as provided in composition 57, wherein the lineage-restricted progenitor cells are well-defined endoderm cells, gastrulation cells, hindbrain cells, pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, immature β cells or mature β cells, and the fully differentiated somatic cells are pancreatic β cells.

[0291] In another composition, namely composition 59, this disclosure provides a composition as provided in composition 58, wherein at least about 50% of these cells express HLA-E.

[0292] In another composition, namely composition 60, this disclosure provides a composition as provided in composition 59, wherein at least about 70% of these cells express HLA-E.

[0293] In another composition, namely composition 61, this disclosure provides a composition as provided in composition 59, wherein at least about 90% of these cells express HLA-E.

[0294] In another composition, namely composition 62, this disclosure provides a composition comprising genetically modified cells having introduced or increased expression of HLA class I histocompatibility antigen α chain E (HLA-E) and disrupted expression of thioredoxin-interacting protein (TXNIP), wherein the genetically modified cells have increased immune evasion and / or cell survival compared to unmodified cells.

[0295] In another composition, namely composition 63, this disclosure provides a composition as provided in composition 62, which comprises an HLA-E-coding nucleotide sequence inserted into or near the gene encoding TXNIP, thereby disrupting the TXNIP gene.

[0296] In another composition, namely composition 64, this disclosure provides a composition as provided in composition 62, wherein the disrupted TXNIP expression includes reduced or eliminated expression.

[0297] In another method, namely method 12, this disclosure provides a method for treating diabetes in a subject in need, the method comprising: obtaining or having obtained a variety of universal donor cells 53 differentiated into pancreatic endoderm cells, pancreatic endocrine cells, immature β cells, mature β cells or pancreatic β cells; and (b) administering such pancreatic endoderm cells, pancreatic endocrine cells, immature β cells, mature β cells or pancreatic β cells to the subject.

[0298] In another method, namely method 13, this disclosure provides a method as provided in method 12, wherein administration includes implanting a device comprising these pancreatic endoderm cells, pancreatic endocrine cells, immature β cells, mature β cells or pancreatic β cells into the subject.

[0299] In another method, namely method 14, this disclosure provides a method as provided in method 12, wherein the subject has type 1 diabetes or type 2 diabetes.

[0300] In another method, namely method 15, this disclosure provides a method as provided in method 12, wherein the subject is a human being.

[0301] In another composition, namely composition 65, this disclosure provides a composition comprising universal donor cells comprising (a) an insertion within or near a gene encoding β-2 microglobulin (B2M) of a nucleotide sequence encoding programmed death-ligand 1 (PD-L1) and (b) an insertion within or near a gene encoding thioredoxin-interacting protein (TXNIP) of a nucleotide sequence encoding HLA class I histocompatibility antigen α chain E (HLA-E), wherein the universal donor cells express PD-L1 and HLA-E and have disrupted B2M and TXNIP expression, and the universal donor cells have increased immune evasion and / or cell survival compared to control cells.

[0302] In another composition, namely composition 66, this disclosure provides a composition as provided in composition 65, wherein the control cells are wild-type cells or cells that do not contain the inserted nucleotide sequence.

[0303] In another composition, namely composition 67, this disclosure provides a composition as provided in composition 65, wherein the disrupted B2M expression comprises reduced or eliminated B2M expression, and the disrupted TXNIP expression comprises reduced or eliminated TXNIP expression.

[0304] In another composition, namely composition 68, this disclosure provides a composition as provided in composition 65, wherein the nucleotide sequence encoding PD-L1 consists substantially of SEQ ID NO:11.

[0305] In another composition, namely composition 69, this disclosure provides a composition as provided in composition 65, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide, the HLA-G presenting peptide being fused to a B2M membrane protein, the B2M membrane protein being fused to HLA-E without its signal peptide.

[0306] In another composition, namely composition 70, this disclosure provides a composition as provided in composition 69, wherein the sequence encoding the HLA-E trimer is substantially composed of SEQ ID NO:55.

[0307] In another composition, namely composition 71, this disclosure provides a composition as provided in composition 65, wherein the nucleotide sequence encoding PD-L1 is operatively linked to a foreign promoter and the nucleotide sequence encoding HLA-E is operatively linked to a foreign promoter.

[0308] In another composition, namely composition 72, this disclosure provides a composition as provided in composition 71, wherein the exogenous promoter is a CAG promoter.

[0309] In another composition, namely composition 73, this disclosure provides a composition as provided in composition 65, wherein the cell is a stem cell.

[0310] In another composition, namely composition 74, this disclosure provides a composition as provided in composition 73, wherein the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.

[0311] In another composition, namely composition 75, this disclosure provides a composition as provided in composition 65, wherein the cell is a differentiated cell or a somatic cell.

[0312] In another composition, namely composition 76, this disclosure provides a composition as provided in composition 65, wherein the cell is capable of differentiating into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0313] In another composition, namely composition 77, this disclosure provides a composition as provided in composition 76, wherein the lineage-restricted progenitor cells are well-defined endoderm cells, gastrulation cells, hindbrain cells, pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, immature β cells or mature β cells, and the fully differentiated somatic cells are pancreatic β cells.

[0314] In another composition, namely composition 78, this disclosure provides a composition comprising a variety of universal donor cells as provided in composition 65.

[0315] In another composition, namely composition 79, this disclosure provides a composition as provided in composition 78, wherein at least about 50% of these cells express PD-L1 and / or at least about 50% of these cells express HLA-E.

[0316] In another composition, namely composition 80, this disclosure provides a composition as provided in composition 78, wherein at least about 70% of these cells express PD-L1 and / or at least about 70% of these cells express HLA-E.

[0317] In another composition, namely composition 81, this disclosure provides a composition as provided in composition 78, wherein at least about 90% of these cells express PD-L1 and / or at least about 90% of these cells express HLA-E.

[0318] In another composition, namely composition 82, this disclosure provides a composition comprising a population of lineage-restricted progenitor cells or fully differentiated somatic cells derived from a variety of universal donor cells as provided in composition 78.

[0319] In another composition, namely composition 83, this disclosure provides a composition as provided in composition 82, wherein the lineage-restricted progenitor cells are well-defined endoderm cells, gastrulation cells, hindbrain cells, pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, immature β cells or mature β cells, and the fully differentiated somatic cells are pancreatic β cells.

[0320] In another composition, namely composition 84, this disclosure provides a composition as provided in composition 83, wherein at least about 50% of these cells express PD-L1 and / or at least about 50% of these cells express HLA-E.

[0321] In another composition, namely composition 85, this disclosure provides a composition as provided in composition 83, wherein at least about 70% of these cells express PD-L1 and / or at least about 70% of these cells express HLA-E.

[0322] In another composition, namely composition 86, this disclosure provides a composition as provided in composition 83, wherein at least about 90% of these cells express PD-L1 and / or at least about 90% of these cells express HLA-E.

[0323] In another composition, namely composition 87, this disclosure provides a composition comprising genetically modified cells having introduced or increased expression of PD-L1 and HLA-E and disrupted expression of B2M and TXNIP, wherein the genetically modified cells have increased immune evasion and / or cell survival compared to unmodified cells.

[0324] In another composition, namely composition 88, this disclosure provides a composition as provided in composition 87, comprising an insertion of a nucleotide sequence encoding PD-L1 within or near a gene encoding B2M, thereby disrupting the B2M gene, and an insertion of a nucleotide sequence encoding HLA-E within or near a gene encoding TXNIP, thereby disrupting the TXNIP gene.

[0325] In another composition, namely composition 89, this disclosure provides a composition as provided in composition 87, wherein the disruption of B2M and TXNIP expression comprises reduced or eliminated B2M and TXNIP expression.

[0326] In another method, namely method 16, this disclosure provides a method for treating diabetes in a subject in need, the method comprising: (a) obtaining or having obtained a variety of universal donor cells of composition 78 differentiated into pancreatic endoderm cells, pancreatic endocrine cells, immature β cells, mature β cells or pancreatic β cells; and (b) administering such pancreatic endoderm cells, pancreatic endocrine cells, immature β cells, mature β cells or pancreatic β cells to the subject.

[0327] In another method, namely method 17, this disclosure provides a method as provided in method 16, wherein administration includes implanting a device comprising these pancreatic endodermal cells, pancreatic endocrine cells, immature β cells, mature β cells or pancreatic β cells into the subject.

[0328] In another method, namely method 18, this disclosure provides a method as provided in method 16, wherein the subject has type 1 diabetes or type 2 diabetes.

[0329] In another approach, namely method 19, this disclosure provides a method as provided in method 16, wherein the subject is a human being.

[0330] In another method, namely method 20, this disclosure provides a method for generating universal donor cells, the method comprising delivering to a cell: (a) a first site-directed nuclease targeting a site within or near a gene encoding a survival factor; and (b) a first nucleic acid containing a nucleotide sequence encoding a first tolerance factor, the flanking of which are (i) nucleotide sequences homologous to a region to the left of the target site in (a) and (ii) nucleotide sequences homologous to a region to the right of the target site in (a), wherein the first site-directed nuclease cleaves the target site in (a), and the first nucleic acid in (b) is inserted at a site that partially overlaps, completely overlaps with, or is contained within the site in (a), thereby generating universal donor cells, wherein the universal donor cells have increased cell survival compared to cells in which the nucleic acid in (b) is not inserted.

[0331] In another method, namely method 21, this disclosure provides a method as provided in method 20, wherein the survival factor is TXNIP, ZNF143, FOXO1, JNK, or MANF.

[0332] In another method, namely method 22, this disclosure provides a method as provided in method 20 or 21, wherein the first tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

[0333] In another approach, namely approach 23, this disclosure provides a method as provided in any one of approaches 20 to 22, wherein the survival factor is TXNIP.

[0334] In another method, namely method 24, this disclosure provides a method as provided in method 23, wherein the first tolerability-inducing factor is HLA-E.

[0335] In another method, namely method 25, this disclosure provides a method as provided in any one of methods 20 to 24, wherein the first site-directed nuclease is a CRISPR system comprising a CRISPR nuclease and a guide RNA (gRNA).

[0336] In another method, namely method 26, this disclosure provides a method as provided in any one of methods 20 to 25, wherein the CRISPR nuclease is a type II Cas9 nuclease or a type V Cfp1 nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal.

[0337] In another method, namely method 27, this disclosure provides a method as provided in any one of methods 20 to 26, wherein the gRNA comprises a spacer subsequence corresponding to the target sequence consisting of SEQ ID NO:15-24.

[0338] In another method, namely method 28, this disclosure provides a method as provided in any one of methods 25 to 27, wherein the nucleotide sequence of (b)(i) is substantially composed of SEQ ID NO:25, and the nucleotide sequence of (b)(ii) is substantially composed of SEQ ID NO:32.

[0339] In another method, namely method 29, this disclosure provides a method as provided in any one of methods 20 to 28, wherein the method further comprises delivering to the cell: (c) a second site-specific nuclease targeting a site within or near a gene encoding one or more of MHC-I or MHC-II human leukocyte antigens or components or transcriptional regulatory factors of the MHC-I or MHC-II complex; and (d) a second nucleic acid comprising a nucleotide sequence encoding a second tolerogenic factor, the nucleotide sequence flanking (iii) the target site located in (c). (iv) A nucleotide sequence homologous to the region to the left of the target site and (c) A nucleotide sequence homologous to the region to the right of the target site, wherein the second tolerogenic factor in (d) is different from the first tolerogenic factor in (b), wherein the second site-directed nuclease cleaves the target site in (c), and the second nucleic acid in (d) is inserted at a site that partially overlaps, completely overlaps with, or is contained within the target site in (c), wherein the universal donor cell has increased immune evasion and / or cell survival compared to cells in which the second nucleic acid in (d) is not inserted.

[0340] In another method, namely method 30, this disclosure provides a method as provided in method 29, wherein the MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of the MHC-I or MHC-II complex is HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, B2M, NLRC5, CIITA, RFX5, RFXAP, or RFXANK.

[0341] In another method, namely method 31, this disclosure provides a method as provided in method 29 or 30, wherein the second tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

[0342] In another method, namely method 32, this disclosure provides a method as provided in any one of methods 29 to 31, wherein the MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of the MHC-I or MHC-II complex is B2M.

[0343] In another method, namely method 33, this disclosure provides a method as provided in method 32, wherein the second tolerability factor is PD-L1.

[0344] In another method, namely method 34, this disclosure provides a method as provided in any one of methods 29 to 33, wherein the second site-directed nuclease is a CRISPR system comprising a CRISPR nuclease and gRNA.

[0345] In another method, namely method 35, this disclosure provides a method as provided in method 34, wherein the CRISPR nuclease is a type II Cas9 nuclease or a type V Cfp1 nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal.

[0346] In another method, namely method 36, this disclosure provides a method as provided in method 34 or 35, wherein the gRNA contains a spacer subsequence corresponding to the target sequence consisting of SEQ ID NO:1-3 or 35-44.

[0347] In another method, namely method 37, this disclosure provides a method as provided in any one of methods 34 to 36, wherein the nucleotide sequence of (d)(iii) consists essentially of SEQ ID NO:7 and the nucleotide sequence of (d)(iv) consists essentially of SEQ ID NO:13.

[0348] In another method, namely method 38, this disclosure provides a method as provided in any one of methods 25 to 28 or 34 to 37, wherein the CRISPR nuclease and the gRNA are present in a molar ratio of 1:3.

[0349] In another method, namely method 39, this disclosure provides a method as provided in any one of methods 20 to 38, wherein the nucleotide sequence encoding the first tolerogenic factor is operatively linked to an exogenous promoter, and the nucleotide sequence encoding the second tolerogenic factor is operatively linked to an exogenous promoter.

[0350] In another method, namely method 40, this disclosure provides a method as provided in method 39, wherein the exogenous promoter is a constitutive promoter, an inducible promoter, a time-specific promoter, a tissue-specific promoter, or a cell type-specific promoter, optionally wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0351] In another method, namely method 41, this disclosure provides a method for generating universal donor cells, the method comprising delivering to the cells: (a) a first site-directed nuclease targeting a site within or near a gene encoding a survival factor; (b) a first nucleic acid containing a nucleotide sequence encoding a first tolerogenic factor, the flanking nucleotide sequence being (i) a nucleotide sequence homologous to a region to the left of the target site in (a) and (ii) a nucleotide sequence homologous to a region to the right of the target site in (a), wherein the first site-directed nuclease cleaves the target site in (a), and the first nucleic acid in (b) is used as a template for inserting the nucleotide sequence encoding the first tolerogenic factor into a site that partially overlaps, completely overlaps with, or is contained within the site in (a), thereby disrupting the gene in (a); and (c) a component or transcription encoding MHC-I or MHC-II human leukocyte antigen or an MHC-I or MHC-II complex. The second site-directed nuclease at a site within or near one or more of the regulatory factors; and (d) a second nucleic acid containing a nucleotide sequence encoding a second tolerance factor, the flanking nucleotide sequence being (iii) a nucleotide sequence homologous to the region to the left of the target site in (c) and (iv) a nucleotide sequence homologous to the region to the right of the target site in (c), wherein the tolerance factor in (d) is different from the tolerance factor in (b), wherein the second site-directed nuclease cleaves the target site in (c), and the second nucleic acid in (d) is used as a template by homologous recombination to insert the nucleotide sequence encoding the second tolerance factor into a site that partially overlaps, completely overlaps with, or is contained within the site in (c), thereby disrupting the gene in (c), thereby producing a universal donor cell, wherein the universal donor cell has increased cell survival compared to a cell in which the first nucleic acid in (b) and the second nucleic acid in (d) are not inserted.

[0352] In another method, namely method 42, this disclosure provides a method as provided in method 41, wherein the survival factor is TXNIP, the first tolerogenic factor is HLA-E, the MHC-I or MHC-II human leukocyte antigen or a component or transcriptional regulator of the MHC-I or MHC-II complex is B2M, and the second tolerogenic factor is PD-L1.

[0353] In another method, namely method 43, this disclosure provides a method as provided in any one of methods 20 to 42, wherein the cell is a mammalian cell, optionally wherein the cell is a human cell.

[0354] In another method, namely method 44, this disclosure provides a method as provided in any one of methods 20 to 43, wherein the cell is a stem cell.

[0355] In another method, namely method 45, this disclosure provides a method as provided in any one of methods 20 to 43, wherein the cell is a pluripotent stem cell, embryonic stem cell, adult stem cell, induced pluripotent stem cell, or hematopoietic stem cell.

[0356] In another method, namely method 46, this disclosure provides a method as provided in any one of methods 20 to 43, wherein the cell is a differentiated cell or a somatic cell.

[0357] In another method, namely method 47, this disclosure provides a method as provided in any one of methods 20 to 43, wherein the universal donor cell is capable of differentiating into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0358] In another method, namely method 48, this disclosure provides a method as provided in method 47, wherein these lineage-restricted progenitor cells are pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells.

[0359] In another method, namely method 49, this disclosure provides a method as provided in method 47, wherein these fully differentiated somatic cells are endocrine cells such as pancreatic β cells, epithelial cells, endoderm cells, macrophages, hepatocytes, adipocytes, kidney cells, blood cells, or immune system cells.

[0360] In another method, namely method 49A, this disclosure provides a method as provided in method 47, wherein these fully differentiated somatic cells are cardiomyocytes or immune system cells.

[0361] In another composition, namely composition 90, this disclosure provides a composition comprising a variety of universal donor cells produced by any one of methods 20 to 49.

[0362] In another composition, namely composition 91, this disclosure provides a composition as provided by composition 90, which is maintained for a time and under conditions sufficient to allow these cells to undergo differentiation.

[0363] In another composition, namely composition 92, this disclosure provides a composition as provided in composition 90 or 91 for treating a subject in need.

[0364] In another composition, namely composition 93, this disclosure provides a composition as provided in composition 92, wherein the subject is a person who has a disease, is suspected of having a disease, or is at risk of having a disease.

[0365] In another method, namely method 50, this disclosure provides a method comprising administering a variety of universal donor cells of composition 90 or 91 to a subject.

[0366] In another method, namely method 51, this disclosure provides a method for treating a subject in need, the method comprising: (a) obtaining or having obtained, a variety of universal donor cells of composition 90 differentiated into lineage-restricted progenitor cells or fully differentiated somatic cells; and (b) administering such lineage-restricted progenitor cells or fully differentiated somatic cells to the subject.

[0367] In another method, namely method 52, this disclosure provides a method for obtaining cells for administration to a subject in need, the method comprising: (a) obtaining or having obtained the universal donor cells as described in claim 31; and (b) maintaining the universal donor cells for a time and under conditions sufficient to differentiate these cells into lineage-restricted progenitor cells or fully differentiated somatic cells.

[0368] In another method, namely method 53, this disclosure provides a method as provided in method 51 or 52, wherein these lineage-restricted progenitor cells are pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells.

[0369] In another method, namely method 54, this disclosure provides a method as provided in method 51 or 52, wherein these fully differentiated somatic cells are endocrine cells such as pancreatic β cells, epithelial cells, endoderm cells, macrophages, hepatocytes, adipocytes, kidney cells, blood cells, or immune system cells.

[0370] In another method, namely method 54A, this disclosure provides a method as provided in method 51 or 52, wherein these fully differentiated somatic cells are cardiomyocytes.

[0371] In another method, namely method 55, this disclosure provides a method as provided in methods 50 to 54, wherein the subject is a person who has a disease, is suspected of having a disease, or is at risk of having a disease.

[0372] In another approach, namely approach 56, this disclosure provides a method as provided in approach 55, wherein the disease is a genetically heritable disease.

[0373] In another composition, namely composition 93, this disclosure provides a guide RNA comprising a spacer subsequence corresponding to the target sequence consisting of SEQ ID NO:15-24.

[0374] In another method, namely method 57, this disclosure provides an in vitro method for generating universal donor cells, the method comprising delivering to stem cells: (a) an RNA-guided nuclease; (b) a guide RNA (gRNA) targeting a target site in the thioredoxin-interacting protein (TXNIP) locus; and (c) a vector containing nucleic acids comprising: (i) a nucleotide sequence encoding a tolerance-inducing factor; (ii) a nucleotide sequence substantially consisting of SEQ ID NO:25 and having sequence homology with a genomic region located to the left of the target site and within 50 nucleotides of the target site; and (iii) a nucleotide sequence substantially consisting of SEQ ID NO:25. NO:32 is composed of a nucleotide sequence that is sequence homologous to a genomic region located to the right of the target site and within 50 nucleotides of the target site, wherein (i) is flanked by (ii) and (iii); wherein the TXNIP locus is cleaved at the target site and the nucleic acid is inserted into the TXNIP locus, thereby disrupting the TXNIP gene and generating universal donor cells, wherein the universal donor cells have increased immune evasion and / or cell survival compared to control cells.

[0375] In another method, namely method 57A, this disclosure provides a method as provided in method 57, wherein the nucleic acid is inserted into the TXNIP locus within 50 base pairs at the target site.

[0376] In another method, namely method 58, this disclosure provides a method as provided in method 57, wherein the control cell is a wild-type cell or a cell that does not contain the inserted nucleic acid.

[0377] In another method, namely method 59, this disclosure provides a method as provided in method 57, wherein the disrupted TXNIP gene has reduced or eliminated TXNIP expression.

[0378] In another method, namely method 60, this disclosure provides a method as provided in method 57, wherein the gRNA comprises a spacer subsequence corresponding to the sequence consisting of SEQ ID NO:15-24.

[0379] In another method, namely method 61, this disclosure provides a method as provided in method 57, wherein the gRNA comprises a spacer subsequence corresponding to the sequence consisting of SEQ ID NO:20.

[0380] In another method, namely method 62, this disclosure provides a method as provided in method 57, wherein the vector is a plasmid vector.

[0381] In another method, namely method 63, this disclosure provides a method as provided in method 57, wherein the tolerogenic factor is HLA class I histocompatibility antigen α chain E (HLA-E).

[0382] In another method, namely method 64, this disclosure provides a method as provided in method 63, wherein the nucleotide sequence encoding HLA-E includes a sequence encoding an HLA-E trimer, the HLA-E trimer including a B2M signal peptide fused to an HLA-G presenting peptide, the HLA-G presenting peptide being fused to a B2M membrane protein, the B2M membrane protein being fused to HLA-E without its signal peptide.

[0383] In another method, namely method 65, this disclosure provides a method as provided in method 63, wherein the sequence encoding the HLA-E trimer consists substantially of SEQ ID NO:55.

[0384] In another approach, namely approach 66, this disclosure provides a method as provided in approach 65, wherein the sequence encoding the HLA-E trimer is operatively linked to an exogenous promoter.

[0385] In another approach, namely approach 67, this disclosure provides a method as provided in approach 66, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0386] In another method, namely method 68, this disclosure provides a method as provided in method 57, wherein the RNA-guided nuclease is a Cas9 nuclease.

[0387] In another method, namely method 69, this disclosure provides a method as provided in method 68, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0388] In another method, namely method 70, this disclosure provides a method as provided in method 69, wherein the Cas9 nuclease and the gRNA are present in a molar ratio of 1:3.

[0389] In another method, namely method 71, this disclosure provides a method as provided in method 57, wherein the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.

[0390] In another method, namely method 72, this disclosure provides a method as provided in method 57, wherein the stem cell is a human stem cell.

[0391] In another method, namely method 73, this disclosure provides an in vitro method for generating universal donor cells, the method comprising delivering to stem cells: (a) an RNA-guided nuclease; (b) a guide RNA (gRNA) targeting a target site in the thioredoxin-interacting protein (TXNIP) locus; and (c) a vector containing nucleic acids comprising: (i) a nucleotide sequence encoding a tolerance-inducing factor; (ii) a nucleotide sequence sequence homologous to a genomic region located to the left of the target site and within 50 nucleotides of the target site; and (iii) a nucleotide sequence sequence homologous to a genomic region located to the right of the target site and within 50 nucleotides of the target site, wherein (i) is flanked by (ii) and (iii), and the vector contains a nucleotide sequence defined by SEQ ID NO. The nucleotide sequence consisting of NO:34 or 56; wherein the TXNIP locus is cleaved at the target site and the nucleic acid is inserted into the TXNIP locus, thereby disrupting the TXNIP gene and generating universal donor cells, wherein the universal donor cells have increased immune evasion and / or cell survival compared to control cells.

[0392] In another method, namely method 73A, this disclosure provides a method as provided in method 73, wherein the nucleic acid is inserted into the TXNIP locus within 50 base pairs at the target site.

[0393] In another method, namely method 74, this disclosure provides a method as provided in method 73, wherein the control cell is a wild-type cell or a cell that does not contain the inserted nucleic acid.

[0394] In another method, namely method 75, this disclosure provides a method as provided in method 73, wherein the disrupted TXNIP gene has reduced or eliminated TXNIP expression.

[0395] In another method, namely method 76, this disclosure provides a method as provided in method 73, wherein the gRNA comprises a spacer subsequence corresponding to the sequence consisting of SEQ ID NO:15-24.

[0396] In another method, namely method 77, this disclosure provides a method as provided in method 73, wherein the gRNA comprises a spacer subsequence corresponding to the sequence consisting of SEQ ID NO:20.

[0397] In another method, namely method 78, this disclosure provides a method as provided in method 73, wherein the vector is a plasmid vector.

[0398] In another method, namely method 79, this disclosure provides a method as provided in method 73, wherein the tolerogenic factor is HLA class I histocompatibility antigen α chain E (HLA-E).

[0399] In another method, namely method 80, this disclosure provides a method as provided in method 73, wherein the RNA-guided nuclease is a Cas9 nuclease.

[0400] In another method, namely method 81, this disclosure provides a method as provided in method 80, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0401] In another method, namely method 82, this disclosure provides a method as provided in method 80, wherein the Cas9 nuclease and the gRNA are present in a molar ratio of 1:3.

[0402] In another method, namely method 83, this disclosure provides a method as provided in method 73, wherein the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.

[0403] In another method, namely method 84, this disclosure provides a method as provided in method 73, wherein the stem cell is a human stem cell.

[0404] In another method, namely method 85, this disclosure provides an in vitro method for generating universal donor cells, the method comprising delivering to stem cells: (a) a first ribonucleoprotein (RNP) complex comprising an RNA-guided nuclease and a guide RNA (gRNA) targeting a target site in the β-2 microglobulin (B2M) locus; (b) a first vector comprising nucleic acid comprising: (i) a nucleotide sequence encoding a first tolerogenic factor; (ii) a nucleotide sequence substantially consisting of SEQ ID NO:7 and having sequence homology with a genomic region located to the left of the target site in the B2M locus and within 50 nucleotides of the target site; and (iii) a nucleotide sequence substantially consisting of SEQ ID NO:7. NO:13 constitutes a nucleotide sequence that is sequence homologous to the genomic region located to the right of the target site at the B2M locus and within 50 nucleotides of the target site, wherein (i) is flanked by (ii) and (iii); wherein the B2M locus is cleaved at the target site, and a nucleic acid containing a nucleotide sequence encoding the first tolerogenic factor is inserted into the B2M locus, thereby disrupting the B2M gene; (c) a second RNP complex comprising an RNA-guided nuclease and a gRNA targeting the target site at the thioredoxin-interacting protein (TXNIP) locus; and (d) a second vector comprising nucleic acid containing: (i) a nucleotide sequence encoding the second tolerogenic factor; (ii) a nucleotide sequence substantially composed of SEQ ID NO:25 and sequence homologous to the genomic region located to the left of the target site at the TXNIP locus and within 50 nucleotides of the target site; and (iii) a nucleotide sequence substantially composed of SEQ ID NO:25. NO:32 is a nucleotide sequence that is sequence homologous to the genomic region located to the right of the target site at the TXNIP locus and within 50 nucleotides of the target site, wherein (i) is flanked by (ii) and (iii); wherein the TXNIP locus is cleaved at the target site and a nucleic acid containing a nucleotide sequence encoding the second tolerogenic factor is inserted into the TXNIP locus, thereby disrupting the TXNIP gene and generating universal donor cells, wherein the universal donor cells have increased immune evasion and / or cell survival compared to control cells.

[0405] In another method, namely method 85A, this disclosure provides a method as provided in method 85, wherein the nucleic acid in (b) is inserted into the B2M locus within 50 base pairs at the target site and / or wherein the nucleic acid in (d) is inserted into the TXNIP locus within 50 base pairs at the target site.

[0406] In another method, namely method 86, this disclosure provides a method as provided in method 85, wherein the control cell is a wild-type cell or a cell that does not contain the inserted nucleic acid.

[0407] In another method, namely method 87, this disclosure provides a method as provided in method 85, wherein the disrupted B2M gene has reduced or eliminated B2M expression, and the disrupted TXNIP gene has reduced or eliminated TXNIP expression.

[0408] In another method, namely method 88, this disclosure provides a method as provided in method 85, wherein the gRNA of the first RNP complex contains a spacer subsequence corresponding to the sequence consisting of SEQ ID NO:1-3 or 35-44, and the gRNA of the second RNP complex contains a spacer subsequence corresponding to the sequence consisting of SEQ ID NO:15-24.

[0409] In another method, namely method 89, this disclosure provides a method as provided in method 85, wherein the gRNA of the first RNP complex comprises a spacer subsequence corresponding to the sequence consisting of SEQ ID NO:2, and the gRNA of the second RNP complex comprises a spacer subsequence corresponding to the sequence consisting of SEQ ID NO:20.

[0410] In another method, namely method 90, this disclosure provides a method as provided in method 85, wherein the first vector is a plasmid vector and the second vector is a plasmid vector.

[0411] In another method, namely method 91, this disclosure provides a method as provided in method 85, wherein the first tolerogenic factor is programmed death-ligand 1 (PD-L1) and the second tolerogenic factor is HLA class I histocompatibility antigen α chain E (HLA-E).

[0412] In another method, namely method 92, this disclosure provides a method as provided in method 91, wherein the nucleotide sequence encoding PD-L1 consists substantially of SEQ ID NO:11.

[0413] In another method, namely method 93, this disclosure provides a method as provided in method 91, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide, the HLA-G presenting peptide being fused to a B2M membrane protein, the B2M membrane protein being fused to HLA-E without its signal peptide, and the sequence encoding the HLA-E trimer is substantially composed of SEQ ID NO:55.

[0414] In another method, namely method 94, this disclosure provides a method as provided in method 85, wherein the nucleotide sequence encoding the first tolerogenic factor is operatively linked to a foreign promoter, and the nucleotide sequence encoding the second tolerogenic factor is operatively linked to a foreign promoter.

[0415] In another approach, namely approach 95, this disclosure provides a method as provided in approach 94, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0416] In another method, namely method 96, this disclosure provides a method as provided in method 85, wherein each of the first RNP complex and the second RNP complex comprises an RNA-guided nuclease to gRNA molar ratio of 1:3.

[0417] In another method, namely method 97, this disclosure provides a method as provided in method 85, wherein the RNA-guided nucleases of the first RNP complex and the second RNP complex are Cas9 nucleases.

[0418] In another method, namely method 98, this disclosure provides a method as provided in method 97, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0419] In another method, namely method 99, this disclosure provides a method as provided in method 85, wherein the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.

[0420] In another method, namely method 100, this disclosure provides a method as provided in method 85, wherein the stem cell is a human stem cell.

[0421] In another method, namely method 101, this disclosure provides an in vitro method for generating universal donor cells, the method comprising delivering to stem cells: (a) a first ribonucleoprotein (RNP) complex comprising an RNA-guided nuclease and guide RNA (gRNA) targeting a target site in a β-2 microglobulin (B2M) locus; (b) a first vector comprising nucleic acid comprising: (i) a nucleotide sequence encoding a first tolerogenic factor; (ii) a nucleotide sequence sequence homologous to a genomic region located to the left of the target site in the B2M locus and within 50 nucleotides of the target site; and (iii) a nucleotide sequence sequence homologous to a genomic region located to the right of the target site in the B2M locus and within 50 nucleotides of the target site, wherein (i) is flanked by (ii) and (iii), and the first vector comprises SEQ The nucleotide sequence consisting of IDNO:33; wherein the B2M locus is cleaved at the target site, and a nucleic acid containing a nucleotide sequence encoding the first tolerogenic factor is inserted into the B2M locus therein, thereby disrupting the B2M gene; (c) a second RNP complex containing an RNA-guided nuclease and gRNA targeting the target site in the thioredoxin-interacting protein (TXNIP) locus; and (d) a second vector containing nucleic acid comprising: (i) a nucleotide sequence encoding the second tolerogenic factor; (ii) a nucleotide sequence sequence homologous to a genomic region located to the left of the target site in the TXNIP locus and within 50 nucleotides of the target site; and (iii) a nucleotide sequence sequence homologous to a genomic region located to the right of the target site in the TXNIP locus and within 50 nucleotides of the target site, wherein (i) is flanked by (ii) and (iii), and the second vector comprises SEQ ID:33. The TXNIP locus is cleaved at the target site by a nucleotide sequence consisting of NO:34 or 56, and a nucleic acid containing a nucleotide sequence encoding the second tolerogenic factor is inserted into the TXNIP locus, thereby disrupting the TXNIP gene and generating universal donor cells, wherein the universal donor cells have increased immune evasion and / or cell survival compared to control cells.

[0422] In another method, namely method 101A, this disclosure provides a method as provided in method 101, wherein the nucleic acid in (b) is inserted into the B2M locus within 50 base pairs at the target site and / or wherein the nucleic acid in (d) is inserted into the TXNIP locus within 50 base pairs at the target site.

[0423] In another method, namely method 102, this disclosure provides a method as provided in method 101, wherein the control cell is a wild-type cell or a cell that does not contain the inserted nucleic acid.

[0424] In another method, namely method 103, this disclosure provides a method as provided in method 10, wherein the disrupted B2M gene has reduced or eliminated B2M expression, and the disrupted TXNIP gene has reduced or eliminated TXNIP expression.

[0425] In another method, namely method 104, this disclosure provides a method as provided in method 101, wherein the gRNA of the first RNP complex contains a spacer subsequence corresponding to the sequence consisting of SEQ ID NO:1-3 or 35-44, and the gRNA of the second RNP complex contains a spacer subsequence corresponding to the sequence consisting of SEQ ID NO:15-24.

[0426] In another method, namely method 105, this disclosure provides a method as provided in method 101, wherein the gRNA of the first RNP complex contains a spacer subsequence corresponding to the sequence consisting of SEQ ID NO:2, and the gRNA of the second RNP complex contains a spacer subsequence corresponding to the sequence consisting of SEQ ID NO:20.

[0427] In another method, namely method 106, this disclosure provides a method as provided in method 101, wherein the first vector is a plasmid vector and the second vector is a plasmid vector.

[0428] In another method, namely method 107, this disclosure provides a method as provided in method 101, wherein the first tolerogenic factor is programmed death-ligand 1 (PD-L1) and the second tolerogenic factor is HLA class I histocompatibility antigen α chain E (HLA-E).

[0429] In another method, namely method 108, this disclosure provides a method as provided in method 101, wherein each of the first RNP complex and the second RNP complex comprises a 1:3 molar ratio of RNA-guided nuclease to gRNA.

[0430] In another method, namely method 109, this disclosure provides a method as provided in method 101, wherein the RNA-guided nucleases of the first RNP complex and the second RNP complex are Cas9 nucleases.

[0431] In another method, namely method 110, this disclosure provides a method as provided in method 109, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0432] In another method, namely method 111, this disclosure provides a method as provided in method 101, wherein the stem cell is an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.

[0433] In another method, namely method 112, this disclosure provides a method as provided in method 101, wherein the stem cell is a human stem cell.

[0434] In the first method, namely method 1, this disclosure provides a method for generating universal donor cells, the method comprising: (a) modifying stem cells by inserting a nucleotide sequence encoding programmed death ligand 1 (PD-L1) into or near a gene encoding β-2 microglobulin (B2M) to generate PD-L1 positive cells; (b) enriching the PD-L1 positive cells; (c) modifying the PD-L1 positive cells from (b) by inserting a nucleotide sequence encoding HLA class I histocompatibility antigen α chain E (HLA-E) into or near a gene encoding thioredoxin interacting protein (TXNIP) to generate PD-L1 and HLA-E double positive cells; (d) enriching the PD-L1 and HLA-E double positive cells; (e) single-cell sorting to select PD-L1 and HLA-E double positive cells; (f) characterizing the cells from (e) as universal donor cells; and (g) freezing these universal donor cells for long-term storage.

[0435] In another method, namely method 2, this disclosure provides a method as provided in method 1, wherein the modification in (a) comprises delivering to the stem cell (1) a first ribonucleoprotein (RNP) complex comprising an RNA-guided nuclease and a guide RNA (gRNA) targeting a target site in the B2M locus and (2) a first vector comprising nucleic acid comprising (i) a nucleotide sequence homologous to a region to the left of the target site in the B2M locus, (ii) the nucleotide sequence encoding PD-L1 and (iii) a nucleotide sequence homologous to a region to the right of the target site in the B2M locus, wherein the B2M locus is cleaved at the target site and the nucleic acid comprising the nucleotide sequence encoding PD-L1 is inserted into the B2M locus, thereby disrupting the B2M gene.

[0436] In another method, namely method 2A, this disclosure provides a method as provided in method 2, wherein the nucleic acid is inserted into the B2M locus within 50 base pairs at the target site.

[0437] In another method, namely method 3, this disclosure provides a method as provided in method 2, wherein the RNA-guided nuclease of the first RNP complex is a Cas9 nuclease, and the gRNA of the first RNP complex contains a spacer subsequence corresponding to the target sequence consisting of SEQ ID NO:2.

[0438] In another method, namely method 4, this disclosure provides a method as provided in method 3, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0439] In another method, namely method 5, this disclosure provides a method as provided in method 2, wherein the first RNP comprises a 3:1 molar ratio of gRNA to RNA-guided nuclease.

[0440] In another method, namely method 6, this disclosure provides a method as provided in method 2, wherein the nucleotide sequence of (a)(2)(i) is substantially composed of SEQ ID NO:7, and the nucleotide sequence of (a)(2)(iii) is substantially composed of SEQ ID NO:13.

[0441] In another method, namely method 7, this disclosure provides a method as provided in method 2, wherein the nucleotide sequence encoding PD-L1 consists substantially of SEQ ID NO:11.

[0442] In another approach, namely approach 8, this disclosure provides a method as provided in approach 2, wherein the nucleotide sequence encoding PD-L1 is operatively linked to the CAG promoter.

[0443] In another method, namely method 9, this disclosure provides a method as provided in method 2, wherein the first vector is a plasmid vector and contains a nucleotide sequence consisting of SEQ ID NO:33.

[0444] In another method, namely method 10, this disclosure provides a method as provided in method 2, wherein the delivery in (a)(1) and (a)(2) includes electroporation.

[0445] In another method, namely method 11, this disclosure provides a method as provided in method 1, wherein the enrichment of PD-L1 positive cells in (b) includes magnetic assist cell sorting (MACS), single-cell cloning, expansion of the PD-L1 positive cells or a combination thereof.

[0446] In another method, namely method 12, this disclosure provides a method as provided in method 1, wherein the modification in (c) comprises delivering to the PD-L1 positive cell (1) a second RNP complex comprising an RNA-guided nuclease and gRNA targeting a target site in the TXNIP locus and (2) a second vector comprising nucleic acid comprising (i) a nucleotide sequence homologous to a region to the left of the target site in the TXNIP locus, (ii) the nucleotide sequence encoding HLA-E and (iii) a nucleotide sequence homologous to a region to the right of the target site in the TXNIP locus, wherein the TXNIP locus is cleaved at the target site and the nucleic acid comprising the nucleotide sequence encoding HLA-E is inserted into the TXNIP locus, thereby disrupting the TXNIP gene.

[0447] In another method, namely method 12A, this disclosure provides a method as provided in method 12, wherein the nucleic acid is inserted into the TXNIP locus within 50 base pairs at the target site.

[0448] In another method, namely method 13, this disclosure provides a method as provided in method 12, wherein the RNA-guided nuclease of the second RNP complex is a Cas9 nuclease, and the gRNA of the second RNP complex contains a spacer subsequence corresponding to the target sequence consisting of SEQ ID NO:20.

[0449] In another method, namely method 14, this disclosure provides a method as provided in method 13, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0450] In another method, namely method 15, this disclosure provides a method as provided in method 12, wherein the second RNP comprises a 3:1 molar ratio of gRNA to RNA-guided nuclease.

[0451] In another method, namely method 16, this disclosure provides the method as provided in method 12, wherein the nucleotide sequence of (c)(2)(i) is substantially composed of SEQ ID NO:25, and the nucleotide sequence of (c)(2)(iii) is substantially composed of SEQ ID NO:32.

[0452] In another method, namely method 17, this disclosure provides a method as provided in method 12, wherein the nucleotide sequence encoding HLA-E includes a sequence encoding an HLA-E trimer, the HLA-E trimer including a B2M signal peptide fused to an HLA-G presenting peptide, the HLA-G presenting peptide being fused to a B2M membrane protein, the B2M membrane protein being fused to HLA-E without its signal peptide.

[0453] In another method, namely method 18, this disclosure provides a method as provided in method 17, wherein the sequence encoding the HLA-E trimer consists substantially of SEQ ID NO:55.

[0454] In another method, namely method 19, this disclosure provides a method as provided in method 12, wherein the nucleotide sequence encoding HLA-E is operatively linked to the CAG promoter.

[0455] In another method, namely method 20, this disclosure provides a method as provided in method 12, wherein the second vector is a plasmid vector and contains a nucleotide sequence consisting of SEQ ID NO: 34 or 56.

[0456] In another method, namely method 21, this disclosure provides a method as provided in method 12, wherein the delivery in (c)(1) and (c)(2) includes electroporation.

[0457] In another method, namely method 22, this disclosure provides a method as provided in method claim 1, wherein the enrichment of PD-L1, HLA-E double-positive cells in (d) includes magnetic-assisted cell sorting, single-cell cloning, amplification of the PD-L1, HLA-E double-positive cells or a combination thereof.

[0458] In another method, namely method 23, this disclosure provides a method as provided in method 1, wherein the single-cell sorting in (e) includes fluorescence activated cell sorting (FACS), single-cell cloning, amplification of the single-cell sorted cells, or a combination thereof.

[0459] In another method, namely method 24, this disclosure provides a method as provided in method 1, wherein the characterization in (f) includes DNA analysis of conjugation and / or insertion / deletion profiles.

[0460] In another method, namely method 25, this disclosure provides a method as provided in method 1, wherein the characterization in (f) includes cell analysis of morphology, viability, karyotype analysis, endotoxin level, mycoplasma level, on-target / off-target analysis, random vector insertion, residual Cas9, residual vector, pluripotency status, differentiation capacity, or combinations thereof.

[0461] In another method, namely method 26, this disclosure provides a method as provided in method 1, wherein the method further includes freezing prior to the characterization in (f).

[0462] In another method, namely method 27, this disclosure provides a method as provided in method 1, which further includes amplifying the generated PD-L1 positive cells in (a), amplifying the generated PD-L1 and HLA-E double positive cells in (c), and amplifying selected PD-L1 and HLA-E double positive cells or combinations thereof in (e).

[0463] In another method, namely method 28, this disclosure provides a method for generating universal donor cells, the method comprising: (a) modifying stem cells by inserting a nucleotide sequence encoding a first tolerance factor into or near a gene encoding a component or transcriptional regulatory factor of the MHC-I or MHC-II human leukocyte antigen or the MHC-I or MHC-II complex, thereby generating first tolerance factor positive cells; (b) enriching the first tolerance factor positive cells; (c) modifying the first tolerance factor positive cells from (b) by inserting a nucleotide sequence encoding a second tolerance factor into or near a gene encoding a survival factor, thereby generating first tolerance factor positive / second tolerance factor positive cells; (d) enriching the first tolerance factor positive / second tolerance factor positive cells; (e) single-cell sorting to select first tolerance factor positive / second tolerance factor positive cells; (f) characterizing these cells from (e) as universal donor cells; and (g) freezing these universal donor cells for long-term storage.

[0464] In another method, namely method 29, this disclosure provides a method as provided in method 28, wherein the enrichment of first tolerance factor positive cells in (b) includes magnetic assist cell sorting (MACS), single-cell cloning, expansion of first tolerance factor positive cells, or a combination thereof.

[0465] In another method, namely method 30, this disclosure provides a method as provided in method 28 or 29, wherein the enrichment of first tolerance factor positive / second tolerance factor positive cells in (d) includes magnetic-assisted cell sorting, single-cell cloning, expansion of first tolerance factor positive / second tolerance factor positive cells or a combination thereof.

[0466] In another method, namely method 31, this disclosure provides a method as provided in any one of methods 28 to 30, the method further comprising (a) amplifying the resulting first tolerance factor positive cells, (c) amplifying the resulting first tolerance factor positive / second tolerance factor positive cells, and (e) amplifying selected first tolerance factor positive / second tolerance factor positive cells, or a combination thereof.

[0467] In another method, namely method 32, this disclosure provides a method as provided in any one of methods 28 to 31, wherein the modification in (a) comprises delivering to these stem cells (1) a first RNA-guided nuclease and a first guide RNA (gRNA) targeting a target site in a component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or the MHC-I or MHC-II complex locus and (2) a first vector containing a first nucleic acid comprising (i) a nucleotide sequence homologous to a region to the left of the target site in a component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or the MHC-I or MHC-II complex locus, (ii) the nucleotide sequence encoding the first tolerogenic factor and (iii) A nucleotide sequence homologous to the region to the right of the target site in a component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or MHC-I or MHC-II complex locus, wherein the component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or MHC-I or MHC-II complex locus is cleaved at the target site, and the first nucleic acid containing the nucleotide sequence encoding the first tolerogenic factor is inserted into a component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or MHC-I or MHC-II complex locus, thereby disrupting the component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or MHC-I or MHC-II complex gene.

[0468] In another method, namely method 32A, this disclosure provides a method as provided in method 32, wherein the nucleic acid is inserted into a component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or the MHC-I or MHC-II complex locus within 50 base pairs at the target site.

[0469] In another method, namely method 33, this disclosure provides a method as provided in method 32, wherein the first RNA-guided nuclease and the first gRNA form a first ribonucleoprotein (RNP) complex.

[0470] In another method, namely method 34, this disclosure provides a method as provided in any one of methods 28 to 33, wherein the modification in (a) comprises delivering to these stem cells (1) a first ribonucleoprotein (RNP) complex comprising a first guide RNA (gRNA) containing a first RNA-guided nuclease and a target site in a component or transcriptional regulator of a MHC-I or MHC-II human leukocyte antigen or MHC-I or MHC-II complex locus and (2) a first vector containing a first nucleic acid comprising (i) a nucleotide sequence homologous to a region to the left of the target site in a component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or MHC-I or MHC-II complex locus and (ii) the nucleus encoding the first tolerogenic factor. The nucleotide sequence and (iii) a nucleotide sequence homologous to the region to the right of the target site in a component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or MHC-I or MHC-II complex locus, wherein the component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or MHC-I or MHC-II complex locus is cleaved at the target site, and the first nucleic acid containing the nucleotide sequence encoding the first tolerogenic factor is inserted into a component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or MHC-I or MHC-II complex locus, thereby disrupting the component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or MHC-I or MHC-II complex gene.

[0471] In another method, namely method 34A, this disclosure provides a method as provided in method 34, wherein the nucleic acid is inserted into a component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or the MHC-I or MHC-II complex locus within 50 base pairs at the target site.

[0472] In another method, namely method 35, this disclosure provides a method as provided in any one of methods 28 to 34, wherein the component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or MHC-I or MHC-II complex gene is HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, B2M, NLRC5, CIITA, RFX5, RFXAP, or RFXANK.

[0473] In another method, namely method 36, this disclosure provides a method as provided in any one of methods 28 to 35, wherein the component or transcriptional regulator of the MHC-I or MHC-II human leukocyte antigen or MHC-I or MHC-II complex gene is B2M.

[0474] In another method, namely method 37, this disclosure provides a method as provided in method 36, wherein the nucleotide sequence of (a)(2)(i) is substantially composed of SEQ ID NO:7, and the nucleotide sequence of (a)(2)(iii) is substantially composed of SEQ ID NO:13.

[0475] In another method, namely method 38, this disclosure provides a method as provided in method 36 or 37, wherein the first gRNA contains a spacer subsequence corresponding to the target sequence consisting of SEQ ID NO:2.

[0476] In another method, namely method 39, this disclosure provides a method as provided in any one of methods 32 to 38, wherein the first RNA-guided nuclease is a Cas9 nuclease.

[0477] In another method, namely method 40, this disclosure provides a method as provided in method 39, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0478] In another method, namely method 41, this disclosure provides a method as provided in any one of methods 32 to 40, wherein the first RNP comprises a 3:1 molar ratio of first gRNA to first RNA-guided nuclease.

[0479] In another method, namely method 42, this disclosure provides a method as provided in any one of methods 28 to 41, wherein the first tolerability-inducing factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

[0480] In another method, namely method 43, this disclosure provides a method as provided in any one of methods 28 to 42, wherein the nucleotide sequence encoding the first tolerogenic factor is operatively linked to a foreign promoter.

[0481] In another approach, namely approach 44, this disclosure provides a method as provided in approach 43, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0482] In another method, namely method 45, this disclosure provides a method as provided in any one of methods 28 to 44, wherein the first tolerability factor is PD-L1.

[0483] In another method, namely method 46, this disclosure provides a method as provided in method 45, wherein the nucleotide sequence encoding PD-L1 consists substantially of SEQ ID NO:11.

[0484] In another method, namely method 47, this disclosure provides a method as provided in method 46, wherein the nucleotide sequence encoding PD-L1 is operatively linked to the CAG promoter.

[0485] In another method, namely method 48, this disclosure provides a method as provided in any one of methods 45 to 47, wherein the first vector comprises a nucleotide sequence consisting of SEQ ID NO:33.

[0486] In another method, namely method 49, this disclosure provides a method as provided in any one of methods 28 to 48, wherein the modification in (c) comprises delivering to these stem cells (1) a second RNA-guided nuclease and a second guide RNA (gRNA) targeting a target site in a survival factor locus and (2) a second vector containing a second nucleic acid comprising (i) a nucleotide sequence homologous to a region to the left of the target site in the survival factor locus, (ii) the nucleotide sequence encoding the second tolerance factor and (iii) a nucleotide sequence homologous to a region to the right of the target site in the survival factor locus, wherein the survival factor locus is cleaved at the target site and the second nucleic acid containing the nucleotide sequence encoding the second tolerance factor is inserted into the survival factor locus to thereby disrupt the survival factor gene.

[0487] In another method, namely method 49A, this disclosure provides a method as provided in method 49, wherein the nucleic acid is inserted into the survival factor locus within 50 base pairs at the target site.

[0488] In another method, namely method 50, this disclosure provides a method as provided in method 49, wherein the second RNA-guided nuclease and the second gRNA form a second ribonucleoprotein (RNP) complex.

[0489] In another method, namely method 51, this disclosure provides a method as provided in any one of methods 28 to 48, wherein the modification in (c) comprises delivering to these first tolerance factor positive cells a second ribonucleoprotein (RNP) complex comprising a second RNA-guided nuclease and a second guide RNA (gRNA) targeting a target site in a survival factor locus, and (2) a second vector comprising a second nucleic acid comprising (i) a nucleotide sequence homologous to a region to the left of the target site in the survival factor locus, (ii) a nucleotide sequence encoding the second tolerance factor, and (iii) a nucleotide sequence homologous to a region to the right of the target site in the second survival factor locus, wherein the survival factor locus is cleaved at the target site, and the second nucleic acid comprising the nucleotide sequence encoding the second tolerance factor is inserted into the survival factor locus to thereby disrupt the survival factor gene.

[0490] In another method, namely method 51A, this disclosure provides a method as provided in method 51, wherein the nucleic acid is inserted into the survival factor locus within 50 base pairs at the target site.

[0491] In another method, namely method 52, this disclosure provides a method as provided in any one of methods 28 to 51, wherein the surviving gene is TXNIP, ZNF143, FOXO1, JNK, or MANF.

[0492] In another method, namely method 53, this disclosure provides a method as provided in method 52, wherein the surviving gene is TXNIP.

[0493] In another method, namely method 54, this disclosure provides a method as provided in method 53, wherein the second gRNA comprises a spacer subsequence corresponding to the target sequence consisting of SEQ ID NO:20.

[0494] In another method, namely method 55, this disclosure provides a method as provided in method 52 or 53, wherein the nucleotide sequence of (c)(2)(i) is substantially composed of SEQ ID NO:25, and the nucleotide sequence of (c)(2)(iii) is substantially composed of SEQ ID NO:32.

[0495] In another method, namely method 56, this disclosure provides a method as provided in any one of methods 49 to 55, wherein the second RNA-guided nuclease is a Cas9 nuclease.

[0496] In another method, namely method 57, this disclosure provides a method as provided in method 56, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

[0497] In another method, namely method 58, this disclosure provides a method as provided in any one of methods 49 to 57, wherein the second RNP comprises a 3:1 molar ratio of second gRNA to second RNA-guided nuclease.

[0498] In another method, namely method 59, this disclosure provides a method as provided in any one of methods 49 to 58, wherein the second tolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47.

[0499] In another method, namely method 60, this disclosure provides a method as provided in any one of methods 28 to 59, wherein the nucleotide sequence encoding the second tolerogenic factor is operatively linked to an exogenous promoter.

[0500] In another approach, namely approach 61, this disclosure provides a method as provided in approach 60, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0501] In another method, namely method 62, this disclosure provides a method as provided in any one of methods 28 to 61, wherein the second tolerability-inducing factor is HLA-E.

[0502] In another method, namely method 63, this disclosure provides a method as provided in method 62, wherein the nucleotide sequence encoding HLA-E includes a sequence encoding an HLA-E trimer, the HLA-E trimer including a B2M signal peptide fused to an HLA-G presenting peptide, the HLA-G presenting peptide being fused to a B2M membrane protein, the B2M membrane protein being fused to HLA-E without its signal peptide.

[0503] In another method, namely method 64, this disclosure provides a method as provided in method 63, wherein the sequence encoding the HLA-E trimer consists substantially of SEQ ID NO:55.

[0504] In another method, namely method 65, this disclosure provides a method as provided in method 63 or 64, wherein the nucleotide sequence encoding HLA-E is operatively linked to the CAG promoter.

[0505] In another method, namely method 66, this disclosure provides a method as provided in any one of methods 62 to 65, wherein the second vector comprises a nucleotide sequence consisting of SEQ ID NO: 34 or 56.

[0506] In another method, namely method 67, this disclosure provides a method as provided in any one of methods 28 to 66, wherein single-cell sorting in (e) includes fluorescence-activated cell sorting (FACS), single-cell cloning, amplification of said single-cell sorted cells, or a combination thereof.

[0507] In another method, namely method 68, this disclosure provides a method as provided in any one of methods 28 to 67, wherein the characterization in (f) includes DNA analysis of conjugation and / or insertion / deletion profiles.

[0508] In another method, namely method 69, this disclosure provides a method as provided in any one of methods 28 to 68, wherein the characterization in (f) includes cell analysis of morphology, viability, karyotype analysis, endotoxin level, mycoplasma level, on-target / off-target analysis, random vector insertion, residual Cas9, residual vector, pluripotency status, differentiation capacity, or combinations thereof.

[0509] In another method, namely method 70, this disclosure provides a method as provided in any one of methods 28 to 69, wherein the method further includes freezing prior to the characterization in (f).

[0510] VII. Examples

[0511] The following examples illustrate the generation and characterization of specific generic donor cells according to this disclosure.

[0512] Example 1: Cell Maintenance and Expansion

[0513] Maintenance of hESC / hiPSC. As described by Schulz et al. (2012) PLoS ONE [PLOS ONE] 7(5):e37004, the human embryonic stem cell line CyT49 (a proprietary hES cell line, ViaCyte, Inc., San Diego, CA) was maintained, cultured, passaged, proliferated, and plated. CyT49 cells were then used… (Stemcell Technologies 07920 or equivalent) dissociation.

[0514] Human induced pluripotent stem cells (hiPSCs) such as the TC1133 cell line (Lonza) were maintained on StemFlex Complete (Life Technologies, A3349401) in tissue culture plates coated with BIOLAMININ 521CTG (BioLamina, catalog number CT521). The plates were pre-coated for 2 hours at 37°C with a 1:10 or 1:20 dilution of BIOLAMININ in DPBS, calcium, and magnesium (Life Technologies, 14040133). Cells were fed daily with StemFlex medium. For cell passage, the same cell density as CyT49 was used. To plate cells as single cells, cells were plated in 1% RevitaCell medium in StemFlex. TM The supplement (100X) (Thermofisher, catalog number A2644501) was plated on a BIOLAMININ-coated plate.

[0515] Single-cell clones of hPSCs. For single-cell clones, in the use of... 3-4 hours before dissociation, hPSCs (hESCs or hiPSCs) were fed StemFlex Complete with Revitacell (final concentration 1X Revitacell). After dissociation, cells were sorted into single cells in each well of a 96-well tissue culture plate coated with Biolamin. Single cells were sorted into wells using a WOLF FACS sorter (Nanocellect). The plate was pre-filled with 100-200 μL of StemFlex Complete with Revitacell. Three days after cell seeding, cells were fed with fresh StemFlex and continued to be fed with 100-200 μL of culture medium every other day. After 10 days of growth, cells were fed with StemFlex daily until day 12-14. At this point, the plate was... The collected cell suspension was dissociated and split 1:2, with half entering a new 96-well plate for maintenance and the other half entering the DNA extraction solution QuickExtract. TM DNA extraction solution (Lucigen). After DNA extraction, PCR was performed to assess the presence or absence of the desired gene editing at the target DNA locus. Sanger sequencing was used to verify the desired editing.

[0516] Amplification of single-cell-derived hPSC clones. For CyT49 (Viastec), successfully targeted clones were passaged into 24-well plates with pure 10% XF KSR A10H10 medium, instead of BIOLAMININ-coated plates. After the 24-well stage, CyT49 clones were passaged as described in Schulz et al. (2012) PLoS ONE 7(5):e37004.

[0517] For hiPSC (TC1133), cells were maintained in StemFlex Complete throughout the entire cloning and periodic maintenance process on BIOLAMININ coated plates with Revitacells during the passage phase.

[0518] Example 2: Generation of B2M knockout (KO) human pluripotent stem cells (hPSCs)

[0519] Guide RNA (gRNA) selection for B2M in hPSCs. Three B2M-targeting gRNAs were designed to target exon 1 of the B2M coding sequence. Based on sequence homology predictions using gRNA design software, these gRNAs had predicted low off-target scores. The target sequences of the gRNAs are presented in Table 1. The gRNAs contain RNA sequences corresponding to the target DNA sequences.

[0520] Table 1. B2M gRNA target sequences

[0521]

[0522] To assess their cleavage efficiency in hPSCs, CyT49 cells (Viastec proprietary hES cell line) were electroporated using a Neon electroporator (Neon transfection system, Thermo Fisher Scientific, catalog number MPK5000) with a ribonucleoprotein (RNP) mixture (125 pmol Cas9 and 375 pmol gRNA) at a molar ratio of Cas9 protein (Biomay) and guide RNA (Synthego) (see gRNA sequences in Table 3). To form the RNP complex, gRNA and Cas9 were combined with R-buffer (Neon transfection system 100 μL kit, Thermo Fisher Scientific, catalog number MPK10096) to a total volume of 25 μL and incubated at room temperature for 15 min. Cells were dissociated and resuspended in DMEM / F12 medium (Gibco, catalog number 11320033), counted, and centrifuged using an NC-200 (Chemometec). A total of 1×10⁶ cells were resuspended in RNP complex. 6 Cells were collected and R-buffer was added to a total volume of 125 μL. The mixture was then electroporated at 1100 V for 30 ms with two pulses. After electroporation, cells were aspirated into Eppendorf tubes filled with StemFlex medium containing RevitaCells. The cell suspension was then plated into tissue culture dishes pre-coated with BIOLAMININ 521CTG at a 1:20 dilution. Cells were incubated in a normoxic incubator (37°C, 8% CO2) for 48 hours. After 48 hours, genomic DNA was harvested from the cells using QuickExtract (Luxigan, Middleton, Wisconsin; catalog number QE09050).

[0523] PCR targeting the B2M sequence was performed, and the cleavage efficiency of the amplified DNA was evaluated by TIDE analysis. PCR of the relevant regions was performed using Platinum Taq Supermix (Invitrogen, catalog numbers 125320176 and 11495017). The sequences of the PCR primers are presented in Table 2; and the cycling conditions are provided in Table 3.

[0524] Table 2. B2M TIDE primers

[0525]

[0526] Table 3. B2M PCR Cycling Parameters

[0527]

[0528] The resulting amplicon was submitted for PCR cleansing and Sanger sequencing. The Sanger sequencing results, along with the guide sequence, were input into Tsunami software. The software calculated the insertion percentage and identity. Then, specific gRNAs were selected based on their insertion / deletion frequencies in hPSCs. Figure 1 The cleavage efficiency of B2M-1, B2M-2, and B2M-3 gRNAs is shown.

[0529] Off-target effects of selected gRNAs in stem cell-derived DNA were assessed using hybridization capture analysis of sequence similarity prediction sites. Neither the B2M-2 nor the B2M-3 guides showed detectable off-target effects. The B2M-2 gRNA was chosen for further cloning due to its high target activity and undetectable off-target activity.

[0530] Generation and characterization of B2M KO hPSC clones. CyT49 hESCs (Viastec) were electroporated using B2M-2gRNA, and single cells were sorted into 96-well plates coated with BIOLAMININ 521CTG containing StemFlex and Revitacells 3 days after electroporation using a WOLF FACS sorter (NanoSelex). The plated single cells were grown in a normoxic incubator (37°C, 8% CO2) with the medium changed every other day until colonies were large enough to be reseeded as single cells. Upon confluence, samples were separated for maintenance and genomic DNA extraction.

[0531] The B2M KO status of the clones was confirmed by PCR and Sanger sequencing. The resulting DNA sequences targeting the B2M region were aligned in Snapgene software to determine insertion / deletion identity and conjugation. Clones with the desired edits were amplified and further validated by flow cytometry assessment of B2M expression (see Table 4 for a list of antibodies used). Clones were evaluated with and without interferon-γ treatment (25 ng / mL, R&D Systems, 285-IF). Figure 2A B2M expression was shown in wild-type cells, and Figure 2B B2M expression in B2M KO cells was presented. Karyotype status of clones was evaluated via Cell Line Genetics Service (Madison, Wisconsin), and normal karyotypes were reported.

[0532] Table 4. Antibodies used in pluripotent flow cytometry

[0533]

[0534] Intracellular flow cytometry targeting the pluripotency markers OCT4 and SOX2 confirmed that the clones retained pluripotency. The confirmed pluripotent clones were differentiated into pancreatic endocrine progenitor cells using a previously established method (Schulz et al. (2012) PLoS ONE [PLOS ONE] 7(5):e37004).

[0535] Example 3: Generation of B2M KO / PD-L1 knock-in (KI) human pluripotent stem cells (hPSCs)

[0536] Design of the B2M KO / PD-L1 KI strategy. Plasmids were designed to insert PD-L1 (CD274) into the B2M locus, such that the start codon of B2M is removed after homology-directed repair (HDR) to insert PD-L1, thereby invalidating any chance of partial B2M expression. Figure 3A schematic diagram of the plasmid is presented, and Table 5 identifies the elements and their locations. The donor plasmid contains PD-L1 cDNA driven by the CAGGS promoter, flanked by 800-base-pair homologous arms that share the same sequence as the B2M locus surrounding exon 1. The complete sequence of this plasmid is contained in SEQ ID NO:33.

[0537] Table 5. Elements of the B2M-CAGGS-PD-L1 donor plasmid

[0538] element Location (size in bp) SEQ ID NO: Left ITR 1-130(130) 6 LHA-B2M 145-944(800) 7 CMV Enhancer 973-1352(380) 8 Chicken β-actin promoter 1355-1630(276) 9 chimeric intron 1631-2639(1009) 10 PD-L1 2684-3556(873) 11 bGH (A) signal 3574-3798(225) 12 RHA-B2M 3805-4604(800) 13 Right ITR 4646-4786(141) 14 intact plasmid 7133bp 33

[0539] B2M-2 gRNA was used to promote the insertion of the PD-L1 transgene at the targeted B2M locus. The PD-L1 donor plasmid was introduced along with an RNP complex consisting of the B2M-targeting gRNA and the Cas9 protein. 4 μg of plasmid DNA was delivered with the RNP per million CyT49 cells (Viastec). Electroporation was performed as described in Example 2. Seven days after electroporation, cells expressing PD-L1 were sorted into 96-well plates coated with BIOLAMININ 521CTG containing StemFlex and Revitacell using a WOLF FACS sorter (NanoSelex). Unedited cells served as a negative control for FACS sorting. PD-L1-positive cells were selected for sorting and single-cell cloning.

[0540] To detect PD-L1 surface expression, an anti-PD-L1 fluorescent antibody was used (see Table 4). Plated single cells were grown in a normoxic incubator (37°C, 8% CO2), with the culture medium changed every other day until colonies were large enough to be re-seeded as single cells. Upon confluence, samples were separated for maintenance and genomic DNA extraction.

[0541] The following primers were used to identify correctly targeted clones via PCR targeting PD-L1 knock-in (KI) insertion. These primers amplified from the outer plasmid homologous arm to the region where the PD-L1 cDNA was inserted, allowing for the amplification of only the KI-integrated DNA. The conjugation of the target insertion was tested by PCR to assess whether KI occurred heterozygous or homozygous. If a heterozygous clone was identified, the KI-negative allele was sent for Sanger sequencing to verify that it contained a B2M-disrupting insertion / deletion in the non-KI allele. Correct KI clones with complete B2M disruption (formed via KI insertion or deletion) were amplified in incremental tissue cultures until a population size of 30 million cells was reached. Approximately 10 clones were amplified in this manner, and their pluripotency was confirmed by intracellular flow cytometry testing for OCT4 and SOX2. Figure 4The clones that passed the above tests were then further tested for karyotype analysis (Cell Line Genetics), as described below. Additionally, the ability of the clones to differentiate into pancreatic endoderm precursors (PECs) was then tested using an established protocol (Schulz et al. (2012) PLoS ONE [PLOS ONE] 7(5):e37004), as described below. The loss of B2M was further confirmed by flow cytometry through the absence of B2M expression with or without interferon-γ treatment (25 ng / mL, R&D Systems, 285-IF). Figure 5A and 5B PD-L1 expression in wild-type and B2M KO / PD-L1 KI cells is shown, respectively.

[0542] Example 4: Karyotype analysis of edited clones

[0543] G-banding karyotype analysis of edited embryonic stem (ES) cells. One million edited ES cells were passaged into T-25 culture flasks containing DMEM / F12 with 10 ng / mL activin and 10 ng / mL modulin, plus 10% Xeno-free KSR. After overnight culture, three T25 culture flasks were transported to the Cytogenetics Laboratory (Cell Line Genetics, Inc.) for karyotype analysis; FISH analysis of chromosomes 1, 12, 17, and 20; and array comparative genomic hybridization (aCGH) analysis using a standard 8x60K array. G-banding results of selected cells electroporated using the non-cutting guide (“NCG”), B2M KO clone, and B2M KO / PD-L1 KI clone (“V1-A”) are shown in Table 6.

[0544] Table 6. Results of G-band karyotype analysis

[0545]

[0546]

[0547] Example 5: Differentiation of edited human embryonic stem cells into pancreatic endoderm cells (PEC)

[0548] Maintenance of edited human embryonic stem cells (ES). Edited human embryonic stem cells from different passages (P38-42) were passaged for 4 days at a rate of 33,000 cells / cm³. 2 Inoculation, or passage at 50,000 cells / cm for 3 days. 2Inoculation was performed using hESM medium (DMEM / F12 + 10% KSR + 10 ng / mL activin A and 10 ng / mL cytokinin) and finally 10% human AB serum.

[0549] The aggregation of edited human embryonic stem cells for PEC differentiation. Edited ES cells were dissociated into single cells, centrifuged, and resuspended at 1 million cells / ml in 2% StemPro (catalog number A1000701, Ingenium Inc., California) in DMEM / F12 medium. A total of 350-400 million cells were then rotated at 8 RPM ± 0.5 RPM in an 850 cm⁻¹ tank. 2 Inoculated in roller bottles (catalog number 431198, Corning, NY) for 18–20 hours, followed by differentiation. As described by Schulz et al. (2012) PLoS ONE 7(5):e37004, roller bottles were used to differentiate ES aggregates from edited human embryonic stem cells into pancreatic lineages.

[0550] Example 6: Characterization of differentiated pancreatic endoderm cells (PEC)

[0551] Flow cytometry was performed on FOXA2 and SOX17 in phase 1 (DE) and CHGA, PDX1, and NKX6.1 in phase PEC. hESC-derived phase 1 aggregates or hESC-derived pancreatic aggregates were washed with PBS and then analyzed using ACCUMAX. TM(Catalogue No. A7089, Sigma, Missouri) The cells were enzymatically dissociated into a single-cell suspension at 37°C. MACS separation buffer (Catalogue No. 130-091-221, Miltenyi Biotec, North Rhine-Westphalia, Germany) was added, and the suspension was passed through a 40 μm filter and precipitated. For intracellular marker staining, cells were fixed in 4% (wt / v) paraformaldehyde for 30 min, washed in FACS buffer (PBS, 0.1% (wt / v) BSA, 0.1% (wt / v) NaN3), and then permeabilized on ice for 30 min with Perm buffer (PBS, 0.2% (v / v) Triton X-100 (catalog number A16046, Alfa Aesar, Massachusetts), 5% (v / v) normal donkey serum, 0.1% (wt / v) NaN3), followed by washing with wash buffer (PBS, 1% (wt / v) BSA, 0.1% (wt / v) NaN3). Cells were incubated overnight at 4°C with primary antibody diluted with blocking buffer (PBS, 0.1% (v / v) Triton X-100, 5% (v / v) normal donkey serum, 0.1% (wt / v) NaN3) (Table 7). Cells were washed in IC50 buffer and then incubated with an appropriate secondary antibody at 4°C for 60 min. Cells were washed again in IC50 buffer and then in FACS buffer. Flow cytometry data were acquired using a NovoCyte flow cytometer (ACEA Biosciences, Brussels). Data were analyzed using FlowJo software (Tree Star, Inc.). Intact cells were identified based on forward (low angle) and lateral (orthogonal, 90°) light scattering. Background was estimated using antibody controls and undifferentiated cells. The figure shows a representative flow cytometry plot for one of the subpopulations. The numbers reported in the figure represent the percentage of total cells from the intact cell phylum.

[0552] Table 7. Antibodies used for flow cytometry characterization of differentiated PECs

[0553]

[0554] During the DE phase, the population of FOXA2 and SOX17 double-positive cells exceeded 90% of the total cells derived from CyT49 wild-type differentiated cells. Compared to wild-type cells, PD-L1 KI / B2M KO and B2M KO cells showed a comparable percentage of DE. Figure 6 and Figure 7 ).

[0555] During the PEC phase, flow cytometry targeting chromogranin (CHGA), PDX1, and NKX6.1 was performed. The heterogeneous population at the PEC phase included pancreatic progenitor cells and early endocrine cells (…). Figure 8 Based on the pie chart of heterogeneous populations ( Figure 9 The distribution of cell populations derived from differentiated edited cells (PD-L1 KI / B2M KO or B2M KO) is very similar to that of wild-type cells.

[0556] Targeted RNA-seq. Targeted RNA-seq for gene expression analysis was performed using Illumina TruSeq and a custom panel of oligonucleotides targeting 111 genes. This panel primarily contained genes that served as markers of developmental stages during pancreatic differentiation. At the end of each differentiation stage, 10 μL of APV (aggregated precipitate volume) was collected and extracted using a Qiagen RNeasy or RNeasy 96 spin column protocol (including on-column DNase treatment). Quantification and quality control were performed using a TapeStation with Qubit binding, or by using Qiagen QIAxcel. 50–200 ng of RNA was processed according to the Illumina TruSeq library preparation protocol, which consists of cDNA synthesis, hybridization to the custom oligonucleotide pool, washing, extension, ligation of bound oligonucleotides, PCR amplification of the library, and library cleansing; the resulting dsDNA library was then quantified and quality controlled using a TapeStation with Qubit binding, or by using Qiagen QIAxcel. The library was then diluted to 4 nM and merged, followed by denaturation, incorporation of a PhiX control, and further dilution to 10⁻¹² pM before loading onto an Illumina MiSeq sequencer. After sequencing run, initial data analysis was automatically performed using BaseSpace to generate raw read counts for each custom probe. For each gene, these read counts for all probes corresponding to that gene were summed, and one read count was added (to prevent downstream portions from reaching zero). Normalization was performed for the SF3B2 gene, and reads were typically visualized as fold changes compared to stage 0. When processing the data for principal component analysis, DEseq methods were used for normalization.

[0557] The expression of the selected genes, such as Figure 10 As shown, the dynamic expression patterns of FOXA2, CHGA, PDX1, and NKX6.1 from PD-L1 KI / B2MKO or B2M KO cells are similar to those in wild-type cells.

[0558] Confirmation of B2M and PD-L1 expression during the PEC phase. During the PEC phase, differentiated aggregates were treated for 48 hours with or without interferon-γ (50 ng / ml). Aggregates were washed with PBS and then subjected to ACCUMAX. TM (Catalogue No. A7089, Sigma-Aldrich, Missouri) Cells were enzymatically dissociated into a single-cell suspension at 37°C. MACS separation buffer (Catalogue No. 130-091-221, Miltenyi Biosciences, North Rhine-Westphalia, Germany) was added, and the suspension was passed through a 40 μm filter and precipitated. For surface marker staining, the dissociated cells were incubated with fluorescently conjugated antibody diluted in MACS separation buffer for 20 min, followed by washing in MACS separation buffer. Cells were resuspended in FACS buffer for flow cytometry acquisition. Flow cytometry data were acquired using a NovoCyte flow cytometer. Figure 11A-11F As shown, B2M expression is derived from B2M KO ( Figure 11B ) or PD-L1 KI / B2M KO( Figure 11C The differentiation of PD-L1 in PEC was below the detection limit, and PD-L1 was derived from PD-L1 KI / B2M KO ( Figure 11F PD-L1 is expressed in differentiated PECs. Typically, over 90% of PECs express PD-L1, indicating a homogeneous population of cells. Typically, transgene expression is lost over time after differentiation of gene-edited stem cells (Hong et al., Mol. Ther. [Molecular Therapy], 2017, 25(1):44-53).

[0559] Immunophenotyping of PEC cells. Differentiation aggregates were treated for 48 hours with or without interferon-γ (50 ng / ml) during the PEC phase. Aggregates were harvested for MHC class I and II staining. No MHC class II expression was observed from wild-type or edited cells (PD-L1 KI / B2M KO and B2M KO) during the PEC phase. Figure 12D-12F HLA-ABC (MHC class I) expression was low (1.3% in wild-type cells) and it was highly regulated upon IFN-γ stimulation. However, even under IFN-γ stimulation, HLA-ABC was not expressed in edited cells (PD-L1 KI / B2M KO and B2M KO). Figures 12A-12C ).

[0560] Example 7: Generation of TXNIP KO Human Pluripotent Stem Cells (hPSCs)

[0561] Guide RNA (gRNA) selection for TXNIP. Ten gRNAs targeting TXNIP were designed to target exons 1 and 2 of the TXNIP coding sequence (Table 8). The PAM sequences are presented in bold in the target sequences presented in Table 8, and the corresponding DNA sequences for the guide sequences are also presented in Table 8. Based on sequence homology predictions using gRNA design software, these gRNAs had predicted low off-target scores.

[0562] Table 8. Selected TXNIP target sequences and gRNA sequences

[0563]

[0564] Generation and characterization of TXNIP KO hiPSC clones. To assess the cleavage efficiency of these gRNAs in hiPSCs, TC1133 hiPSC cells were electroporated using a Neon electroporator (Neon transfection system, Thermo Fisher Scientific, catalog number MPK5000) with an RNP mixture (125 pmol Cas9 and 375 pmol gRNA) at a molar ratio of Cas9 protein (Bio-European) and guide RNA (Synthego). To form the RNP complex, gRNA and Cas9 were combined with R-buffer to a total volume of 25 μL and incubated at room temperature for 15 min. Cells were dissociated and resuspended in DMEM / F12 medium (Gibco, catalog number 11320033), counted, and centrifuged using an NC-200 (Krommet). A total of 1 × 10⁻⁶ cells were resuspended in RNP complex. 6 RevitaCells were collected, and R-buffer was added to a total volume of 125 μL. The mixture was then electroporated using the following parameters: 2 pulses, 30 ms, 1100 V. After electroporation, cells were aspirated into Eppendorf tubes filled with StemFlex medium containing RevitaCells. The cell suspension was then plated into tissue culture dishes pre-coated with BIOLAMININ 521CTG. Cells were incubated in a normoxic incubator (37°C, 8% CO2) for 48 hours. After 48 hours, genomic DNA was harvested from the cells using QuickExtract.

[0565] PCR targeting the TXNIP sequence was performed, and the amplified DNA was sequenced using Sanger sequencing. TIDE analysis was used to analyze the insertion percentage of the output sequencing data using Tsunami software. Figure 13 The cleavage efficiency of TXNIP gRNAs is shown. gRNAs were then selected based on their insertion / deletion frequencies in hPSCs.

[0566] Off-target effects of the most efficient cleavage gRNAs in stem cell-derived DNA were assessed using hybridization capture analysis of sequence similarity predicted sites. Further experiments were conducted with TXNIP gRNA T5, as it showed no detectable off-target effects and demonstrated high on-target activity.

[0567] Generation and characterization of TXNIP KO hPSC clones. CyT49 hESCs (Viastec) were electroporated using TXNIP gRNA T5, and single cells were sorted into BIOLAMININ 521CTG 96-well plates equipped with StemFlex and Revitacells 3 days after electroporation using a WOLF FACS sorter (NanoSelex). The plated single cells were grown in a normoxic incubator (37°C, 8% CO2), with the medium changed every other day, until the colonies were large enough to be reseeded as single cells. Upon confluence, the samples were separated for maintenance and genomic DNA extraction.

[0568] The TXNIP KO status of the clones was confirmed by PCR and Sanger sequencing. The obtained DNA sequences of the target TXNIP region were aligned in Snapgene software to determine insert / delete identity and conjugation. Clones with the desired edits were amplified and further validated by flow cytometry assessment targeting TXNIP expression. The karyotype status of the clones was evaluated using Cell Line Genetics service, and normal karyotypes were reported (Table 9).

[0569] Table 9. Karyotype Analysis

[0570] cell lines Passing down Karyotype analysis FISH Analysis aCGH array analysis TXNIPKO#2 P31 normal normal pass TXNIPKO#13 P31 normal normal pass

[0571] Intracellular flow cytometry targeting the pluripotency markers OCT4 and SOX2 confirmed that the clones retained pluripotency. The confirmed pluripotent clones were differentiated into pancreatic endocrine progenitor cells using a previously established method (Schulz et al. (2012) PLoS ONE [PLOS ONE] 7(5):e37004).

[0572] Targeted RNA-seq for gene expression analysis was performed using Illumina TruSeq and a custom panel for oligonucleotides, as described above. The expression of the selected genes was as follows: Figure 20As shown. The kinetic expression patterns of FOXA2, CHGA, PDX1, and NKX6.1 from TXNIP KO cells were similar to those in wild-type cells. Flow cytometry targeting chromogranin (CHGA), PDX1, and NKX6.1 was also performed at the PEC stage. The heterogeneous population at the PEC stage included 30.6% pancreatic progenitor cells (i.e., CHGA). - / NKX6.1 + / PDX1 + ()( Figure 21 ).

[0573] Example 8: Generation of human pluripotent stem cells (hPSCs) from B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI

[0574] Design of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI strategies. Cells were generated in which the PD-L1 coding sequence was inserted into the B2M locus (thus knocking out the B2M gene) and the HLA-E coding sequence was inserted into the TXNIP locus (thus knocking out the TXNIP gene).

[0575] Example 3 describes the plasmid design for inserting PD-L1 (CD274) into the B2M locus. The donor plasmid contains PD-L1 cDNA driven by the CAGGS promoter, flanked by 800-base-pair homologous arms that share the same sequence as the B2M locus surrounding exon 1. B2M-2 gRNA was used to facilitate the insertion of the PD-L1 transgene into the targeted B2M locus. The PD-L1 donor plasmid was introduced along with an RNP complex consisting of the B2M-targeting gRNA and the Cas9 protein. 4 μg of plasmid DNA was delivered per million CyT49 cells (Viastec). Electroporation was performed as described in Example 2. Seven days after electroporation, Miltenyi reagent (anti-mouse IgG beads, catalog number 130-048-401; LS column, catalog number 130-042-401; and MidiMACS separator, catalog number 130-042-302) or Thermofisher reagent (DynaMed) was used. TM -15 magnet, catalog number 12301D; Cellection TM Pan mouse IgG kit, catalog number 11531D; Dynabeads TM Pan mouse IgG (catalog number 11042) enriched PD-L1 positive cells in cells via magnetic-assisted cell sorting (MACS).

[0576] Following amplification of the enriched PD-L1 positive population, HLA-E trimer cDNA transgenes were inserted into the TXNIP genomic locus via CRISPR-induced HDR using a donor plasmid containing the HLA-E sequence. The HLA-E trimer cDNA consists of a B2M signal peptide fused to an HLA-G presenting peptide, which is fused to a B2M membrane protein, which is fused to an HLA-E protein without its signal peptide. This trimer design has been previously published (Gornalusse et al. (2017) Nat. Biotechnol. [Nature Biotechnology] 35(8):765-772). The HLA-E trimer coding sequence (including the adapter) is SEQ ID NO:55 (i.e., SEQ ID NO:26-31). The donor plasmid used for HLA-E delivery contains the CAGGS promoter that drives HLA-E trimer expression, flanked by 800-base-pair homologous arms that share the same sequence as the TXNIP locus around exon 1. Figure 14 (See Tables 10 and 11). In some embodiments, the donor plasmid comprises SEQ ID NO: 34 or 56.

[0577] Table 10. Elements of TXNIP-CAGGS-HLA-E donor plasmid 1

[0578] element Location (size in bp) SEQ ID NO: Left ITR 1-130(130) 6 LHA-TXNIP 145-944(800) 25 CMV Enhancer 973-1352(380) 8 Chicken β-actin promoter 1355-1630(276) 9 chimeric intron 1631-2639(1009) 10 B2M signal sequence 2684-2743(60) 26 HLA-G peptide 2744-2770(27) 27 GS connector 2771-2815(45) 28 B2M membrane protein 2816-3112(297) 29 GS connector 3113-3172(60) 30 HLA-E 3173-4183(1011) 31 bGH (A) signal 4204-4428(225) 12 RHA-TXNIP 4435-5234(800) 32 Right ITR 5276-5416(141) 14 intact plasmid 7763bp 34

[0579] Table 11. Elements of TXNIP-CAGGS-HLA-E donor plasmid 2

[0580] element Location (size in bp) SEQ ID NO: Left ITR 1-130(130) 6 LHA-TXNIP 145-944(800) 25 CMV Enhancer 973-1352(380) 8 Chicken β-actin promoter 1355-1630(276) 9 Chimeric introns (truncated) 1631-2336(706) 57 B2M signal sequence 2381-2440(60) 26 HLA-G peptide 2441-2467(27) 27 GS connector 2468-2512(45) 28 B2M membrane protein 2513-2809(297) 29 GS connector 2810-2869(60) 30 HLA-E 2870-3880(1011) 31 bGH (A) signal 3901-4125(225) 12 RHA-TXNIP 4132-4931(800) 32 Right ITR 4973-5113(141) 14 intact plasmid 7460bp 56

[0581] TXNIP-T5 gRNA was used to promote the insertion of the HLA-E transgene at the targeted TXNIP locus. The HLA-E donor plasmid was introduced along with an RNP complex consisting of TXNIP-T5 gRNA and Cas9 protein. 4 μg of HLA-E plasmid DNA (SEQ ID NO: 56) was delivered with the RNP per million PD-L1+ cells. Alternatively, HLA-E donor plasmid DNA (SEQ ID NO: 34) could be used. Electroporation was performed as described in Example 2. Seven days after electroporation, HLA-E-positive cells were enriched via MACS using Miltenyi's reagent or Thermofisher's reagent. After HLA-E enrichment, single cells were sorted into 96-well plates coated with BIOLAMININ521CTG containing StemFlex and Revitacell using a WOLF FACS sorter (NanoSelex). Single cells plated were grown in a normoxic incubator (37°C, 8% CO2) with the medium changed every other day until colonies were large enough to be reseeded as single cells. Upon confluence, samples were separated for maintenance and genomic DNA extraction. Anti-PD-L1 and anti-HLA-E antibodies (Table 4) were used for MACS enrichment and FACS sorting into 96-well plates, with gating set for HLA-E and PD-L1 double-positive cells. Unedited cells served as negative controls for FACS sorting.

[0582] Correctly targeted clones were identified using PCR targeting PD-L1 KI insertion and HLA-E KI insertion, amplifying the region from the plasmid homologous arm to the PD-L1 cDNA insertion or HLA-E cDNA insertion, respectively, thus enabling the amplification of only the KI-integrated DNA. The conjugation of the target insertion was tested by PCR to assess whether KI occurred heterozygous or homozygous. If a heterozygous clone was identified, the KI-negative allele was sent for Sanger sequencing to verify that it contained either a B2M-disrupting insertion / deletion or a TXNIP-disrupting insertion / deletion, respectively. Correctly targeted KI clones with complete B2M and TXNIP disruption (formed via KI insertion or deletion) were amplified in incremental tissue cultures until a population size of 30 million cells was reached. Approximately 10 clones were amplified in this manner, and their pluripotency was confirmed by intracellular flow cytometry testing for OCT4 and SOX2. Figure 15 ).

[0583] The clones that passed the above tests were then further tested for karyotype analysis (Cell Line Genetics), as described above. The G-banding results of the selected B2M KO / PD-L1 KI+TXNIP KO / HLA-E KI (“V1-B”) clones are shown in Table 12. Additionally, the ability of the V1-B clones to differentiate into pancreatic endodermal precursors (PECs) was then tested.

[0584] Table 12. G-band Results

[0585] cell lines Passing down Karyotype analysis FISH Analysis aCGH array analysis V1-B003 P37 normal normal pass V1-B007 P37 normal normal pass V1-B008 P36 normal normal pass

[0586] According to a previously reported pancreatic endocrine protocol (Rezania et al. (2014) Nat. Biotechnol. [Nature Biotechnology] 32(11):1121-1133), PD-L1 and HLA-E continued to be expressed after differentiation into stage 6 cells. Figure 16 The population of differentiated cells is homogeneous in terms of transgene expression; for example, 94.4% of cells express PD-L1 and 97.0% of cells express HLA-E. Figure 22 A shows the similar morphology of various clones (“S6-V1B-H9”, “S6-V1B-3B11”, “S6-V1B-1G7”, and “S6-V1B-3C2”) differentiated to stage 6 compared to wild-type and uncut guide control cells. Selected gene expression in the B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones is shown in Figure 1. Figures 23A to 23F As shown, the kinetic expression patterns of INS, NKX6.1, GCK, GCG, and SST from B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones were similar to those in wild-type cells. Figure 23A ). INS (phase 6 markers) from various differentiated B2MKO / PD-L1 KI and TXNIP KO / HLA-EKI clones (“S6-V1B-H9”, “S6-V1B-3B11”, “S6-V1B-1G7”, and “S6-V1B-3C2”). Figure 23B ), NKX6.1 Figure 23C ), GCG Figure 23D ), SST Figure 23E ) and GCK Figure 23F The expression level of ) was similar to that in stage 6 wild-type cells and wild-type islets. Undifferentiated B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones (“ES-V1B-H9”) were used as negative controls.

[0587] Figures 24A-24B The expression of INS and GCG in stage 6 cells differentiated from B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones is shown. Figure 24A ) and INS and NKX6.1 expressions ( Figure 24B Flow cytometry evaluation. Figures 25A-25B The image shows INS expression in stage 6 cells differentiated from two B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI clones (“S6-V1B003” and “V1B-H9”). Figure 25A ) and NKX6.1 expression ( Figure 25B The percentage of expression in both was similar to that in wild-type and uncut guide control cells.

[0588] During the PEC phase, flow cytometry targeting chromogranin (CHGA), PDX1, and NKX6.1 was performed. The heterogeneous population in the PEC phase included pancreatic progenitor cells and early endocrine cells (…). Figure 17 ). Targeted RNA-seq was performed as described above for gene expression analysis. The expression of selected genes from the TXNIP KO clone was as follows: Figure 18A As shown, and the expression of selected genes in the V1-B clone is as follows: Figure 18B As shown, the kinetic expression patterns of FOXA2, CHGA, PDX1, and NKX6.1 from V1-B or TXNIP KO clones are similar to those in wild-type cells.

[0589] Cells were generated using an HLA-E donor vector containing the nucleotide sequence of SEQ ID NO:56, wherein the HLA-E coding sequence was inserted into the TXNIP locus (thus knocking out the TXNIP gene). Targeted RNA-seq was performed as described above for gene expression analysis. The expression of selected genes from the TXNIP KO / HLA-E KI clones is shown below. Figure 28 As shown, the dynamic expression patterns of FOXA2, CHGA, PDX1, and NKX6.1 from TXNIP KO / HLA-E KI cells are similar to those in wild-type cells.

[0590] Alternatively, HLA-E donor vectors containing the nucleotide sequence of SEQ ID NO:34 were used to generate cells in which the HLA-E coding sequence was inserted into the TXNIP locus. A large number of edited cells differentiated into the PEC stage and expressed HLA-E in at least 75% of the cell population (data not shown). Flow cytometry assessment of PDX1 and NKX6.1 expression in PEC cells differentiated from TXNIP KO cells was similar to that in PEC cells differentiated from wild-type cells (data not shown).

[0591] Example 9: T cell activation / proliferation assay

[0592] The ability of PEC-differentiated cells to trigger immune responses was tested using an in vitro human T cell activation / proliferation assay. Fresh donor PBMCs were purchased from Hemacare, and CD3+ T cells were purified using a human pan-T cell isolation kit (Mitentech, catalog number 130-096-535). The isolated T cells were then processed using CellTrace according to the manufacturer's instructions. TM The cells were labeled according to the CFSE cell proliferation kit protocol (Thermo Fisher Scientific, catalog number C34554) and co-incubated with differentiated PEC cells for 5 days. Dynabeads for T cell expansion and activation were then used. TM Human T activating factor CD3 / CD28 (Thermo Fisher Scientific, catalog number 11161D) was used as a positive control to activate T cells. Individual T cells were labeled with CFSE and used as a negative control. The percentage of CD3+CFSE+ cells was measured to assess the percentage of T cell proliferation. Figures 19A-19B WT PEC triggered higher T cell proliferation than the T cell-only control. B2M KO, B2M KO / PD-L1 KI, and B2M KO / PD-L1 KI+TXNIP KO / HLA-E KI CyT49-derived PECs did not trigger higher T cell proliferation than the T cell-only control, demonstrating the low immunogenicity of the edited cells.

[0593] Example 10: In vivo efficacy study of gene-targeted clone lines

[0594] Pancreatic endoderm cells were derived from the CyT49-derived clonal hES cell line, possessing the following genetic modifications: 1) target deletion of B2M expression and forced expression of PD-L1, 2) target deletion of B2M expression and forced expression of HLA-E, or 3) target deletion of TXNIP. Additionally, unmodified clonal cell lines were obtained by transfection with non-cleaved guide RNA (NCG).

[0595] Following standard procedures, pancreatic endoderm aggregates derived from the indicated clone line are loaded into a perforation device (PD) to produce either a test sample or a control. The PD allows for direct vascularization during subcutaneous transplantation, and the encapsulated pancreatic progenitor cells mature in vivo into functional pancreatic endocrine cells, including glucose-responsive insulin-producing cells.

[0596] As summarized in Table 13, two products were subcutaneously implanted into five groups of athymic nude rats. Each product contained approximately 7 × 10⁻⁶ cells derived from the four clonal lines mentioned above. 6 One pancreatic endoderm cell or wild-type CyT49 hES (Viastec) cells.

[0597] Table 13. Research Design

[0598]

[0599] Efficacy was evaluated in all surviving animals starting at week 12 using a glucose-stimulated insulin secretion (GSIS) test. Blood samples were obtained from non-fasted animals before and after intraperitoneal administration of 3 g / kg glucose. Serum concentrations of human C-peptide were determined using a standard enzyme-linked immunosorbent assay (ELISA).

[0600] GSIS testing was performed at weeks 12, 16, and 20. Results showed no significant differences between experimental groups, especially beyond week 12. In group 3, 2 out of 6 animals showed elevated C-peptide levels (TXNIP KO, mean 1.5 nM) compared to the control group (group 1, <40 pM to 2.0 nM, mean 1.1 nM). The other groups, group 2 (NCG, mean 0.5 nM), group 4 (B2M KO / PD-L1 KI, mean 0.5 nM), and group 5 (B2M KO / HLA-E KI, mean 0.4 nM), showed similar C-peptide level ranges compared to the control group, but more animals approached the lower limit of this range. However, these differences were not statistically significant. These results suggest that the introduced genetic modifications or manipulations required to generate clonal lines do not affect the ability of the cell lines in question to differentiate into pancreatic endoderm cells in vitro and subsequently generate functional β-cells in vivo.

[0601] At week 20, after the GSIS test, the animals were euthanized, and the explanted test specimens were fixed in neutral buffered formalin, processed into glass slides, stained with H&E, and insulin and glucagon were measured by immunohistochemistry.

[0602] In vivo efficacy evaluation using the GSIS assay showed no significant difference between the unedited control and the edited test product formulated with pancreatic endoderm cells derived from clonal cell lines carrying their respective genetically modified subgroups. The results indicate that the individual genetic modifications and their introduction are tolerable in vivo.

[0603] Example 11. In vivo efficacy study of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI cell lines

[0604] Essentially, as described in Example 8 above, four clonal lines were generated and loaded into a perforation device to form the test sample. The control sample contained unmodified CyT49 cells (Viastec). It will contain approximately 7 × 10⁶ cells. 6 Two pancreatic endoderm cell products were subcutaneously implanted into athymic nude rats (2 products / rat, 8 rats / group).

[0605] At weeks 12, 16, 20, and 24, glucose-stimulated insulin secretion (GSIS) was tested in all surviving animals. Blood samples were obtained from fasted animals before and after intraperitoneal administration of 3 g / kg glucose. Serum concentrations of human C-peptide were determined by standard enzyme-linked immunosorbent assay (ELISA). Serum C-peptide was detectable in most animals at week 12 post-implantation. Serum C-peptide levels at weeks 16, 20, and 24 post-implantation are presented in Table 14. No statistically significant differences were observed between the groups implanted with gene-edited cells and those implanted with control cells.

[0606] Table 14. Serum C-peptide levels in vivo.

[0607]

[0608] At week 25, surviving animals will undergo an insulin challenge (insulin tolerance test, ITT) to assess changes in serum human C-peptide in response to decreased glucose levels during fasting. Blood samples will be obtained from the fasting animals at multiple time points (15, 30, and 60 minutes) before and after intraperitoneal administration of 1 unit of insulin per kg body weight. Serum concentrations of human C-peptide will be determined by standard enzyme-linked immunosorbent assay (ELISA).

[0609] At week 26, surviving animals will be euthanized, and the explanted specimens will be processed into slides and stained with H&E. Insulin and glucagon levels will be measured by immunohistochemistry (IHC) to identify human pancreatic endocrine cells. Additional IHC will be performed on the human-specific nuclear marker NuMA1 to identify potential sites of graft-derived cells outside the lumen of the explant.

[0610] Example 12. In vivo efficacy study of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI cell lines.

[0611] B2M KO / PD-L1 KI, TXNIP KO / HLA-E KI pancreatic endoderm cells (containing approximately 7 × 10⁻⁶ cells) 6 The test sample was formulated from aggregates of cells. Two test samples were subcutaneously implanted into each of the forty-six athymic nude rats. GSIS, ITT, and non-fasting blood glucose (NFBG) in the study animals were evaluated. Ten animals from each group were euthanized at the planned termination time points of weeks 13, 17, 26, and 39, while six additional animals were studied to consider possible early unplanned termination. Two explanted test samples were randomly assigned from each animal for histological evaluation or assessment of total C-peptide content. Table 15 presents the study design.

[0612] Table 15. Research Design

[0613]

[0614] Efficacy will be evaluated in all surviving animals at weeks 12, 16, 20, 24, 30, and 36 by glucose-stimulated insulin secretion (GSIS) assay. Blood samples will be obtained from fasted animals before and after intraperitoneal administration of 3 g / kg glucose. Serum concentrations of human C-peptide will be determined by standard enzyme-linked immunosorbent assay (ELISA).

[0615] At weeks 25 and 33, surviving animals underwent an insulin challenge (insulin tolerance test, ITT) to assess changes in serum human C-peptide in response to decreased glucose levels during fasting. Blood samples were obtained from the fasting animals at multiple time points (15, 30, and 60 minutes) before and after intraperitoneal administration of 1 unit of insulin per kg body weight. Serum concentrations of human C-peptide were determined by standard enzyme-linked immunosorbent assay (ELISA).

[0616] Non-fasting blood glucose (NFBG) will be measured at approximately 12, 16, 20, 24, 25, 30, 33, and 36 weeks before starting fasting GSIS and ITT tests.

[0617] At the planned endpoints identified in Table 13, the animals will be euthanized. Euthanasia will be performed via CO2 inhalation followed by bilateral thoracotomy. Gross autopsies will be performed on all planned and unplanned terminations, and macroscopic abnormalities will be recorded.

[0618] The designated explants were frozen, and the lumen contents were then homogenized. The total C-peptide content of the homogenate was determined by standard enzyme-linked immunosorbent assay (ELISA). The total C-peptide content of the explants will be used to design clinical dosing.

[0619] The designated explant specimens were fixed in neutral buffered formalin, prepared into glass slides, and stained with H&E. Insulin and glucagon were measured by immunohistochemistry (IHC) to identify human pancreatic endocrine cells. Additional IHC was performed on the human-specific nuclear marker NuMA1 to identify the potential location of graft-derived cells outside the lumen of the explant.

[0620] Example 13: Production of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI in human iPSCs

[0621] A human iPSC (iPSC 0025) was generated, in which the PD-L1 coding sequence was inserted into the B2M locus. The RNP complex was formed by combining B2M-2 gRNA (SEQ ID NO:2) and Cas9 protein in a 3:1 molar ratio (gRNA:Cas9). To form the RNP complex, gRNA and Cas9 were combined with R-buffer in a container to a total volume of 25 μL and incubated at room temperature for 15 min. Cells were dissociated and resuspended in DMEM / F12 medium (Gibco, catalog number 11320033), counted, and centrifuged using an NC-200 (Krommet). A total of 1 × 10⁻⁶ cells were resuspended in RNP complex. 6 Cells. Add 4 μg B2M-CAGGS-PD-L1 donor plasmid (SEQ ID NO:33) and R-buffer to a total volume of 125 μL. Then electroporate the mixture using the following parameters: 2 pulses, 30 ms, 1100 V. Seven days after electroporation, PD-L1 positive cells were enriched via MACS using Miltenyi reagent or Thermofisher reagent, essentially as described in Example 8.

[0622] Following expansion of the enriched PD-L1 positive population, cells were electroporated essentially as described above using an RNP complex containing TXNIP-T5 gRNA (SEQ ID NO:20) and Cas9 protein at a molar ratio of 3:1 (gRNA:Cas9) and 4 μg of TXNIP-CAGGS-HLA-E donor plasmid 2 (SEQ ID NO:56). Seven days after electroporation, HLA-E positive cells were enriched via MACS using Miltenyi's reagent or Thermofisher's reagent. After HLA-E enrichment, single cells were sorted into 96-well plates coated with BIOLAMININ521CTG containing StemFlex and Revitacell using a WOLF FACS sorter (NanoSelex). The plated single cells were grown in a normoxic incubator (37°C, 8% CO2), with the medium changed every other day, until the colonies were large enough to be reseeded as single cells. Upon pooling, samples were separated for maintenance and genomic DNA extraction. Anti-PD-L1 and anti-HLA-E antibodies (Table 4) were used for MACS enrichment and FACS sorting into 96-well plates, with gating set for HLA-E and PD-L1 double-positive cells. Unedited cells served as negative controls for FACS sorting.

[0623] The following primers were used to identify correctly targeted clones via PCR targeting PD-L1 KI insertion and HLA-E KI insertion. These primers amplified the region from the plasmid homologous arm to the PD-L1 cDNA insertion or HLA-E cDNA insertion, respectively, allowing for the amplification of only the KI-integrated DNA. The conjugation of the target insertion was tested by PCR to assess whether KI occurred heterozygous or homozygous. If a heterozygous clone was identified, the KI-negative allele was sent for Sanger sequencing to verify that it contained either a B2M-disrupting insertion / deletion or a TXNIP-disrupting insertion / deletion, respectively. Correctly targeted KI clones with complete B2M and TXNIP disruption (formed via KI insertion or deletion) were amplified in incremental tissue cultures until a population size of 30 million cells was reached. The selected clones were amplified in this manner, and their pluripotency was confirmed by intracellular flow cytometry testing for OCT4 and SOX2.

[0624] Four edited hiPSC clones (VI-B) were differentiated using the pancreatic endocrine protocol described by Rezania et al. (Nat Biotechnol. [Nature Biotechnology] 2014 Nov; 32(11):1121-33). In stage 4, flow cytometry targeting chromogranin (CHGA), PDX1, and NKX6.1 was performed. Results of PDX1 and NKX6.1 in clones (clone 1) seeded at different representative densities are shown below. Figure 26A As shown. All four clones were CHGA negative. Flow cytometry for PD-L1 and HLA-E was also performed. PD-L1 and HLA-E results for clone (clone 1) are shown below. Figure 26B As shown.

[0625] Example 14: Method for manufacturing a cryopreservation cell bank of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI human pluripotent stem cells (hPSCs)

[0626] CyT49 hESCs (Viastec) were electroporated for 30 ms with two pulses at 1100V using an RNP complex containing B2M-2 gRNA (SEQ ID NO:2) and Cas9 protein at a molar ratio of 3:1 (gRNA:Cas9) and 4 μg of B2M-CAGGS-PD-L1 donor plasmid (SEQ ID NO:33). After electroporation, cells were aspirated into Eppendorf tubes filled with StemFlex medium containing RevitaCell. The cell suspension was then plated into tissue culture dishes pre-coated with BIOLAMININ 521CTG at a 1:20 dilution. Cells were cultured in a normoxic incubator (37°C, 8% CO2).

[0627] Seven days after electroporation, anti-PD-L1 antibody labeled with Alexa-488 and magnetic beads were used. Pan mouse IgG (Thermo Fisher Scientific) was used to enrich PD-L1 positive cells in cells via MACS. PD-L1 positive cells were expanded by culturing in XF-KSR amplification medium (Gibco) for 7 days.

[0628] PD-L1 positive cells were then electroporated for 30 ms with two pulses at 1100V using an RNP complex containing TXNIP-T5 gRNA (SEQ ID NO:20) and Cas9 protein at a molar ratio of 3:1 (gRNA:Cas9) and 4 μg of TXNIP-CAGGS-HLA-E donor plasmid 2 (SEQ ID NO:56). After electroporation, cells were aspirated into Eppendorf tubes filled with StemFlex medium containing RevitaCell. The cell suspension was then plated into tissue culture dishes pre-coated with BIOLAMININ 521CTG at a 1:20 dilution. Cells were cultured in a normoxic incubator (37°C, 8% CO2).

[0629] Seven days after electroporation, PE-labeled anti-HLA-E antibody and magnetic beads were used. Pan mouse IgG (Thermo Fisher Scientific) was used to enrich HLA-E positive cells via MACS. PD-L1 and HLA-E double-positive cells were expanded by culturing in XF-KSR amplification medium (Gibco) for approximately 5 days.

[0630] Single-cell sorting was performed on PD-L1 and HLA-E double-positive cells. For this purpose, in using... 3-4 hours before dissociation, cells were fed with StemFlex Complete containing Revitacell (final concentration 1X Revitacell). After dissociation, single cells were sorted into individual wells of a 96-well tissue culture plate coated with Biolamin. A WOLF FACS sorter (NanoCelec) was used to sort single cells into the wells using the aforementioned anti-PD-L1 and anti-HLA-E antibodies. The plate was pre-filled with 100-200 μL of StemFlex Complete containing Revitacell. Three days after cell seeding, cells were fed with fresh StemFlex and continued to be fed with 100-200 μL of culture medium every other day. After 10 days of growth, cells were fed with StemFlex daily until day 12-14. At this time, the plate was... The collected cell suspension was dissociated and separated into two 96-well plates at a 1:2 ratio and cultured for approximately 4 days.

[0631] A portion of the cells were harvested for visual analysis (morphology) and DNA analysis (PCR and DNA sequencing for conjugation analysis and insertion / deletion profiling), and the remaining cells were cultured and amplified for further culture in T175 flasks. After approximately two weeks of culture, clones were selected for cryopreservation. Cell morphology, viability, endotoxin levels, mycoplasma count, karyotype, pluripotency, differentiation capacity, on-target / off-target analysis, random plasmid integration, and residual Cas9 / plasmids were characterized using standard procedures before and after cryopreservation. Cells were frozen in cryopreservation medium and stored in cryovials at -80°C or liquid nitrogen.

[0632] Specific B2M KO / PD-L1KI+TXNIP KO / HLA-E KI clones (“seed clones”) were prepared and isolated using the method described above. The seed clones were differentiated to the PEC stage and characterized. Figure 27A The morphology of seed clones at the PEC stage is similar to that of wild-type cells. Figure 27B The dynamic expression patterns of FOXA2, CHGA, PDX1, and NKX6.1 in cells differentiated from seed clones during the differentiation process were similar to those in wild-type cells. Figure 27C CHGA is shown - / NKX6.1 + / PDX1 + Percentage of cells in the differentiated population.

Claims

1. A method for generating genetically modified cells, the method comprising delivering to stem cells: (a) A first site-directed nuclease targeting a site within or near a gene encoding TXNIP, wherein the first site-directed nuclease is a CRISPR system comprising a CRISPR nuclease and a guide RNA (gRNA), and wherein the gRNA comprises a spacer sequence containing an RNA sequence corresponding to any one of SEQ ID NO: 15-24; and (b) A first nucleic acid containing a nucleotide sequence encoding HLA-E, flanked by (i) a nucleotide sequence homologous to the region to the left of the target site in (a) and (ii) a nucleotide sequence homologous to the region to the right of the target site in (a), wherein the nucleotide sequence in (b)(i) contains the sequence of SEQ ID NO: 25, and the nucleotide sequence in (b)(ii) contains the sequence of SEQ ID NO: 32; The first site-specific nuclease cleaves the target site of (a), and the first nucleic acid of (b) is inserted at a site that partially overlaps, completely overlaps with, or is contained within the site of (a). (c) A second site-specific nuclease targeting a site within or near a gene encoding B2M, wherein the second site-specific nuclease is a CRISPR system comprising a CRISPR nuclease and gRNA, and wherein the gRNA comprises a spacer sequence containing an RNA sequence corresponding to any one of SEQ ID NO: 1-3 or 35-44; and (d) A second nucleic acid containing a nucleotide sequence encoding PD-L1, flanked by (iii) a nucleotide sequence homologous to the region to the left of the target site in (c) and (iv) a nucleotide sequence homologous to the region to the right of the target site in (c), wherein the nucleotide sequence in (d) and (iii) contains the sequence of SEQ ID NO: 7, and the nucleotide sequence in (d)(iv) contains the sequence of SEQ ID NO: 13; The second site-specific nuclease cleaves the target site of (c), and the second nucleic acid of (d) is inserted at a site that partially overlaps, completely overlaps with, or is contained within the site of (c). This results in genetically modified cells that have increased immune evasion and / or cell survival compared to cells without the insertion of (b) the first nucleic acid and (d) the second nucleic acid.

2. The method of claim 1, wherein the CRISPR nuclease is a type II Cas9 nuclease or a type V Cfp1 nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal.

3. The method of claim 2, wherein the CRISPR nuclease and the gRNA are present in a molar ratio of 1:

3.

4. The method of claim 1 or 2, wherein the nucleotide sequence encoding HLA-E is operatively linked to a foreign promoter, and the nucleotide sequence encoding PD-L1 is operatively linked to a foreign promoter.

5. The method of claim 4, wherein the exogenous promoter is a constitutive promoter, an inducible promoter, a time-specific promoter, a tissue-specific promoter, or a cell type-specific promoter, optionally wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

6. A method for generating genetically modified cells, the method comprising delivering to stem cells: (a) A first site-directed nuclease targeting a site within or near a gene encoding TXNIP, wherein the first site-directed nuclease is a CRISPR system comprising a CRISPR nuclease and a guide RNA (gRNA), and wherein the gRNA comprises a spacer subsequence containing an RNA sequence corresponding to any one of SEQ ID NO: 15-24; (b) A first nucleic acid containing a nucleotide sequence encoding a first tolerance factor, the flanking nucleotide sequence being (i) a nucleotide sequence homologous to the region to the left of the target site in (a) and (ii) a nucleotide sequence homologous to the region to the right of the target site in (a), wherein the first tolerance factor is HLA-E, wherein the first site-directed nuclease cleaves the target site in (a), and the first nucleic acid in (b) is used as a template by homologous recombination to insert the nucleotide sequence encoding the first tolerance factor into a site that partially overlaps, completely overlaps with, or is contained within the site in (a), thereby disrupting the gene in (a), wherein the nucleotide sequence in (b) (i) contains the sequence of SEQ ID NO: 25, and the nucleotide sequence in (b) (ii) contains the sequence of SEQ ID NO: 32; (c) A second site-specific nuclease targeting a site within or near a gene encoding B2M, wherein the second site-specific nuclease is a CRISPR system comprising a CRISPR nuclease and gRNA, and wherein the gRNA comprises a spacer sequence containing an RNA sequence corresponding to any one of SEQ ID NO: 1-3 or 35-44; and (d) A second nucleic acid containing a nucleotide sequence encoding a second tolerance factor, flanked by (iii) nucleotide sequences homologous to the region to the left of the target site in (c) and (iv) nucleotide sequences homologous to the region to the right of the target site in (c), wherein the second tolerance factor is PD-L1, wherein the second site-directed nuclease cleaves the target site in (c), and through homologous recombination, the second nucleic acid in (d) is used as a template for inserting the nucleotide sequence encoding the second tolerance factor into a site that partially overlaps, completely overlaps with, or is contained within the site in (c), thereby disrupting the gene in (c), wherein the nucleotide sequence in (d) and (iii) contains the sequence of SEQ ID NO: 7, and the nucleotide sequence in (d) and (iv) contains the sequence of SEQ ID NO:

13. This results in the genetically modified cell, which has increased cell survival compared to cells in which the first nucleic acid (b) and the second nucleic acid (d) are not inserted, wherein the disruption includes reducing or eliminating the expression of TXNIP and B2M.

7. The method of any one of claims 1 to 3 and 6, wherein the stem cell is a mammalian cell, optionally wherein the stem cell is a human cell.

8. The method according to any one of claims 1 to 3 and 6, wherein the stem cell is a pluripotent stem cell, embryonic stem cell, adult stem cell, induced pluripotent stem cell, or hematopoietic stem cell.

9. The method of any one of claims 1 to 3 and 6, wherein the genetically modified cell is capable of differentiating into lineage-restricted progenitor cells or fully differentiated somatic cells.

10. The method of claim 9, wherein these lineage-restricted progenitor cells are pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells.

11. The method of claim 9, wherein the fully differentiated somatic cells are pancreatic β cells, epithelial cells, endoderm cells, macrophages, hepatocytes, adipocytes, kidney cells, blood cells, cardiomyocytes, or immune system cells.

12. A plurality of genetically modified cells, wherein the plurality of genetically modified cells are produced by the method of any one of claims 1 to 11.

13. The plurality of genetically modified cells as described in claim 12, wherein the plurality of genetically modified cells are maintained under time and conditions sufficient to allow the cells to undergo differentiation.

14. The plurality of genetically modified cells as described in claim 12 or 13, wherein the plurality of genetically modified cells are used to treat a subject in need.

15. The plurality of genetically modified cells as described in claim 14, wherein the subject is a person who has a disease, is suspected of having a disease, or is at risk of having a disease.

16. A method for obtaining cells for administration to a subject in need, the method comprising: (a) Obtaining or having obtained, a plurality of genetically modified cells as described in claim 12; as well as (b) Maintain these multiple genetically modified cells under time and conditions sufficient to allow them to differentiate into lineage-restricted progenitor cells or fully differentiated somatic cells.

17. The method of claim 16, wherein the lineage-restricted progenitor cells are pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells.

18. The method of claim 16, wherein the fully differentiated somatic cells are pancreatic β cells, epithelial cells, endoderm cells, macrophages, hepatocytes, adipocytes, kidney cells, blood cells, cardiomyocytes, or immune system cells.

19. The method of any one of claims 16 to 18, wherein the subject is a person who has a disease, is suspected of having a disease, or is at risk of having a disease.

20. The method of claim 19, wherein the disease is a genetically heritable disease.

21. A method for producing genetically modified cells, the method comprising: (a) Modifying stem cells by inserting a nucleotide sequence encoding a first tolerance factor into or near a gene encoding B2M, thereby generating first tolerance factor-positive cells, wherein the first tolerance factor is PD-L1, wherein the modification in (a) comprises delivering to these stem cells (1) a first RNA-guided nuclease and a first guide RNA (gRNA) targeting a target site in the B2M locus, wherein the first gRNA comprises a spacer sequence containing an RNA sequence corresponding to the sequence of SEQ ID NO: 2, and (2) a first vector containing a first nucleic acid comprising (i) a nucleotide sequence homologous to a region to the left of the target site in the B2M locus, (ii) the nucleotide sequence encoding the first tolerance factor, and (iii) A nucleotide sequence homologous to the region to the right of the target site in the B2M locus, wherein the B2M locus is cleaved at the target site, and the first nucleic acid containing the nucleotide sequence encoding the first tolerogenic factor is inserted into the B2M locus to disrupt the B2M gene, wherein the disruption includes reducing or eliminating the expression of B2M, and wherein the nucleotide sequence of (a) (2) (i) contains the sequence of SEQ ID NO: 7, and the nucleotide sequence of (a) (2) (iii) contains the sequence of SEQ ID NO: 13; (b) Enrichment of cells positive for the first tolerance factor; (c) Modifying the first tolerance factor-positive cells from (b) by inserting a nucleotide sequence encoding a second tolerance factor within or near the gene encoding TXNIP, wherein the second tolerance factor is HLA-E, thereby generating first tolerance factor-positive / second tolerance factor-positive cells, wherein the modification in (c) comprises delivering to these first tolerance factor-positive cells (1) a second RNA-guided nuclease and a second guide RNA (gRNA) targeting a target site in the TXNIP locus, wherein the second gRNA contains a spacer sequence containing an RNA sequence corresponding to the sequence of SEQ ID NO: 20, and (2) a second vector containing a second nucleic acid comprising (i) a nucleotide sequence homologous to a region to the left of the target site in the TXNIP locus, (ii) the nucleotide sequence encoding the second tolerance factor, and (iii) A nucleotide sequence homologous to the region to the right of the target site in the TXNIP locus, wherein the TXNIP locus is cleaved at the target site, and a second nucleic acid containing a nucleotide sequence encoding the second tolerogenic factor is inserted into the TXNIP locus to disrupt the TXNIP gene, wherein the disruption includes reducing or eliminating the expression of TXNIP, wherein the nucleotide sequence of (c) (2) (i) contains the sequence of SEQ ID NO: 25, and the nucleotide sequence of (c) (2) (iii) contains the sequence of SEQ ID NO: 32; (d) Enrichment of cells positive for the first tolerance factor / positive for the second tolerance factor; (e) Single-cell sorting to select cells positive for the first tolerance factor / the second tolerance factor; (f) Characterize these cells from (e) as genetically modified cells; and (g) Freeze these genetically modified cells for long-term storage.

22. The method of claim 21, wherein the enrichment of the first tolerance factor-positive cells in (b) comprises magnetic-assisted cell sorting (MACS), single-cell cloning, expansion of the first tolerance factor-positive cells, or a combination thereof.

23. The method of claim 21 or 22, wherein the enrichment of cells positive for the first tolerance factor / second tolerance factor in (d) comprises magnetic-assisted cell sorting, single-cell cloning, amplification of cells positive for the first tolerance factor / second tolerance factor or a combination thereof.

24. The method of claim 21 or 22, further comprising (a) amplifying the resulting first tolerance-inducing factor positive cells, (c) amplifying the resulting first tolerance-inducing factor positive / second tolerance-inducing factor positive cells, and (e) amplifying selected first tolerance-inducing factor positive / second tolerance-inducing factor positive cells, or a combination thereof.

25. The method of claim 21, wherein the first RNA-guided nuclease and the first gRNA form a first ribonucleoprotein (RNP) complex.

26. The method of claim 21, wherein the first RNA-guided nuclease is a Cas9 nuclease.

27. The method of claim 26, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

28. The method of claim 25, wherein the first RNP comprises a 3:1 molar ratio of first gRNA to first RNA-guided nuclease.

29. The method of claim 21 or 22, wherein the nucleotide sequence encoding the first tolerogenic factor is operatively linked to an exogenous promoter.

30. The method of claim 29, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

31. The method of claim 30, wherein the nucleotide sequence encoding the first tolerogenic factor comprises the sequence of SEQ ID NO:

11.

32. The method of claim 31, wherein the nucleotide sequence encoding the first tolerogenic factor is operatively linked to the CAG promoter.

33. The method of claim 21, wherein the first vector comprises a nucleotide sequence containing the sequence of SEQ ID NO:

33.

34. The method of claim 21, wherein the second RNA-guided nuclease and the second gRNA form a second ribonucleoprotein (RNP) complex.

35. The method of claim 21, wherein the second RNA-guided nuclease is a Cas9 nuclease.

36. The method of claim 35, wherein the Cas9 nuclease is linked to at least one nuclear localization signal.

37. The method of claim 34, wherein the second RNP comprises a 3:1 molar ratio of second gRNA to second RNA-guided nuclease.

38. The method of claim 21 or 22, wherein the nucleotide sequence encoding the second tolerogenic factor is operatively linked to an exogenous promoter.

39. The method of claim 38, wherein the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

40. The method of claim 39, wherein the nucleotide sequence encoding the second tolerogenic factor comprises a sequence encoding an HLA-E trimer, the HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide, the HLA-G presenting peptide fused to a B2M membrane protein, the B2M membrane protein fused to HLA-E without its signal peptide.

41. The method of claim 40, wherein the sequence encoding the HLA-E trimer comprises the sequence of SEQ ID NO:

55.

42. The method of claim 40 or 41, wherein the nucleotide sequence encoding the second tolerogenic factor is operatively linked to the CAG promoter.

43. The method of claim 21, wherein the second vector comprises a nucleotide sequence containing the sequence of SEQ ID NO: 34 or 56.

44. The method of claim 21 or 22, wherein the single-cell sorting in (e) comprises fluorescence-activated cell sorting (FACS), single-cell cloning, amplification of the single-cell sorted cells, or a combination thereof.

45. The method of claim 21 or 22, wherein the characterization in (f) includes DNA analysis of conjugation and / or insertion / deletion profiles.

46. ​​The method of claim 21 or 22, wherein the characterization in (f) comprises cellular analysis of morphology, viability, karyotype analysis, endotoxin levels, mycoplasma levels, on-target / off-target analysis, random vector insertion, residual Cas9, residual vector, pluripotency status, differentiation capacity, or combinations thereof.

47. The method of claim 21 or 22, wherein the method further comprises freezing prior to the characterization in (f).

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