Universal donor cells

Abstract

Provided herein are genetically modified cells, e.g., universal donor cells, that are compatible with multiple subjects, and methods for producing the genetically modified cells. The universal donor cells contain at least one genetic modification within or near at least one gene encoding a survival factor, where the genetic modification comprises the insertion of a polypeptide encoding a tolerogenic factor. The 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 a component or transcriptional regulator of an MHC-I or MHC-II complex, where the genetic modification comprises the insertion of a polypeptide encoding a second tolerogenic factor.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 62 / 896,477, filed on September 5, 2019, and U.S. Provisional Patent Application No. 62 / 979,756, filed on February 21, 2020, the respective disclosures of which are incorporated herein by reference in whole.

[0002] Inclusion by referencing the sequence list This application includes a sequence listing submitted via EFS-Web in ASCII format, which is incorporated herein by reference in whole. The ASCII copy, created on 2 September 2020, is named CT123-PCT-100867-666513-Sequence-Listing_ST25.txt and is approximately 53,000 bytes in size.

[0003] The present invention relates to the field of gene editing, and in some embodiments, to gene modification for the purpose of producing cells suitable for multiple targets, such as universal donor cells. [Background technology]

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

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

[0006] To generate such a lineage of universal donor cells, one preceding method involved functionally disrupting the expression of MHC-I and MHC-II class genes. This can be activated, for example, by disrupting the gene alleles of both the MHC-I light chain and the B2M-encoding gene. The resulting B2M-deficient cell lines and their derivatives are expected to exhibit a significant reduction in surface MHC-I, thereby reducing immunogenicity to allogeneic CD8+ T cells. Using a method that targets transcription activator-like effector nucleases (TALENs), B2M-deficient hESC lines have been generated by deleting several nucleotides in exon 2 of the B2M gene (Lu, P. et al., “Generating hypoimmunogenic human embryonic stem cells by the disruption of beta 2-microglobulin”, Stem Cell Rev. 2013, 9:806-813). Although B2M-targeted hESC strains appeared to be surface HLA-I deficient, they were found to still contain B2M and MHC-I specific mRNAs. B2M and MHC-I mRNAs were expressed at levels comparable to those of untargeted hESCs (both constitutive and IFN-γ-inducible). Therefore, there is concern that these TALEN B2M-targeted hESC strains may express residual cell surface MHC-I sufficient to induce immune rejection, as 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 TALEN B2M-targeted hESC strains were not tested for off-target cleavage, the occurrence of nonspecific cleavage when using TALENs remains a significant issue that poses major safety concerns for their clinical use (Grau, J. et al. “TALEN offer: genome-wide TALEN off-target prediction”, Bioinformatics, 2013, 29:2931-2932; Guilinger JP et al. “Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity”, Nat Methods 2014, 11:429-435). Furthermore, another report described how knocking out the first B2M allele and knocking in the HLA-E gene in the second B2M allele resulted in the creation of iPS cells that evaded allogeneic recognition by inducing surface expression of HLA-E dimers or trimers while lacking surface expression of HLA-A, HLA-B, or HLA-C (Gornalusse, GGet al., “HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells”, Nature Biotechnology, 2017, 35, 765-773). [Overview of the project] [Problems that the invention aims to solve]

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

[0008] In some embodiments, the present disclosure comprises a method for producing universal donor cells. The method comprises delivering to a cell a site-specific nuclease that targets a site within or near a gene encoding a survival factor, and a nucleic acid comprising a nucleotide sequence encoding an immunotolerogenic factor adjacent to a nucleotide sequence homologous to a region located to the left of the target site in (i)(a), and a nucleotide sequence homologous to a region located to the right of the target site in (ii)(a), wherein the site-specific nuclease cleaves the target site in (a), and the nucleic acid of (b) is inserted at a site that partially overlaps, completely overlaps, or contains the site in (a), thereby producing a universal donor cell having increased cell viability compared to a cell in which the nucleic acid of (b) is not inserted.

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

[0010] In some embodiments, the method further comprises delivering to cells a site-specific nuclease that targets a site within or near a gene encoding one or more MHC-I or MHC-II human leukocyte antigens or components of the MHC-I or MHC-II complex or transcription regulators, and a nucleic acid comprising a nucleotide sequence encoding an immunotolerogenic factor adjacent to a nucleotide sequence homologous to a region located to the left of the target site in (c), and an immunotolerogenic factor in (d) that, unlike immunotolerogenic factor (b), the site-specific nuclease cleaves the target site in (c), and the nucleic acid of (d) is inserted at a site that partially overlaps, completely overlaps, or contains the site in (c), and the universal donor cells have increased immune evasion and / or cell survival compared to cells in which the nucleic acid of (d) is inserted.

[0011] In some embodiments, the gene encoding one or more MHC-I or MHC-II human leukocyte antigens or components or transcription factors 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 immunotolerogenic factor is PD-L1, HLA-E, HLA-G, CTLA-4, or CD47. In a particular embodiment, the gene encoding one or more MHC-I or MHC-II human leukocyte antigens or components or transcription factors of the MHC-I or MHC-II complex is B2M, and the immunotolerogenic factor is PD-L1. In embodiments where the site-specific nuclease is a CRISPR system comprising a CRISPR nuclease and a gRNA, the CRISPR nuclease is a type II Cas9 nuclease or a type V Cfp1 nuclease, and the CRISPR nuclease is linked to at least one nuclear localization signal. In some embodiments, the gRNA targets a polynucleotide sequence selected from SEQ ID NOs: 1-3 or 35-44, and is essentially composed of (iii) the nucleotide sequence of SEQ ID NO: 7 and (iv) the nucleotide sequence of SEQ ID NO: 13.

[0012] In some embodiments, the nucleotide sequences encoding immunotolerogenic factors (b) and (d) are operably ligated to an exogenous promoter. The exogenous promoter may be selected from constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters. In some embodiments, the exogenous promoter is the CMV, EFla, PGK, CAG, or UBC promoter. In certain embodiments, the exogenous promoter is the CAG promoter.

[0013] This disclosure also includes universal donor cells produced by the methods disclosed herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are stem cells. In some embodiments, the cells are pluripotent stem cells (PSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), or hematopoietic stem cell progenitor cells (HSPCs) (also called hematopoietic stem cells (HSCs)). In some embodiments, the cells are differentiated cells. In some embodiments, the cells are somatic cells.

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

[0015] Further embodiments of this disclosure provide a method for administering a treatment to a subject in need thereof, comprising obtaining or having obtained universal donor cells as disclosed herein after differentiation into lineage-limited progenitor cells or fully differentiated somatic cells, and administering the lineage-limited progenitor cells or fully differentiated somatic cells to the subject. Also provided is a method for obtaining cells for administration to a subject in need thereof, comprising obtaining or having obtained universal donor cells as disclosed herein, and maintaining the universal donor cells for a period of time and under conditions sufficient for the cells to differentiate into lineage-limited progenitor cells or fully differentiated somatic cells. In some embodiments, the subject is a human being who has, is suspected of having, or is at risk of having a disease. In some embodiments, the disease is a genetically inherited disease.

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

[0017] While the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and are described in detail herein. However, the drawings and detailed description presented herein are not intended to limit the present disclosure to the particular embodiments disclosed, but on the contrary, are intended to cover all modifications, equivalents, and alternative forms falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0018] Other features and advantages of the present disclosure will become apparent in the following detailed description of embodiments of the invention when taken in conjunction with the accompanying drawings.

Brief Description of the Drawings

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

[0020] I. Definition Deletion: As used herein, the term “deletion,” which may be used interchangeably with the terms “gene deletion” or “knockout,” generally refers to a genetic modification in which a site or region of genomic DNA is removed by any molecular biological method, e.g., the methods described herein, e.g., 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, a deletion includes 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 twenty-five nucleotides. In some embodiments, a deletion includes the removal of 10–50, 25–75, 50–100, 50–200, or more than 100 nucleotides. In some embodiments, a deletion includes the removal of an entire target gene, e.g., the B2M gene. In some embodiments, a deletion includes the removal of a portion of a target gene, e.g., all or part of the promoter and / or coding sequence of the B2M gene. In some embodiments, the deletion includes the removal of a transcription regulator, such as the promoter region of a target gene. In some embodiments, the deletion includes the removal of all or part of a coding region such that the product normally expressed by the coding region is no longer expressed, expressed as a truncated form, or expressed at a low level. In some embodiments, the deletion results in reduced gene expression in unmodified cells.

[0021] Endonucleases: As used herein, the term “endonuclease” generally refers to enzymes that cleave phosphodiester bonds within polynucleotides. In some embodiments, endonucleases specifically cleave phosphodiester bonds within DNA polynucleotides. In some embodiments, endonucleases are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), homing endonucleases (HEs), meganucleases, MegaTAL, or CRISPR-related endonucleases. In some embodiments, endonucleases are RNA-inducible endonucleases. In certain embodiments, RNA-inducible endonucleases are CRISPR nucleases, such as type II CRISPR Cas9 endonucleases or type V CRISPR Cpf1 endonucleases. In some embodiments, the endonucleases are 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, Cm These include r3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonucleases or their homologs, recombinants of their naturally occurring molecules, their codon-optimized versions or modified versions 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).

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

[0023] Guide RNA (gRNA): As used herein, the term “guide RNA” or “gRNA” generally refers to a short ribonucleic acid that can interact with, for example, an endonuclease and bind to or hybridize with a target genomic site or region. In some embodiments, the gRNA is a single-molecule guide RNA (sgRNA). In some embodiments, the gRNA may include a spacer elongation region. In some embodiments, the gRNA may include a tracrRNA elongation region. In some embodiments, the gRNA is single-stranded. In some embodiments, the gRNA contains naturally occurring nucleotides. In some embodiments, the gRNA is a chemically modified gRNA. In some embodiments, the chemically modified gRNA is a gRNA containing at least one nucleotide having a chemical modification, e.g., a 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 a 2'-O-methyl-phosphorothioate residue. In some embodiments, the gRNA may pre-complex with a DNA endonuclease.

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

[0025] Major Histocompatibility Complex Class I (MHC-I): As used herein, the term “Major Histocompatibility Complex Class I” or “MHC-I” generally refers to a class of biomolecules found on the cell surface of all nucleated cells in mammals, including vertebrates such as humans; and to the function of presenting non-self or foreign antigen peptides, such as proteins, from the cell (i.e., cytosol) to cytotoxic T cells, such as CD8+ T cells, in order to stimulate an immune response. In some embodiments, the MHC-I biomolecule is the MHC-I gene or the MHC-I protein. Complexation of the MHC-I protein with the beta-2 microglobulin (B2M) protein is required for the cell surface expression of all MHC-I proteins. In some embodiments, reducing the expression of MHC-I human leukocyte antigen (HLA) in unmodified cells includes a decrease (or reduction) in the expression of the MHC-I gene. In some embodiments, reducing the expression of MHC-I human leukocyte antigen (HLA) in unmodified cells includes a decrease (or reduction) in the cell surface expression of the MHC-I protein. In some embodiments, the MHC-I biomolecule is HLA-A (NCBI gene ID number: 3105), HLA-B (NCBI gene ID number: 3106), HLA-C (NCBI gene ID number: 3107), or B2M (NCBI gene ID number: 567).

[0026] Major Histocompatibility Complex Class II (MHC-II): As used herein, the term “Major Histocompatibility Complex Class II” or “MHC-II” generally refers to a class of biomolecules typically found on the cell surface of antigen-presenting cells in mammals, including vertebrates such as humans; and the function of presenting peptides, such as proteins, of non-self or foreign antigens from outside the cell (extracellularly) to cytotoxic T cells, such as CD8+ T cells, in order to stimulate an immune response. In some embodiments, the antigen-presenting cell is a dendritic cell, macrophage, or B cell. In some embodiments, the MHC-II biomolecule is an MHC-II gene or MHC-II protein. In some embodiments, reducing the expression of MHC-II human leukocyte antigen (HLA) in unmodified cells includes a decrease (or reduction) in the expression of the MHC-II gene. In some embodiments, reducing the expression of MHC-II human leukocyte antigen (HLA) in unmodified cells includes a decrease (or reduction) in the cell surface expression of the MHC-II protein. In some embodiments, the MHC-II biomolecule is HLA-DPA (NCBI gene ID number: 3113), HLA-DPB (NCBI gene ID number: 3115), HLA-DMA (NCBI gene ID number: 3108), HLA-DMB (NCBI gene ID number: 3109), HLA-DOA (NCBI gene ID number: 3111), HLA-DOB (NCBI gene ID number: 3112), HLA-DQA (NCBI gene ID number: 3117), HLA-DQB (NCBI gene ID number: 3119), HLA-DRA (NCBI gene ID number: 3122), or HLA-DRB (NCBI gene ID number: 3123).

[0027] 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 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 250, at least 500, or any number of nucleotides. For example, a polynucleotide may contain at least 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, at least about 1000 nucleotides, at least about 2000 nucleotides, at least about 3000 nucleotides, at least about 4000 nucleotides, at least about 4500 nucleotides, or at least about 5000 nucleotides. A polynucleotide may be a DNA or RNA molecule or a hybrid DNA / RNA molecule. A polynucleotide may be single-stranded or double-stranded. In some embodiments, a polynucleotide is a site or region of genomic DNA. In some embodiments, the polynucleotide is an endogenous gene contained within the genome of an unmodified cell or a universal donor cell. In some embodiments, the polynucleotide is an exogenous polynucleotide not integrated into genomic DNA. In some embodiments, the polynucleotide is an exogenous polynucleotide integrated into genomic DNA. In some embodiments, the polynucleotide is a plasmid or an adeno-associated virus vector. In some embodiments, the polynucleotide is a cyclic or linear molecule.

[0028] Safe harbor loci: As used herein, the term “safe harbor loci” generally refers to any location, site, or region of genomic DNA in which a gene insertion can be accommodated without adverse effects on the cell. In some embodiments, safe harbor loci are intra-genetic or extra-genetic regions. In some embodiments, safe harbor loci are typically regions of genomic DNA that are transcriptionally silent. In some embodiments, safe harbor loci are AAVS1(PPP1R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX(F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1, TF, or TTR loci. 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, pages 51–58.

[0029] Safety switch: As used herein, the term “safety switch” generally refers to a biomolecule that induces apoptosis in cells. In some embodiments, the safety switch is a protein or a gene. In some embodiments, the safety switch is a suicide gene. In some embodiments, the safety switch, such as herpes simplex virus thymidine kinase (HSV-tk), induces apoptosis in cells by metabolizing a prodrug, such as ganciclovir. In some embodiments, the presence of an overexpressed safety switch induces apoptosis in cells. In some embodiments, the safety switch is a p53 molecule, HSV-tk, or inducible caspase-9.

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

[0031] Survival Factors: As used herein, the term “survival factors” generally refers to proteins (expressed, for example, by polynucleotides as described herein) that, when increased or decreased in a cell, enable cells, such as universal donor cells, to survive with a higher viability rate after transplantation or engraftment into a host subject compared to unmodified cells. In some embodiments, the survival factors are human survival factors. In some embodiments, the survival factors are members of a critical pathway involved in cell survival. In some embodiments, the critical pathway involved in cell survival has effects on hypoxia, reactive oxygen species, nutrient deficiencies 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 after engraftment than unmodified cells, e.g., 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 number: 7702), TXNIP (NCBI gene ID number: 10628), FOXO1 (NCBI gene ID number: 2308), JNK (NCBI gene ID number: 5599), or MANF (NCBI gene ID number: 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, insertion of the polynucleotide encoding MANF allows cells, e.g., universal donor cells, to survive with a higher viability rate after transplantation or engraftment into a host target compared to unmodified cells. In some embodiments, deletion or insertion-deletion mutations within or near the ZFN143, TXNIP, FOXO1, or JNK genes allow cells, e.g., universal donor cells, to survive with a higher viability rate after transplantation or engraftment into a host target compared to unmodified cells.

[0032] Immunotogenic Factors: As used herein, the term “immunotogenic factor” generally refers to proteins (expressed, for example, by polynucleotides as described herein) that, when increased or decreased in cells, enable cells, such as universal donor cells, to inhibit or avoid immune rejection at a higher rate after transplantation or engraftment into a host target compared to unmodified cells. In some embodiments, the immunotolerogenic factor is a human immunotolerogenic factor. In some embodiments, genetic modification of at least one immunotolerogenic factor (e.g., insertion or deletion of at least one immunotolerogenic factor) enables cells, such as universal donor cells, to inhibit or avoid immune rejection at a rate at least 1.05, at least 1.1, at least 1.25, at least 1.5, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, or at least 50 times higher after engraftment than unmodified cells. In some embodiments, the immunotolerogenic 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 immunotolerogenic factor is inserted into cells, e.g., universal donor cells. In some embodiments, the immunotolerogenic factor is deleted from cells, e.g., universal donor cells. In some embodiments, insertion of polynucleotides encoding HLA-E, HLA-G, CTLA-4, CD47, and / or PD-L1 allows cells, e.g., universal donor cells, to inhibit or evade immune rejection after transplantation or engraftment into a host target.

[0033] 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 modulates, for example, increases or decreases, the expression of MHC-I and / or MHC-II human leukocyte antigens. In some embodiments, the biomolecule is a polynucleotide, for example, a gene or a protein. In some embodiments, the MHC-I or MHC-II transcriptional regulator will increase or decrease the cell surface expression of at least one MHC-I or MHC-II protein. In some embodiments, the MHC-I or MHC-II transcriptional regulator will 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 number: 4261) or NLRC5 (NCBI gene ID number: 84166). In some embodiments, deletion or reduction of CIITA or NLRC5 expression will decrease the expression of at least one MHC-I or MHC-II gene.

[0034] Universal donor cells: As used herein, the term “universal donor cells” generally refers to genetically modified cells that, compared to unmodified cells, are less likely to cause allogeneic rejection during cell transplantation and / or demonstrate increased post-transplant survival. 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 cell progenitor cells (HSPCs) (also called 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 subjects who have, are suspected of having, or are at risk of having a disease. In some embodiments, universal donor cells can differentiate into lineage-limited progenitor cells or fully differentiated somatic cells. In some embodiments, lineage-limited progenitor cells are pancreatic endodermal progenitor cells, pancreatic endocrine progenitor cells, mesenchymal progenitor cells, muscle progenitor cells, blast cells, hematopoietic progenitor cells, or neural progenitor cells. In some embodiments, fully differentiated somatic cells are endocrine cells such as pancreatic beta cells, epithelial cells, endodermal cells, macrophages, hepatocytes, adipocytes, renal cells, blood cells, or immune system cells. In some embodiments, fully differentiated somatic cells are cardiomyocytes.

[0035] Unmodified Cells: As used herein, the term “unmodified cells” refers to cells that have not been genetically modified and contain polynucleotides or genes encoding MHC-I, MHC-I, MHC-I or MHC-II transcription regulators, survival factors and / or immunotolerogenic factors. 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 cell progenitor cells (HSPCs) (also called 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, e.g., T cells, e.g., CD8+ T cells). When universal donor cells are compared “to unmodified cells,” universal donor cells and unmodified cells are of the same cell type or share a common parental cell line, for example, universal donor iPSCs are compared to unmodified iPSCs.

[0036] Intra-gene or near a gene: As used herein, the term “intra-gene or near a gene” means a site or region of genomic DNA that is an intron or extron component of the gene or is located adjacent to the gene. In some embodiments, a site of genomic DNA is intra-gene if it contains at least a portion of the intron or exon of the gene. In some embodiments, a site of genomic DNA located near a gene may be at the 5' or 3' end of the gene (e.g., the 5' or 3' end of the coding region of the gene). In some embodiments, a site of genomic DNA located near a gene may be a promoter region or repressor region that regulates the expression of the gene. In some embodiments, a site of genomic DNA located near a gene may be on the same chromosome as the gene. In some embodiments, a site or region of genomic DNA is near a gene if it is within 50Kb, 40Kb, 30Kb, 20Kb, 10Kb, 5Kb, 1Kb or more of the 5' or 3' end of the gene (e.g., the 5' or 3' end of the coding region of the gene).

[0037] II. Genome Editing Methods Genome editing generally refers to the process of modifying the nucleotide sequence of a genome in a preferred, precise, or predetermined manner. In some embodiments, using genome editing methods as described herein, for example, a CRISPR-endonuclease system, cells may be genetically modified as described herein, for example, to produce universal donor cells. In some embodiments, using genome editing methods as described herein, for example, a CRISPR-endonuclease system, cells may be genetically modified as described herein, for example, to introduce at least one gene modification in 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 in unmodified cells; to introduce at least one gene modification that increases the expression of at least one polynucleotide encoding an immunotolerogenic factor in unmodified cells; and / or to introduce at least one gene modification that increases or decreases the expression of at least one gene encoding a survival factor in unmodified cells.

[0038] Examples of genome editing methods described herein include methods that use site-specific nucleases to cleave deoxyribonucleic acid (DNA) at precise target locations in the genome, thereby creating single-strand or double-strand DNA breaks at specific locations within the genome. Such breaks may be repaired, and are regularly repaired, by innate endogenous cellular processes such as homologous recombination repair (HDR) and non-homologous end ligation (NHEJ), as described in Cox et al., “Therapeutic genome editing: prospects and challenges”, Nature Medicine, 2015, 21(2), 121-31. These two major DNA repair processes consist of families of alternative pathways. NHEJ directly ligates the DNA ends resulting from double-strand breaks, which may involve deletions or additions of nucleotide sequences and can disrupt or enhance gene expression. HDR utilizes homologous or donor sequences as templates for inserting predetermined DNA sequences at the break points. Homologous sequences may be present in endogenous genomes, such as sister chromatids. Alternatively, the donor sequence may be a foreign polynucleotide such as a plasmid, single-stranded oligonucleotide, double-stranded oligonucleotide, or virus, which has regions of high homology to the nuclease cleavage site (e.g., left and right homology arms) but may also contain additional sequences or sequence changes, including deletions that can be incorporated into the target site to be cleaved. A third repair mechanism may be a microhomology-mediated end junction (MMEJ), also called an "alternative NHEJ," in that the genetic outcome is similar to an NHEJ in that some deletions and insertions may occur at the cleavage site.MMEJs can promote more favorable DNA end ligation repair outcomes by utilizing homologous sequences of a few base pairs adjacent to the DNA break site, and recent reports have further elucidated the molecular mechanisms of this process; see, for example, Cho and Greenberg, Nature, 2015, 518, 174-76; Kent et al., Nature Structural and Molecular Biology, 2015, 22(3):230-7; Mateos-Gomez et al., Nature, 2015, 518, 254-57; Ceccaldi et al., Nature, 2015, 528, 258-62. In some cases, it may be possible to predict repair outcomes based on an analysis of potential microhomology at the DNA break site.

[0039] Each of these genome editing mechanisms can be used to create a desired gene modification. The steps in the genome editing process may involve creating one or two DNA breaks, the latter being a double-strand break or two single-strand breaks at a target locus near the intended mutation site. This can be achieved through the use of endonucleases as described and shown herein.

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

[0041] crRNA typically facilitates sequence recognition and specificity of the CRISPR-endonuclease complex through Watson-Crick base pairing with approximately 20 nucleotide (nt) sequences in target DNA. By altering the 5'20nt sequence in the crRNA, targeting of the CRISPR-endonuclease complex to specific loci becomes possible. The CRISPR-endonuclease complex simply binds to a DNA sequence containing a sequence that matches the first 20nt of a single guide RNA (sgRNA) if the target sequence is followed by a specific short DNA motif (containing the sequence NGG) called a protospacer adjacent motif (PAM).

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

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

[0044] In some embodiments, the endonuclease is Cas9 (CRISPR-related protein 9). In some embodiments, the Cas9 endonuclease is derived from Streptococcus pyogenes, but other Cas9 homologs, such as S. aureus Cas9, N. meningitidis Cas9, S. thermophilus CRISPR1 Cas9, S. thermophilus CRISPR3 Cas9, or T. denticola Cas9, may be used. In other cases, the CRISPR endonuclease is Cpf1, such as L. bacterium ND2006 Cpf1 or Acidaminococcus sp. BV3L6 Cpf1. In some embodiments, the endonucleases are 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, These are 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 endonucleases. In some embodiments, wild-type variants may be used. In some embodiments, modified versions of the aforementioned endonucleases (e.g., their homologs, recombinants of their naturally occurring molecules, their codon-optimized forms, or modified versions thereof) may be used.

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

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

[0047] Zinc finger nuclease Zinc finger nucleases (ZFNs) are modular proteins composed of an engineered zinc finger DNA-binding domain linked to the catalytic domain of the type II endonuclease FokI. Because FokI functions only as a dimer, a pair of ZFNs must be designed to bind to identical target "half-site" sequences on opposite DNA strands at precise intervals to enable the formation of catalytically active FokI dimers. When the FokI domain, which itself does not possess sequence specificity, dimerizes, a DNA double-strand break occurs between the half-sites of the ZFN, which serves as the initiation step of genome editing.

[0048] The DNA-binding domain of each ZFN typically consists of 3–6 zinc fingers with abundant Cys2-His2 structures. Each finger primarily recognizes triplets of nucleotides on a single strand of the target DNA sequence, although cross-chain interactions with a fourth nucleotide may also be important. Amino acid modifications of the fingers at the sites that make critical contact with the DNA alter the sequence specificity of a given finger. Thus, a 4-finger zinc finger protein will selectively recognize a 12 bp target sequence, which consists of triplet priorities given by each finger, although these triplets can be influenced to varying degrees by adjacent fingers. A key aspect of ZFNs is that they can be readily retargeted to most genomic addresses simply by modifying the individual fingers. In most ZFN applications, 4–6 finger proteins are used, each recognizing 12–18 bp. Therefore, a pair of ZFNs will typically recognize a combined target sequence of 24–36 bp that does not include the typical 5–7 bp spacers between half-sites. The binding site can be further separated by a larger spacer containing 15–17 bp. Target sequences of this length are thought to be unique within the human genome, and it is assumed that repeating sequences or gene homologs are excluded during the design process. Nevertheless, because the protein-DNA interactions of ZFNs are not absolute in their specificity, off-target binding and cleavage phenomena occur as heterodimers between two ZFNs or as homodimers of one or the other ZFN. The latter possibility was efficiently eliminated by manipulating the dimerization interface of the FokI domain to create “plus” and “minus” variants, also known as absolute heterodimer variants, that can dimerize to each other and not to themselves. By forcing absolute heterodimerization, homodimer formation is prevented. This significantly enhanced the specificity not only of ZFNs but also of any other nucleases employing these FokI variants.

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

[0050] Transcription activator-like effector nucleases (TALENs) TALENs, like ZFNs, represent another form of modular nuclease in which an engineered DNA-binding domain is ligated to a FokI nuclease domain, and pairs of TALENs work in series to achieve cleavage of targeted DNA. The main differences from ZFNs are the nature of the DNA-binding domain and the characteristics of the target DNA sequence recognition to which they associate. The TALEN DNA-binding domain originates from the TALE protein, which was first described in the plant pathogenic microorganism Xanthomonas sp. TALE consists of a series sequence of 33-35 amino acid repeats, each repeat typically recognizing one base pair in a target DNA sequence up to 20 bp in length, giving a total target sequence length of up to 40 bp. The nucleotide specificity of each repeat is determined by a repeating variable duo (RVD) containing two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine, and thymine are primarily recognized by four RVDs: Asn-Asn, Asn-Ile, His-Asp, and Asn-Gly, respectively. This results in a much simpler recognition code than zinc fingers, and therefore is more advantageous than zinc fingers in nuclease design. Nevertheless, like ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs have also benefited from the use of absolute heterodimer variants of the FokI domain, which reduces off-target activity.

[0051] Additional variants of the FokI domain, which is inactivated in catalytic function, have been created. If one of the TALEN or ZFN pair contains an inactive FokI domain, only single-strand DNA breaks (nicking) occur at the target site, rather than DSBs. This result is equivalent to using a "nickase" variant of CRISPR / Cas9 or CRISPR / Cpf1 (in which one of the Cas9 cleavage domains is inactivated). While DNA nicks can be used to drive HDR-based genome editing, they are less efficient than those using DSBs. The main advantage is that off-target nicks are repaired rapidly and accurately, unlike DSBs, which are prone to misrepair mediated by NHEJs.

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

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

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

[0055] MegaTAL / Tev-mTALEN / MegaTev As further examples of hybrid nucleases, the MegaTAL platform and the Tev-mTALEN platform utilize a fusion of the DNA-binding domain of TALE and the catalytically active HE, leveraging both the tunable DNA-binding and specificity of TALE as well as 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.

[0056] In further variants, the MegaTev structure is a fusion of the meganuclease (Mega) with the nuclease domain derived from the GIY-YIG homing endonuclease I-TevI ​​(Tev). The two active sites are located approximately 30 bp apart on the DNA substrate, generating two double-segment breaks (DSBs) with mismatched sticky ends; see, e.g., Wolfs et al., Nucleic Acids Res., 2014, 42, 8816-29. Other combinations of existing nuclease-based methods are expected to develop and be useful in realizing the targeted genome modifications described herein.

[0057] dCas9-FokI or dCpf1-Fok1 and other nucleases Combining the structural and functional properties of the nuclease platforms described above provides further genome editing techniques that may overcome some of their inherent shortcomings. For example, the CRISPR genome editing system typically uses a single Cas9 endonuclease to induce double-sequence branching (DSB). Targeting specificity is facilitated by 20 or 24 nucleotide sequences in the guide RNA that initiate Watson-Crick base pairing with the target DNA (and, in the case of Cas9 derived from S. pyogenes, an additional two bases in the adjacent NAG or NGG PAM sequence). While such sequences are long enough to be unique in the human genome, the specificity of RNA / DNA interactions is not absolute, and significant randomness may be tolerated, particularly in the 5' half of the target sequence, efficiently reducing the number of bases that facilitate specificity. One solution to this is to completely inactivate the catalytic function of Cas9 or Cpf1 (retaining only the RNA-guided DNA binding function) and instead fuse the FokI domain to the inactivated Cas9; see, 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 required to tether the two FokI fusions proximal to form the dimer and cleave the DNA. This essentially doubles the number of bases at the combined target site, thereby increasing the precision of targeting by CRISPR-based systems.

[0058] As a further example, the fusion of the TALE DNA-binding domain to catalytically active HE such as I-TevI ​​is expected to further reduce off-target cleavage, utilizing both the tunable DNA-binding and specificity of TALE and the cleavage sequence specificity of I-TevI.

[0059] RNA-induced endonucleaseThe RNA-induced endonuclease systems used herein may include amino acid sequences having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid sequence identity with a wild-type exemplary endonuclease, such as Cas9 from S. pyogenes (Sequence ID 8 in U.S. Patent Application Publication No. 2014 / 0068797 or Sapranauskas 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 derived from S. pyogenes, as described above) across 10 consecutive amino acids. The endonuclease may contain up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with the wild-type endonuclease (e.g., Cas9 derived from S. pyogenes, as described above) across 10 consecutive amino acids. 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 derived from S. pyogenes, as described above) across 10 consecutive amino acids in the HNH nuclease domain of the endonuclease. The endonuclease may contain up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with the wild-type endonuclease (e.g., Cas9 derived from S. pyogenes, as described above) across 10 consecutive amino acids in the HNH nuclease domain of the endonuclease.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 derived from S. pyogenes, as described above) across 10 consecutive amino acids in the RuvC nuclease domain of the endonuclease. The endonuclease may contain up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with the wild-type endonuclease (e.g., Cas9 derived from S. pyogenes, as described above) across 10 consecutive amino acids in the RuvC nuclease domain of the endonuclease.

[0060] Endonucleases may include modified forms of the wild-type exemplary endonucleases. Modified forms of the wild-type exemplary endonucleases may include mutations that reduce the nucleic acid cleavage activity of the endonucleases. Modified forms of the 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 the wild-type exemplary endonucleases (e.g., Cas9 from S. pyogenes, above). Modified forms of endonucleases may not have substantial nucleic acid cleavage activity. When an endonuclease is a modified form that does not have substantial nucleic acid cleavage activity, it is referred to herein as “enzyme-inactive.”

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

[0062] Guide RNA This disclosure provides guide RNA (gRNA) capable of directing the activity of an endonucleases relevant to a specific target site within a polynucleotide. The guide RNA may include at least one spacer sequence that hybridizes to a target nucleic acid sequence of interest and a CRISPR repeat sequence. In a type II CRISPR system, the gRNA also includes a second RNA called a tracrRNA sequence. In a type II CRISPR guide RNA (gRNA), the CRISPR repeat sequence and the tracrRNA sequence hybridize to each other to form a double helix. In a type V CRISPR system, the gRNA includes a crRNA that forms the double helix. In some embodiments, the gRNA can bind to an endonucleases, resulting in the formation of a complex between the gRNA and the endonucleases. The gRNA can provide target specificity to the complex for association with the endonucleases. Thus, genome-targeted nucleic acids can direct the activity of endonucleases.

[0063] Exemplary guide RNAs include spacer sequences containing 15–200 nucleotides, and the gRNAs target genomic locations 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.

[0064] gRNA can be a bimolecule guide RNA. gRNA can be a single-molecule guide RNA.

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

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

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

[0068] In some embodiments, the sgRNA includes a spacer extension sequence containing another portion (e.g., a stability control sequence, an endoribonuclease binding sequence, or a ribozyme). The portion can reduce or increase the stability of the nucleic acid being targeted. The portion may be a transcriptional terminator segment (i.e., a transcription termination sequence). The portion may function in eukaryotic cells. The portion may function in prokaryotic cells. The portion may function in both eukaryotic and prokaryotic cells. Non-limiting examples of preferred parts include 5' caps (e.g., 7-methylguanylate caps (m7 G)), riboswitch sequences (e.g., enabling regulation of stability and / or contactability by proteins and protein complexes), sequences that form dsRNA double helix (i.e., hairpins), sequences that target RNA to intracellular locations (e.g., nucleus, mitochondria, chloroplasts, etc.), modifications or sequences that provide tracking (e.g., direct conjugation to fluorescent molecules, conjugation to regions that facilitate fluorescence detection, sequences that enable 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.).

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

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

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

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

[0073] In some cases, the complementarity percentage between the spacer sequence and the target nucleic acid is at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100%. In some cases, the complementarity percentage between the spacer sequence and the target nucleic acid is up to approximately 30%, up to approximately 40%, up to approximately 50%, up to approximately 60%, up to approximately 65%, up to approximately 70%, up to approximately 75%, up to approximately 80%, up to approximately 85%, up to approximately 90%, up to approximately 95%, up to approximately 97%, up to approximately 98%, up to approximately 99%, or 100%. In some examples, the complementarity percentage between the spacer sequence and the target nucleic acid is 100% over the majority of the six consecutive 5' nucleotides of the target sequence in the complementary strand of the target nucleic acid. The complementarity percentage 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 may differ by 1 to 6 nucleotides, which can be considered as one or more bulges.

[0074] TracrRNA sequences may contain nucleotides that hybridize to minimal CRISPR repeat sequences in cells. Minimal tracrRNA sequences and minimal CRISPR repeat sequences may form a double-stranded structure, i.e., a base-paired double-stranded structure. Together, minimal tracrRNA sequences and minimal CRISPR repeats can bind to RNA-induced endonucleases. At least a portion of the minimal tracrRNA sequence can hybridize to minimal CRISPR repeat sequences. Minimal tracrRNA sequences may be at least approximately 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to minimal CRISPR repeat sequences.

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

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

[0077] The double helix between the smallest CRISPR RNA and the smallest tracrRNA may contain a double helix. The double helix between the smallest CRISPR RNA and the smallest tracrRNA may 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 may contain up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.

[0078] A double helix can contain mismatches (i.e., the two strands of a double helix are not 100% complementary). A double helix can contain at least approximately 1, 2, 3, 4, or 5 mismatches. A double helix can contain up to approximately 1, 2, 3, 4, or 5 mismatches. A double helix can contain 2 or fewer mismatches.

[0079] 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 S. pyogenes).

[0080] In some embodiments, the gRNA may include a tracrRNA extension sequence. The tracrRNA extension sequence may have a length of about 1 nucleotide to about 400 nucleotides. The tracrRNA extension sequence may have a length of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or more than 200 nucleotides. The tracrRNA extension sequence may have a length of about 20 to about 5000 or more nucleotides. The tracrRNA extension sequence may have a length of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or less than 100 nucleotides. The tracrRNA extension sequence may have a nucleotide length of less than 10. The tracrRNA extension sequence may be 10 to 30 nucleotides long. The tracrRNA extension sequence can be 30 to 70 nucleotides long.

[0081] The tracrRNA extension sequence may contain a functional moiety (e.g., a stability control sequence, a ribozyme, or an endoribonuclease binding sequence). The functional moiety may contain a transcription terminator segment (i.e., a transcription termination sequence). The functional moiety may have a total length of approximately 10 nucleotides (nt) to approximately 100 nucleotides, approximately 10 nt to approximately 20 nt, approximately 20 nt to approximately 30 nt, approximately 30 nt to approximately 40 nt, approximately 40 nt to approximately 50 nt, approximately 50 nt to approximately 60 nt, approximately 60 nt to approximately 70 nt, approximately 70 nt to approximately 80 nt, approximately 80 nt to approximately 90 nt, or approximately 90 nt to approximately 100 nt, approximately 15 nt to approximately 80 nt, approximately 15 nt to approximately 50 nt, approximately 15 nt to approximately 40 nt, approximately 15 nt to approximately 30 nt, or approximately 15 nt to approximately 25 nt.

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

[0083] The linker may contain any of a variety of sequences, but in some examples, the linker will not contain a sequence that has extensive regions homologous to other parts of the guide RNA, which could cause intramolecular binding that would interfere with other functional regions of the guide. In Jinek et al., a simple 4-nucleotide sequence—GAAA—was used (Jinek et al., Science, 2012, 337(6096):816-821), but many other sequences, including longer sequences, could be used similarly.

[0084] A linker sequence may contain functional moieties. For example, a linker sequence may contain one or more features, including aptamers, ribozymes, protein-interaction hairpins, protein binding sites, CRISPR arrays, introns, or exons. A linker sequence may contain at least about 1, 2, 3, 4, or 5 or more functional moieties. In some examples, a linker sequence may contain up to about 1, 2, 3, 4, or 5 or more functional moieties.

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

[0086] sgRNA can be chemically modified. In some embodiments, the chemically modified gRNA is a gRNA containing at least one nucleotide having a chemical modification, such as a 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 a 2'-O-methyl-phosphorothioate residue. In some embodiments, the chemical modification enhances stability, reduces the likelihood or degree of innate immune response, and / or enhances other properties, as described in the Art.

[0087] In some embodiments, the modified gRNA may include a modified backbone, such as a phosphorothioate, phosphotriester, morpholino, methylphosphonate, short alkyl or cycloalkyl intersaccharide bonds, or short heteroatoms or heterocyclic intersaccharide bonds.

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

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

[0090] In some embodiments, the modified gRNA may contain one or more substituted sugar moieties at the 2' position, for example: OH, SH, SCH3, F, OCN, OCH3, OCH3O(CH2)nCH3, O(CH2)nNH2 or O(CH2)nCH3 (where n is 1 to about 10); C1-C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S - or N-alkyl; O-, S- or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkalyl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; interfering substance; 2'-O-(2-methoxyethyl); 2'-methoxy(2'-O-CH3); 2'-propoxy(2'-OCH2CH2CH3); and 2'-fluoro(2'-F). Similar modifications can be made at other positions on gRNA, particularly at the 3' position of the sugar on the 3' terminal nucleotide and at the 5' position of the 5' terminal nucleotide. In some examples, both the sugar and nucleoside bonds of the nucleotide unit, i.e., the backbone, can be replaced with novel groups.

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

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

[0093] Genome-targeted nucleic acid and endonuclease complex gRNA interacts with endonucleases (such as RNA-induced nucleases like Cas9) to form a complex. The gRNA directs the endonuclease to a target polynucleotide.

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

[0095] Nucleic acid coding system components This disclosure provides nucleic acids comprising nucleotide sequences encoding the genome-targeted nucleic acids of this disclosure, endonucleases of this disclosure, and / or any nucleic acid or proteinaceous molecules necessary to carry out embodiments of the methods of this disclosure. The encoding nucleic acid may be RNA, DNA, or a combination thereof.

[0096] The nucleic acids encoding the genome-targeted nucleic acids of this disclosure, the endonucleases of this disclosure, and / or any nucleic acids or protein molecules necessary to carry out embodiments of the methods of this disclosure may include vectors (e.g., recombinant expression vectors).

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

[0098] In some cases, vectors can direct the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” or, more simply, “expression vectors” that perform the same function.

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

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

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

[0102] Non-limiting examples of suitable eukaryotic promoters (i.e., promoters that function in eukaryotic cells) include those derived from the earliest cytomegalovirus (CMV), herpes simplex virus (HSV) thymidine kinase, early and late SV40, retroviral terminal repeat sequences (LTRs), human elongation factor-1α promoter (EF1α), chicken vertebra-actin promoter (CBA), ubiquitin C promoter (UBC), hybrid constructs (CAG) containing a cytomegalovirus enhancer fused to the chicken vertebra-actin promoter, promoters, hybrid constructs (CAG or CAGGS) containing a cytomegalovirus enhancer fused to the first exon and the first intron of the chicken vertebra-actin gene, mouse stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I promoter.

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

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

[0105] III. Strategies to evade immune responses and increase survival This specification describes strategies that enable genetically modified cells, i.e., universal donor cells, to survive or increase their viability after engraftment in a subject and / or to evade immune responses. In some embodiments, these strategies enable universal donor cells to survive with a higher success rate than unmodified cells and / or to evade immune responses. In some embodiments, the genetically modified cells include the introduction of at least one genetic modification in or near at least one gene encoding a survival factor, wherein the genetic modification includes the insertion of a polypeptide encoding an immunotolerogenic factor. The universal donor cells further include at least one genetic modification in or near a gene encoding one or more MHC-I or MHC-II human leukocyte antigens or components of the MHC-I or MHC-II complex or transcription regulators, wherein the genetic modification includes the insertion of a polypeptide encoding a second immunotolerogenic factor.

[0106] In some embodiments, genetically modified cells include, in addition to unmodified cells, at least one gene modification in or near at least one gene that reduces the expression of one or more MHC-I and MHC-II human leukocyte antigens; at least one gene modification that increases the expression of at least one polynucleotide encoding an immunotolerogenic factor; and at least one gene modification that alters the expression of at least one gene encoding a survival factor. In other embodiments, genetically modified cells include, in addition to unmodified cells, at least one deletion or insertion-deletion mutation in or near at least one gene that alters the expression of one or more MHC-I and MHC-II human leukocyte antigens; and at least one insertion of a polynucleotide encoding at least one immunotolerogenic factor at a site that partially overlaps, completely overlaps with, or is contained within a deletion site of a gene that alters the expression of one or more MHC-I and MHC-II HLA. In yet another embodiment, genetically modified cells include, in addition to unmodified cells, at least one gene modification that alters the expression of at least one gene encoding a survival factor.

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

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

[0109] In some embodiments, the cells include genetic modifications of one or more MHC-I or MHC-II genes. In some embodiments, the cells include genetic modifications of one or more polynucleotide sequences that regulate the expression of MHC-I and / or MHC-II. In some embodiments, the genetic modifications of this disclosure are carried out using any gene editing method, including but not limited to the methods described herein.

[0110] In some embodiments, reducing the expression of one or more MHC-I and MHC-II human leukocyte antigens in unmodified cells is achieved, for example, by targeting for direct gene 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 in unmodified cells is achieved, for example, by targeting the CIITA gene for gene deletion. In some embodiments, reducing the expression of one or more MHC-I and MHC-II human leukocyte antigens in unmodified cells is achieved, for example, by targeting at least one transcription factor of MHC-I or MHC-II for gene deletion. In some embodiments, the transcription factor of MHC-I or MHC-II is NLRC5 or the CIITA gene. In some embodiments, the MHC-I or MHC-II transcription regulators are the RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, IRF-1, and / or TAP1 genes.

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

[0112] In some embodiments, the cell genome is modified to reduce the expression of beta-2-microglobulin (B2M). B2M is a non-polymorphic gene that encodes a shared protein subunit required for the surface expression of all polymorphic MHC class I heavy chains. The HLA-I protein directly associates with B2M in the endoplasmic reticulum, which is essential for forming a functional cell surface-expressed HLA-I molecule. In some embodiments, the gRNA targets a site in the B2M gene containing the 5'-GCTACTCTCTCTTTCTGGCC-3' sequence (SEQ ID NO: 1). In some embodiments, the gRNA targets a site in the B2M gene containing the 5'-GGCCGAGATGTCTCGCTCCG-3' sequence (SEQ ID NO: 2). In some embodiments, the gRNA targets a site in 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: 43), or 5'-ACTCTCTCTTTCTGGCCTGG-3' (SEQ ID NO: 44). In some embodiments, the gRNA contains one of the polynucleotide sequences 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 a target site in the B2M locus.Repair of double-strand breaks by NHEJ may result in deletions and / or insertions of at least one nucleotide, thereby disrupting or eliminating B2M expression. Alternatively, the B2M locus may be targeted by at least two CRISPR systems, each containing a different gRNA, resulting in breaks at two sites within the B2M locus leading to sequence deletions between the two breaks, thereby eliminating B2M expression.

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

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

[0115] In some embodiments, the cell genome is modified to reduce the expression of CARD domain-containing 5 (NLRC5), a member of the NLR family. NLRC5 is a crucial regulator of the MHC-I mediated immune response, and like CIITA, NLRC5 is highly induceable 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.

[0116] In some embodiments, immunotolerogenic factors can be inserted or reinserted into genetically modified cells to produce immune-privileged universal donor cells. In some embodiments, the universal donor cells disclosed herein are further modified to express one or more immunotolerogenic factors. Exemplary immunotolerogenic 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 immunotolerogenic factor allows universal donor cells to inhibit or evade immune rejection at a rate 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 higher than unmodified cells after engraftment. In some embodiments, insertion of polynucleotides encoding HLA-E, HLA-G, CTLA-4, CD47, and / or PD-L1 allows universal donor cells to inhibit or evade immune rejection after transplantation or engraftment into a host target.

[0117] Polynucleotides encoding immunotolerogenic factors generally include left and right homology arms adjacent to the sequence encoding the immunotolerogenic factor. These homology arms have substantial sequence homology to the genomic DNA located at or near the targeted insertion site. For example, the left homology arm may be a nucleotide sequence homologous to a region located to the left or upstream of the target or cleavage site, and the right homology arm may be a nucleotide sequence homologous to a region located to the right or downstream of the target or cleavage site. The proximal end of each homology arm may be homologous to a genomic DNA sequence adjacent to the cleavage site. Alternatively, the proximal end of each homology arm may be homologous to a genomic DNA sequence located up to approximately 10, 20, 30, 40, 50, 60, or 70 nucleic acid bases away from the cleavage site. Therefore, polynucleotides encoding immunotolerogenic factors may be inserted into targeted gene loci within approximately 10, 20, 30, 40, 50, 60, or 70 base pairs of the cleavage site, and further genomic DNA adjacent to the cleavage site (and not homologous to the homology arm) may be deleted. The homology arm may range in length from approximately 50 to several thousand nucleotides. In some embodiments, the homology arm may range in length from approximately 500 to approximately 1000 nucleotides. Substantial sequence homology between the homology arm and the genomic DNA may be at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, or at least approximately 99%.

[0118] In some embodiments, the homology arm is used with a B2M guide (e.g., a gRNA containing the nucleotide sequences of SEQ ID NOs. 1-3, 35-44). In some embodiments, the homology arm is designed to be used with any B2M guide that will remove the start site of the B2M gene. In some embodiments, the B2M homology arm may or may be composed of the polynucleotide sequence of SEQ ID NOs. 7 or 13 or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 7 or 13. In some embodiments, the left B2M homology arm may or may be composed of the polynucleotide sequence of SEQ ID NOs. 7 or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 7. In some embodiments, the right B2M homology arm may or may be composed of the polynucleotide sequence of SEQ ID NOs. 13 or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 13.

[0119] In some embodiments, the homology arm is used in conjunction with a TXNIP guide (e.g., a gRNA containing the nucleotide sequences of SEQ ID NOs. 15–24). In some embodiments, the homology arm is designed to be used in conjunction with any TXNIP guide that targets exon 1 of TXNIP (e.g., a gRNA containing the nucleotide sequences of SEQ ID NOs. 15–20). In some embodiments, the TXNIP homology arm may or may be comprised of the polynucleotide sequence of SEQ ID NOs. 25 or 32, or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 25 or 32. In some embodiments, the left TXNIP homology arm may or may be comprised of the polynucleotide sequence of SEQ ID NOs. 25, or a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity with SEQ ID NOs. 25. In some embodiments, the right TXNIP homology arm may include, or may be essentially, a polynucleotide sequence having at least 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 32 or the sequence of SEQ ID NO: 32.

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

[0121] In some embodiments, at least one polynucleotide encoding at least one immunotolerogenic factor is inserted into a safe harbor locus, for example, the AAVS1 locus. In some embodiments, at least one polynucleotide encoding at least one immunotolerogenic factor is inserted into a site or region of genomic DNA that partially, completely, or contains (i.e., is in or near) an MHC-I gene, an MHC-II gene, or a transcription regulator of MHC-I or MHC-II.

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

[0123] In some embodiments, the polynucleotide encoding HLA-E is inserted at or near the B2M gene locus. In some embodiments, the polynucleotide encoding HLA-E is inserted at a site corresponding to, or following, a deletion of all or part of, the B2M gene or its promoter, within or near the B2M gene locus. The polynucleotide encoding HLA-E is operably ligated to an exogenous promoter. The exogenous promoter may be the CMV promoter. In some embodiments, the polynucleotide includes the nucleotide sequence of SEQ ID NOs. 26, 27, 28, 29, 30 and / or 30. In some embodiments, the polynucleotide includes the nucleotide sequence of SEQ ID NOs. 55.

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

[0125] In some embodiments, the polynucleotide encoding CD47 is inserted within or near the CIITA gene locus. In some embodiments, the polynucleotide encoding CD47 is inserted at a site corresponding to, or following, a deletion in the CIITA gene or its promoter, within or near the CIITA gene locus.

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

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

[0128] In some embodiments, the cells include an increase or decrease in the expression of one or more survival factors. In some embodiments, the cells include an insertion of one or more polynucleotide sequences encoding a survival factor. In some embodiments, the cells include a deletion of one further survival factor. In some embodiments, the genetic modification of this disclosure is carried out using any gene editing method, including but not limited to the methods described herein. In some embodiments, the cells include an increase or decrease in the expression of at least one survival factor compared to unmodified cells. In some embodiments, the survival factor is a member or decisive pathway involved in cell survival, e.g., hypoxia, reactive oxygen species, nutrient deficiency and / or oxidative stress. In some embodiments, genetic modification of at least one survival factor allows universal donor cells to survive for a longer period after engraftment than unmodified cells, e.g., 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.

[0129] In some embodiments, the cells include an insertion of a polynucleotide encoding MANF, enabling universal donor cells to survive with a higher viability rate after transplantation or engraftment into a host target compared to unmodified cells. In some embodiments, the polynucleotide encoding MANF is inserted into a safe harbor locus. In some embodiments, the polynucleotide encoding MANF is inserted into a gene belonging to an MHC-I, MHC-II, or MHC-I or MHC-II transcription regulator.

[0130] In some embodiments, the cell genome is modified to delete all or part of the ZNF143, TXNIP, FOXO1, and / or JNK genes. In some embodiments, the cell genome is modified to delete all or part of the promoter regions of the ZNF143, TXNIP, FOXO1, and / or JNK genes.

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

[0132] In certain 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 MHC-I / II surface expression are further edited to have PD-L1 expression, for example, an insertion of a polynucleotide encoding PD-L1. In yet another embodiment, cells lacking MHC-I / II surface expression are further edited to have PD-L1 expression, for example, an insertion of a polynucleotide encoding PD-L1, and are also genetically modified to increase or decrease the expression of at least one gene encoding a survival factor compared to unmodified cells.

[0133] In some embodiments, the cell further includes increased or decreased expression of one or more further genes, for example, by genetic modification, which are not necessarily related to either immune evasion after engraftment or cell survival. In some embodiments, the cell further includes increased expression of one or more safety switch proteins compared to unmodified cells. In some embodiments, the cell includes increased expression of one or more further 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, at least one polynucleotide encoding a safety switch is inserted into the genome, for example, at a safe harbor locus. In some other embodiments, the one or more further genes to be genetically modified encode one or more safety switch proteins; targeted modalities; receptors; signaling molecules; transcription factors; pharmaceutically active proteins or peptides; drug target candidates; and proteins that promote their engraftment, transport, migration, viability, self-renewal, persistence and / or survival.

[0134] One aspect of the present invention provides a method for producing genome-modified universal donor cells, the universal donor cells comprising at least one targeted genome modification at one or more selected sites in the genome, the method comprising: genetically engineering the cells to a cell type as described herein by introducing one or more constructs into the cells that enable targeted modifications at the selected sites; introducing one or more double-strand breaks into the cells at the selected sites using one or more endonucleases capable of recognizing the selected sites; culturing the cells edited to enable endogenous DNA repair for producing targeted insertions or deletions at the selected sites; and thereby obtaining genome-modified universal donor cells. The genome-modified donor cells may undergo a series of rounds of genome modifications so that multiple sites are targeted and modified. The genome-modified cells are cultured, characterized, selected, and grown using techniques well known in the art. Universal donor cells produced by this method will contain at least one functionally targeted genome modification, and the genome-modified cells can differentiate into progenitor cells or fully differentiated cells, if they are stem cells.

[0135] In some other embodiments, the genome-engineered universal donor cells include 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 cells are HLA class I and / or class II deficiencies. In some embodiments, the genome-engineered universal donor cells include B2M null or insufficient B2M. In some embodiments, the genome-engineered universal donor cells include embedded or unembedded exogenous polynucleotides encoding one or more of HLA-E, HLA-G, and PD-L1. In some embodiments, the introduced expression is an increase in expression from any of the genes present in the cells that are not expressed or are insufficiently expressed. In some embodiments, the unembedded exogenous polynucleotides are introduced using Sendai virus, AAV, episome, or plasmid. In some embodiments, the universal donor cells are B2M null and include the introduction of expression of one or more of HLA-E, HLA-G, and PD-L1, and an increase or decrease in the expression of at least one safety switch protein. In another embodiment, the universal donor cells are HLA-A, HLA-B, and HLA-C null, and involve the introduction of expression of one or more HLA-E, HLA-G, PD-L1, and at least one safety switch protein. In some embodiments, the universal donor cells are B2M null, and involve the introduction of expression of one or more HLA-E, HLA-G, and PD-L1, and an increase or decrease in the expression of at least one survival factor, such as MANF. The method for producing any of the genetically modified cells described herein is intended to be carried out using at least one of the gene editing methods described herein.

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

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

[0138] Stem cells possess both the ability to proliferate and to generate further progenitor cells, and these have the ability to produce a number of parent cells that can give rise to differentiated daughter cells or differentiateable daughter cells. The daughter cells themselves can be induced to proliferate and subsequently differentiate into one or more mature cell types, but they also retain one or more cells that have the parental developmental potential. The term “stem cell” then refers to a cell that has the ability or potential to differentiate into a more specific phenotype or differentiated phenotype under certain circumstances, and that retains the ability to proliferate substantially without differentiation under certain circumstances. In one aspect, the terms progenitor cell or stem cell refer to a general parent cell, whose offspring (progeny) often specialize in different directions by acquiring completely distinct characteristics, as seen in differentiation, for example, the gradual diversification of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can originate from pluripotent cells, which themselves are derived from pluripotent cells, etc. Each of these pluripotent cells can be considered a stem cell, but the range of cell types that each can give rise to can vary considerably. Some differentiated cells also possess the ability to produce cells with higher developmental potential. Such ability may be naturally occurring or can be artificially induced through treatment with various factors. In many biological examples, stem cells can also be "pluripotent," meaning they can produce offspring of two or more different cell types, but this is not necessary for them to be "stem cells."

[0139] A "differentiated cell" is a cell that has progressed further in its developmental pathway than the cell being compared. Therefore, stem cells can differentiate into lineage-limited precursor cells (such as muscle cell precursors), which can then differentiate into other types of precursor cells in more advanced pathways (such as muscle cell precursors), and subsequently into final-stage differentiated cells such as muscle cells, which may or may not retain the ability to play a characteristic role in certain tissue types and to proliferate further. In some embodiments, the differentiated cell may be pancreatic beta cells.

[0140] embryonic stem cells The cells described herein may be embryonic stem cells (ESCs). ESCs originate from undifferentiated germ cells of mammalian embryos, can differentiate into any cell type, and can proliferate rapidly. ESCs are also thought to possess a normal karyotype, maintain high telomerase activity, and exhibit remarkable long-term proliferative potential, making them excellent candidates for use as universal donor cells.

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

[0142] induced pluripotent stem cells The cells described herein may be induced pluripotent stem cells (iPSCs). iPSCs can be directly generated from adult human cells by introducing genes encoding key transcription factors involved in pluripotency, such as OCT4, SOX2, cMYC, and KLF4. The iPSCs may originate from the same subject to which subsequent progenitor cells are administered. That is, somatic cells may be obtained from a subject, reprogrammed into induced pluripotent stem cells, and then redifferentiated into progenitor cells (e.g., autologous cells) to be administered to the subject. However, in the case of autologous cells, risks of post-engraft immune responses and insufficient viability remain.

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

[0144] Cell differentiation into other cell types Another step of the method of the present disclosure may include differentiating cells into differentiated cells. The differentiation step may be carried out according to any method known in the art. For example, human iPSCs are differentiated into embryonic endoderm using various treatments including activin and B27 supplement (Life Technologies). Embryonic endoderm are further differentiated into hepatocytes, the treatments including FGF4, HGF, BMP2, BMP4, oncostatin M, dexamethasone, etc. (Duan et al, Stem Cells, 2010;28:674-686; Ma et al, Stem Cells Translational Medicine, 2013;2:409-419). In another embodiment, the differentiation step may be carried out according to Sawitza et al, Sci Rep. 2015;5:13320. Differentiated cells may be any somatic cells of a mammal, e.g., human. In some embodiments, somatic cells may be exocrine epithelial cells (e.g., salivary gland mucinous cells, prostate cells), hormone-secreting cells (e.g., anterior pituitary cells, intestinal cells, pancreatic islets), keratinizing epithelial cells (e.g., epidermal keratinocytes), moist stratified barrier epithelial cells, sensory conversion cells (e.g., photoreceptors), autonomic nerve cells, sensory organ and peripheral neuron supporting cells (e.g., Schwann cells), central nervous system neurons, glial cells (e.g., astrocytes, oligodendrocytes), lenticular cells, adipocytes, renal 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., spermatids), nurse cells, or stromal cells.

[0145] In general, populations of universal donor cells disclosed herein maintain the 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 universal donor cells express one or more immunotolerogenic factors. Furthermore, populations of lineage-limited cells or fully differentiated cells derived from universal donor cells disclosed herein maintain the 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 lineage-limited cells or fully differentiated cells express one or more immunotolerogenic factors.

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

[0147] Guide RNA and / or polynucleotides may be formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, and diluents, depending on the specific mode of administration and dosage form. Guide RNA and / or polynucleotide compositions may be formulated to reach a pH range of physiologically compatible pH and pH range of about 3 to about 11, or pH range of about 3 to about 7, depending on the formulation and route of administration. In some cases, the pH may be adjusted to a range of about pH 5.0 to about pH 8. In some cases, the composition may contain at least one compound in a therapeutically effective amount as described herein, together with one or more pharmaceutically acceptable excipients. Optionally, the composition may contain combinations of compounds described herein, or a second active ingredient (but not limited to, antibacterial or antimicrobial agents) useful in treating or preventing bacterial growth, or a combination of reagents of the Disclosure.

[0148] Suitable excipients include, for example, carrier molecules containing large, slowly metabolizing polymers such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive virus particles. Other exemplary excipients include antioxidants (but not limited to, ascorbic acid), chelating agents (but not limited to, EDTA), carbohydrates (but not limited to, dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose), stearic acid, liquids (but not limited to, oil, water, saline solution, glycerol, and ethanol), wetting agents or emulsifiers, and pH buffering agents.

[0149] Guide RNA polynucleotides (RNA or DNA) and / or endonuclease polynucleotides (RNA or DNA) may be delivered by viral or nonviral delivery vehicles known in the art. Alternatively, endonuclease polypeptides may be delivered by viral or nonviral delivery vehicles known in the art, such as electroporation or lipid nanoparticles. In a further alternative embodiment, DNA endonucleases may be delivered as one or more polypeptides, either alone or in a pre-complexed state with one or more crRNAs combined with one or more guide RNAs or tracrRNAs.

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

[0151] With respect to the polynucleotides of this disclosure, formulations may be selected from, for example, those taught in International Application PCT / U.S. Patent Application Publication No. 2012 / 069610.

[0152] Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding endonucleases, can be delivered to cells or targets by lipid nanoparticles (LNPs).

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

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

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

[0156] LNPs can be generated using any lipid or combination of lipids known in the art. 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.

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

[0158] Recombinant adeno-associated virus (AAV) vectors may be used for delivery. Methods for generating rAAV particles, which provide a packaged AAV genome containing the delivered polynucleotides, rep and cap genes, and helper virus function to a cell, 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, AAV rep and cap genes isolated from (i.e., not contained within) the rAAV genome, and helper virus function. The AAV rep and cap genes may be derived from any AAV serotype, which may be derived from a recombinant virus and may be derived from an AAV serotype different from the rAAV genome ITR, including but not limited to the AAV serotypes described herein. The generation of pseudotyped rAAV is disclosed, for example, in brochure International Publication No. 01 / 83692.

[0159] Formulation and administration of cells, such as universal donor cells. Genetically modified cells, such as universal donor cells, as described herein, can be formulated and administered to a subject in any manner known in the art.

[0160] The terms “administer,” “introduce,” “implant,” “engraft,” and “transplant” are interchangeable in context in relation to the placement of cells, such as progenitor cells, into a target by a method or route that results in at least partial localization of introduced cells at a desired site. Cells, such as progenitor cells or their differentiated progeny, may be administered by any suitable route that results in delivery to a desired location in the target, in which at least some of the implanted cells or components of the cells remain viable. The survival period of cells after administration to a target can range from a short period of a few hours (e.g., 24 hours) to several days, several years, or even the lifespan of the target (i.e., long-term engraftment).

[0161] Genetically modified cells, such as universal donor cells, as described herein, may be able to survive for a longer period after administration to a subject than unmodified cells.

[0162] In some embodiments, compositions comprising cells as described herein may be administered by preferred routes, which may include, for example, large doses over a period of time or intravenous administration by continuous infusion. In some embodiments, intravenous administration may be carried out by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, synovial, 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 sprayed or lyophilized. In some embodiments, the sprayed or lyophilized form may be reconstituted with an aqueous or liquid solution.

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

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

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

[0166] In some embodiments, the cell-containing composition may be administered to subjects who have, are suspected of having, or are at risk of having, a disease, such as human subjects. In some embodiments, the composition may be administered to subjects who do not have, are not suspected of having, or are not at risk of having, a disease. In some embodiments, the subject is a healthy human. In some embodiments, the subject has, is suspected of having, or is at risk of having, a genetically inherited disease, such as human subjects. In some embodiments, the subject suffers from or is at risk of developing symptoms of the disease. In some embodiments, the disease is diabetes, such as type 1 diabetes or type 2 diabetes.

[0167] VI. Specific compositions and methods of this disclosure Therefore, this disclosure relates in particular to the following non-limiting compositions and methods.

[0168] The present disclosure provides a composition comprising a universal donor cell containing a nucleotide sequence encoding a first immunotolerogenic factor, which is inserted into or near a gene encoding a survival factor, wherein the universal donor cell expresses the immunotolerogenic factor and has disrupted expression of the survival factor, and the universal donor cell has increased immune evasion and / or cell survival compared to a control cell.

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

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

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

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

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

[0174] In another composition, composition 7, the 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, which includes a B2M signal peptide fused to an HLA-G presenting peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide.

[0175] In another composition, composition 8, the disclosure provides a composition as provided in composition 7, wherein the sequence encoding the HLA-E trimer essentially consists of sequence number 55.

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

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

[0178] In another composition, composition 11, the present disclosure further comprises a nucleotide sequence encoding a second immunotolerogenic factor, which is inserted into or near a gene encoding an MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor, and provides a composition as provided in any one of claims 1 to 10, wherein universal donor cells express the immunotolerogenic factor and have disrupted expression of the MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor.

[0179] In another composition, composition 12, the present disclosure provides a composition as provided in composition 11, which includes disrupted expression of MHC-I or MHC-II human leukocyte antigen or components of the MHC-I or MHC-II complex or transcription regulators, or reduced or absent expression.

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

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

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

[0183] In another composition, composition 16, the present disclosure provides a composition as provided in composition 15, wherein the nucleotide sequence encoding PD-L1 is essentially derived from sequence number 11.

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

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

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

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

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

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

[0190] In other compositions, composition 23, the present disclosure provides compositions as provided in any one of compositions 1 to 19, wherein cells can differentiate into lineage-limited progenitor cells or fully differentiated somatic cells.

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

[0192] In another composition, composition 25, the present disclosure provides a composition as provided in composition 23, wherein the fully differentiated somatic cells are pancreatic beta cells, epithelial cells, endodermal cells, macrophages, hepatocytes, adipocytes, renal cells, hematopoietic cells, cardiomyocytes, or immune system cells.

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

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

[0195] In another composition, composition 28, the present disclosure provides a composition as provided in composition 27, wherein the lineage-limited 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 beta cells, epithelial cells, endodermal cells, macrophages, hepatocytes, adipocytes, renal cells, blood cells, cardiomyocytes, or immune system cells.

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

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

[0198] In another composition, composition 31, the present disclosure provides a composition as provided in composition 30, wherein the lineage-limited progenitor cells are embryonic endoderm cells, primitive intestinal cells, posterior foregut cells, pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, immature beta cells or mature beta cells, and the fully differentiated somatic cells are pancreatic beta cells.

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

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

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

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

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

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

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

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

[0207] In another composition, composition 40, the disclosure provides a composition as provided in composition 39, wherein the subject has type 1 diabetes or type 2 diabetes.

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

[0209] In the first method, Method 1, the Disclosure provides a method for obtaining cells for administration to a subject in need thereof, 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 sufficient amount of time and under conditions to differentiate into lineage-limited progenitor cells or fully differentiated somatic cells.

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

[0211] In another method, Method 3, the Disclosure provides a method as provided in Method 2, which involves administering and implanting a device containing lineage-limited progenitor cells or fully differentiated somatic cells.

[0212] In another method, Method 4, the present disclosure provides a method as provided in any one of Methods 1 to 3, wherein the lineage-limited 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 beta cells, epithelial cells, endodermal cells, macrophages, hepatocytes, adipocytes, renal cells, blood cells, cardiomyocytes or immune system cells.

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

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

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

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

[0217] In another method, Method 9, the present disclosure provides a method as provided in Method 8, which involves implanting a device containing pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, mature beta cells, or pancreatic beta cells.

[0218] In another method, Method 10, the Disclosure provides a method as provided in Method 8 or 9, wherein the subject has type 1 diabetes or type 2 diabetes.

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

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

[0221] In another composition, composition 42, the present 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.

[0222] In another composition, composition 43, the Disclosure provides a composition as provided in composition 41, wherein the disrupted expression of TXNIP includes a reduced or absent expression.

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

[0224] In another composition, composition 45, the present disclosure provides a composition as provided in composition 44, wherein the sequence encoding the HLA-E trimer essentially consists of sequence number 55.

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

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

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

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

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

[0230] In another composition, composition 51, the present disclosure provides a composition as provided in composition 41, in which cells can differentiate into lineage-limited progenitor cells or fully differentiated somatic cells.

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

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

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

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

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

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

[0237] In another composition, composition 58, the present disclosure provides a composition as provided in composition 57, wherein the lineage-limited progenitor cells are embryonic endoderm cells, primitive intestinal cells, posterior foregut cells, pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, immature beta cells or mature beta cells, and the fully differentiated somatic cells are pancreatic beta cells.

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

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

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

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

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

[0243] In another composition, composition 64, the disclosure provides a composition as provided in composition 62, in which the disrupted expression of TXNIP includes a reduced or absent expression.

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

[0245] In another method, Method 13, the present disclosure provides a method as provided in Method 12, which involves implanting a device containing pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, mature beta cells, or pancreatic beta cells.

[0246] In another method, Method 14, the Disclosure provides a method as provided in Method 12, wherein the subject has type 1 diabetes or type 2 diabetes.

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

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

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

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

[0251] In another composition, composition 68, the present disclosure provides a composition as provided in composition 65, wherein the nucleotide sequence encoding PD-L1 essentially consists of sequence number 11.

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

[0253] In another composition, composition 70, the present disclosure provides a composition as provided in composition 69, wherein the sequence encoding the HLA-E trimer essentially consists of sequence number 55.

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

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

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

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

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

[0259] In another composition, composition 76, the present disclosure provides a composition as provided in composition 65, in which cells can differentiate into lineage-limited progenitor cells or fully differentiated somatic cells.

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

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

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

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

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

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

[0266] In another composition, composition 83, the present disclosure provides a composition as provided in composition 82, wherein the lineage-limited progenitor cells are embryonic endoderm cells, primitive intestinal cells, posterior foregut cells, pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, immature beta cells or mature beta cells, and the fully differentiated somatic cells are pancreatic beta cells.

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

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

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

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

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

[0272] In another composition, composition 89, the Disclosure provides a composition as provided in composition 87, wherein the disrupted expression of B2M and TXNIP includes a reduced or absent expression of B2M and TXNIP.

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

[0274] In another method, Method 17, the present disclosure provides a method as provided in Method 16, which involves implanting a device containing pancreatic endoderm cells, pancreatic endocrine cells, immature beta cells, mature beta cells, or pancreatic beta cells.

[0275] In another method, Method 18, the Disclosure provides a method as provided in Method 16, wherein the subject has type 1 diabetes or type 2 diabetes.

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

[0277] In another method, Method 20, the Disclosure provides a method for producing universal donor cells, comprising: (a) a first site-specific nuclease that targets a site in or near a gene encoding a survival factor; and (b) delivering a first nucleic acid to cells comprising a nucleotide sequence encoding a first immunotolerogenic factor adjacent to a nucleotide sequence homologous to a region to the left of the target site in (i)(a) and a nucleotide sequence homologous to a region to the right of the target site in (ii)(a), wherein the first site-specific nuclease cleaves the target site in (a) and the first nucleic acid in (b) is inserted at a site that partially overlaps, completely overlaps with, or contains within the site in (a), thereby producing universal donor cells, the universal donor cells having increased cell viability compared to cells in which the nucleic acid in (b) is not inserted.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0294] In another method, Method 37, the Disclosure provides a method as provided in any one of Methods 34 to 36, wherein the nucleotide sequence of (d)(iii) is essentially derived from Sequence ID No. 7 and the nucleotide sequence of (d)(iv) is essentially derived from Sequence ID No. 13.

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

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

[0297] In another method, Method 40, the present disclosure provides a method as provided in Method 39, wherein the exogenous promoter is a constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoter, and optionally the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

[0298] In another method, Method 41, the Disclosure relates to a method for producing universal donor cells, comprising: (a) a first site-specific nuclease that targets a site within or near a gene encoding a survival factor; (b) a first nucleic acid comprising a nucleotide sequence encoding a first immunotolerogenic factor adjacent to a nucleotide sequence homologous to a region located to the left of the target site in (a), and (ii) a nucleotide sequence homologous to a region located to the right of the target site in (a) (the first site-specific nuclease cleaves the target site in (a), and through the process of homologous recombination, the first nucleic acid in (b) is used as a template to disrupt the gene in (a) by inserting the nucleotide sequence encoding the first immunotolerogenic factor into a site that partially, completely, or contains the site in (a); (c) a site within a gene encoding one or more MHC-I or MHC-II human leukocyte antigens or components of the MHC-I or MHC-II complex or transcription regulators (d) is a second site-specific nuclease that targets the site in its vicinity; and (iv) is a nucleotide sequence encoding a second immunotolerogenic factor adjacent to a nucleotide sequence homologous to the region located to the left of the target site of (iii)(c), and a nucleotide sequence homologous to the region located to the right of the target site of (c). Unlike immunotolerogenic factor (b), the second site-specific nuclease cleaves the target site of (c) and, through the process of homologous recombination, the second immunotolerogenic factor of (d) The method provides delivering a second nucleic acid to a cell (in which a nucleotide sequence encoding a second immunotolerogenic factor is inserted into a site in (c) that partially, completely, or contains a nucleotide sequence encoding a second immunotolerogenic factor, thereby being used as a template for disrupting the gene in (c)), thereby producing universal donor cells, the universal donor cells having increased cell viability compared to cells in which the first nucleic acid in (b) and the second nucleic acid in (d) have not been inserted.

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

[0300] In another method, Method 43, the present disclosure provides a method as provided in any one of Methods 20 to 42, wherein the cells are mammalian cells, and optionally, the cells are human cells.

[0301] In another method, Method 44, the present disclosure provides a method as provided in any one of Methods 20 to 43, wherein the cells are stem cells.

[0302] In another method, Method 45, the present disclosure provides a method as provided in any one of Methods 20 to 43, wherein the cells are pluripotent stem cells, embryonic stem cells, adult stem cells, induced pluripotent stem cells, or hematopoietic stem cells.

[0303] In another method, Method 46, the present disclosure provides a method as provided in any one of Methods 20 to 43, wherein the cells are differentiated cells or somatic cells.

[0304] In another method, Method 47, the present disclosure provides a method, as provided in any one of Methods 20 to 43, in which universal donor cells can be differentiated into lineage-limited progenitor cells or fully differentiated somatic cells.

[0305] In another method, Method 48, the present disclosure provides a method as provided in Method 47, wherein the lineage-specific 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.

[0306] In another method, Method 49, the present disclosure provides a method as provided in Method 47, wherein the fully differentiated somatic cells are endocrine cells such as pancreatic beta cells, epithelial cells, endodermal cells, macrophages, hepatocytes, adipocytes, renal cells, blood cells, or immune system cells.

[0307] In another method, Method 49A, the Disclosure provides a method as provided in Method 47, wherein the fully differentiated somatic cells are cardiomyocytes or immune system cells.

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

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

[0310] In another composition, composition 92, the Disclosure provides a composition as provided in composition 90 or 91 for use in administering a treatment to a subject in need thereof.

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

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

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

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

[0315] In another method, Method 53, the present disclosure provides a method as provided by Method 51 or 52, wherein the lineage-limited 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.

[0316] In another method, Method 54, the present disclosure provides a method as provided by Method 51 or 52, wherein the fully differentiated somatic cells are endocrine cells such as pancreatic beta cells, epithelial cells, endodermal cells, macrophages, hepatocytes, adipocytes, renal cells, blood cells, or immune system cells.

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

[0318] In another method, Method 55, the Disclosure provides a method as provided by Methods 50-54, wherein the subject is a human being who has, is suspected of having, or is at risk of having a disease.

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

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

[0321] In another method, Method 57, the present disclosure provides an in vitro method for producing universal donor cells, comprising delivering a vector ((i) adjacent to (ii) and (iii)) to stem cells, the TXNIP gene locus being cleaved at the target site and the nucleic acid being inserted into the TXNIP gene locus, thereby disrupting the TXNIP gene and producing universal donor cells, the universal donor cells having increased immune evasion and / or cell viability compared to control cells.

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

[0323] In another method, Method 58, the present disclosure provides a composition as provided by Method 57, wherein the control cells are wild-type cells or cells that do not contain the inserted nucleic acid.

[0324] In another method, Method 59, the present disclosure provides a method, as provided by Method 57, in which the disrupted TXNIP gene has reduced or absent expression of TXNIP.

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

[0326] In another method, Method 61, the present disclosure provides a method as provided by Method 57, wherein the gRNA includes a spacer sequence corresponding to the sequence consisting of Sequence ID No. 20.

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

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

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

[0330] In another method, Method 65, the present disclosure provides a method as provided by Method 63, wherein the sequence encoding the HLA-E trimer is essentially derived from Sequence ID No. 55.

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

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

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

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

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

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

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

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

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

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

[0341] In another method, Method 75, the present disclosure provides a method, as provided by Method 73, in which the disrupted TXNIP gene has reduced or absent expression of TXNIP.

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

[0343] In another method, Method 77, the present disclosure provides a method as provided by Method 73, wherein the gRNA includes a spacer sequence corresponding to the sequence consisting of Sequence ID No. 20.

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

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

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

[0347] In another method, Method 81, the present disclosure provides a method as provided by Method 80, in which a Cas9 nuclease is coupled to at least one nuclear localization signal.

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

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

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

[0351] In another method, Method 85, the present disclosure provides an in vitro method for producing universal donor cells, comprising: (a) a first ribonucleoprotein (RNP) complex comprising an RNA-induced nuclease and guide RNA (gRNA) that targets a target site in the beta-2 microglobulin (B2M) gene locus; (b) a nucleic acid comprising (i) a nucleotide sequence encoding a first immunotolerogenic factor; and (ii) a genomic region essentially consisting of Sequence ID No. 7 and located to the left and within 50 nucleic acid bases of the target site in the B2M gene locus. (i) a nucleotide sequence having sequence homology to (ii) and (iii) a first vector comprising a nucleic acid having sequence homology to a genomic region that is essentially composed of SEQ ID NO: 13 and located to the right and within 50 nucleic acid bases of the target site in the B2M gene locus ((i) adjacent to (ii) and (iii); the B2M gene locus is cleaved at the target site, and the nucleic acid comprising the nucleotide sequence encoding the first immunotolerogenic factor is inserted into the B2M gene locus, thereby disrupting the B2M gene); (c) thioredoxin interaction A second RNP complex comprising an RNA-induced nuclease and gRNA that targets a target site in the protein (TXNIP) gene locus; and (d) nucleic acids comprising (i) a nucleotide sequence encoding a second immunotolerogenic factor; (ii) a nucleotide sequence essentially comprising SEQ ID NO: 25 and having sequence homology with a genomic region located to the left and within 50 nucleic acid bases of the target site in the TXNIP gene locus; and (iii) a nucleotide sequence essentially comprising SEQ ID NO: 32 and located to the right and within 50 nucleic acid bases of the target site in the TXNIP gene locus The present invention provides a method for producing universal donor cells, comprising delivering a second vector containing a nucleic acid having sequence homology with a genomic region ((i) is adjacent to (ii) and (iii); the TXNIP gene locus is cleaved at the target site, and a nucleic acid containing a nucleotide sequence encoding a second immunotolerogenic factor is inserted into the TXNIP gene locus, thereby disrupting the TXNIP gene) to stem cells, wherein the universal donor cells have increased immune evasion and / or cell survival compared to control cells.

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

[0353] In another method, Method 86, the present disclosure provides a method as provided by Method 85, wherein the control cells are wild-type cells or cells that do not contain the inserted nucleic acid.

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

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

[0356] In another method, Method 89, the present disclosure provides a method as provided by Method 85, wherein the gRNA of a first RNP complex includes a spacer sequence corresponding to the sequence of Sequence ID No. 2, and the gRNA of a second RNP complex includes a spacer sequence corresponding to the sequence of Sequence ID No. 20.

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

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

[0359] In another method, Method 92, the present disclosure provides a method as provided by Method 91, wherein the nucleotide sequence encoding PD-L1 essentially comes from Sequence ID No. 11.

[0360] In another method, Method 93, the present disclosure provides a method as provided by Method 91, wherein the nucleotide sequence encoding HLA-E comprises a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presenting peptide fused to a B2M membrane protein fused to HLA-E without its signal peptide, and the sequence encoding the HLA-E trimer is essentially derived from Sequence ID No. 55.

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

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

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

[0364] In another method, Method 97, the present disclosure provides a method as provided by Method 85, wherein the RNA-inducible nucleases of the first RNP complex and the second RNP complex are Cas9 nucleases.

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

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

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

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

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

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

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

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

[0373] In another method, Method 105, the present disclosure provides a method as provided by Method 101, wherein the gRNA of a first RNP complex includes a spacer sequence corresponding to the sequence of Sequence ID No. 2, and the gRNA of a second RNP complex includes a spacer sequence corresponding to the sequence of Sequence ID No. 20.

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

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

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

[0377] In another method, Method 109, the present disclosure provides a method as provided by Method 101, wherein the RNA-inducible nucleases of the first RNP complex and the second RNP complex are Cas9 nucleases.

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

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

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

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

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

[0383] In another process, process 2A, the present disclosure provides a method, as provided by process 2, in which a nucleic acid is inserted into a B2M gene locus no more than 50 base pairs from the target site.

[0384] In another process, process 3, the disclosure provides a process as provided in process 2, wherein the RNA-inducible nuclease of the first RNP complex is a Cas9 nuclease, and the gRNA of the first RNP complex includes a spacer sequence corresponding to the target sequence of sequence number 2.

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

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

[0387] In another process, process 6, the disclosure provides a process as provided in process 2, wherein the nucleotide sequence of (a)(2)(i) is essentially derived from sequence number 7 and the nucleotide sequence of (a)(2)(iii) is essentially derived from sequence number 13.

[0388] In another process, process 7, the present disclosure provides a process as provided in process 2, wherein the nucleotide sequence encoding PD-L1 essentially comes from sequence number 11.

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

[0390] In another process, process 9, the disclosure provides a process as provided in process 2, wherein the first vector is a plasmid vector and comprises the nucleotide sequence consisting of sequence number 33.

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

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

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

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

[0395] In another process, process 13, the disclosure provides a process as provided in process 12, wherein the RNA-inducible nuclease of the second RNP complex is a Cas9 nuclease, and the gRNA of the second RNP complex includes a spacer sequence corresponding to the target sequence of sequence number 20.

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

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

[0398] In another process, process 16, the disclosure provides a process as provided in process 12, wherein the nucleotide sequence of (c)(2)(i) is essentially derived from sequence number 25 and the nucleotide sequence of (c)(2)(iii) is essentially derived from sequence number 32.

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

[0400] In another process, process 18, the present disclosure provides a process as provided in process 17, wherein the sequence encoding the HLA-E trimer essentially derives from sequence number 55.

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

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

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

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

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

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

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

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

[0409] In another process, process 27, the present disclosure provides a process as provided in process 1, further comprising (a) growing the prepared PD-L1-positive cells, (c) growing the prepared PD-L1, HLA-E bipositive cells, and (e) growing selected PD-L1, HLA-E bipositive cells or a combination thereof.

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

[0411] In another process, process 29, the present disclosure provides a process as provided in process 28, wherein the enrichment of first immunotolerogenic factor-positive cells in (b) includes magnetically assisted cell sorting (MACS), single-cell cloning, proliferation of the first immunotolerogenic factor-positive cells, or a combination thereof.

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

[0413] In another process, process 31, the present disclosure provides a process as provided in any one of processes 28 to 30, further comprising (a) growing first immunotolerogenic factor-positive cells produced in (a), (c) growing first immunotolerogenic factor-positive / second immunotolerogenic factor-positive cells produced in (a), (e) growing selected first immunotolerogenic factor-positive / second immunotolerogenic factor-positive cells, or a combination thereof.

[0414] In another process, process 32, the Disclosure provides that the modification in (a) (1) a first RNA-induced nuclease and a first guide RNA (gRNA) that targets a target site in a locus of MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor gene locus, and (2) a first nucleic acid comprising (i) a nucleotide sequence homologous to a region located to the left of the target site in a locus of MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor gene locus, (ii) a nucleotide sequence encoding a first immunotolerogenic factor, and (iii) a MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor gene The present invention provides a process as provided in any one of processes 28 to 31, comprising delivering a first vector to stem cells containing a first nucleic acid containing a nucleotide sequence homologous to a region located to the right of a target site in a locus, wherein a locus of MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor gene locus is cleaved at the target site, and the first nucleic acid containing a nucleotide sequence encoding a first immunotolerogenic factor is inserted into the locus of MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor gene, thereby disrupting the MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor gene.

[0415] In another process, process 32A, the present disclosure provides a method, as provided by process 32, in which a nucleic acid is inserted into a target site of no more than 50 base pairs of MHC-I or MHC-II human leukocyte antigen or a component of an MHC-I or MHC-II complex or a transcription factor gene locus.

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

[0417] In another process, process 34, the Disclosure relates to a modification in (a) a first ribonucleoprotein (RNP) complex comprising a first RNA-induced nuclease and a first guide RNA (gRNA) that targets a target site in a locus of MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor gene locus, and (2) a first nucleic acid comprising (i) a nucleotide sequence homologous to a region located to the left of a target site in a locus of MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor gene locus, (ii) a nucleotide sequence encoding a first immunotolerogenic factor, and (iii) a component of MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or The present invention provides a process as provided in any one of processes 28 to 33, comprising delivering a first vector to a stem cell, the first vector containing a first nucleic acid containing a nucleotide sequence homologous to a region located to the right of a target site in a transcription factor gene locus, wherein the MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor gene locus is cleaved at the target site, and the first nucleic acid containing a nucleotide sequence encoding a first immunotolerogenic factor is inserted into the MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor gene locus, thereby disrupting the MHC-I or MHC-II human leukocyte antigen or a component of the MHC-I or MHC-II complex or a transcription factor gene.

[0418] In another process, process 34A, the present disclosure provides a method, as provided by process 34, in which a nucleic acid is inserted into a target site of no more than 50 base pairs of MHC-I or MHC-II human leukocyte antigen or a component of an MHC-I or MHC-II complex or a transcription factor gene locus.

[0419] In another process, process 35, the present disclosure provides a process as provided in any one of processes 28 to 34, wherein the MHC-I or MHC-II human leukocyte antigen or component of the MHC-I or MHC-II complex or transcription factor 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.

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

[0421] In another process, process 37, the Disclosure provides a process as provided in process 36, wherein the nucleotide sequence of (a)(2)(i) is essentially derived from sequence number 7 and the nucleotide sequence of (a)(2)(iii) is essentially derived from sequence number 13.

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

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

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

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

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

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

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

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

[0430] In another process, process 46, the present disclosure provides a process as provided in process 45, wherein the nucleotide sequence encoding PD-L1 essentially comes from sequence number 11.

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

[0432] In another process, process 48, the present disclosure provides a process in which the first vector comprises a nucleotide sequence consisting of sequence number 33, as provided in any one of processes 45 to 47.

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

[0434] In another process, process 49A, the present disclosure provides a method, as provided by process 49, in which a nucleic acid is inserted into a survival factor gene locus no more than 50 base pairs from a target site.

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

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

[0437] In another process, process 51A, the present disclosure provides a method, as provided by process 51, in which a nucleic acid is inserted into a survival factor gene locus no more than 50 base pairs from a target site.

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

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

[0440] In another process, process 54, the disclosure provides a process as provided in process 53, wherein the second gRNA includes a spacer sequence corresponding to the target sequence consisting of sequence number 20.

[0441] In another process, process 55, the Disclosure provides a process as provided in process 52 or 53, wherein the nucleotide sequence of (c)(2)(i) is essentially derived from sequence number 25 and the nucleotide sequence of (c)(2)(iii) is essentially derived from sequence number 32.

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

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

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

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

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

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

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

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

[0450] In another process, process 64, the present disclosure provides a process as provided in process 63, wherein the sequence encoding the HLA-E trimer essentially derives from sequence number 55.

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

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

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

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

[0455] In a separate process, process 69, the present disclosure provides a process as provided in processes 28-68, wherein the characterization in (f) includes cellular analysis for morphology, viability, karyotype analysis, endotoxin levels, mycoplasma levels, on / off-target analysis, random vector insertion, residual Cas9, residual vector, pluripotency, differentiation potential, or a combination thereof.

[0456] In another process, process 70, the Disclosure provides a process as provided in processes 28-69, which further includes freezing before characterization in (f). [Examples]

[0457] The following examples describe the preparation and characterization of specific universal donor cells according to this disclosure.

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

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

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

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

[0462] Regarding hiPSC(TC1133), the cells were maintained in StemFlex Complete throughout the entire process of regular maintenance on BIOLAMININ-coated plates containing Revitacell during the cloning and passage stages.

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

[0464] [Table 1]

[0465] To evaluate the cleavage efficiency of these proteins in hPSCs, CyT49 cells (a ViaCyte exclusive hES cell line) were electroporated using a Neon electroporator (Neon transfection system ThermoFisher Cat#MPK5000) with a ribonucleoprotein (RNP) mixture of Cas9 protein (Biomay) and guide RNA (Synthego) (see Table 3 for gRNA sequence) in a 3:1 (gRNA:Cas9) molar ratio, having absolute values ​​of 125 pmol Cas9 and 375 pmol gRNA. To form the RNP complex, the gRNA and Cas9 were combined in a single container containing R-buffer (Neon transfection system 100 μL kit ThermoFisher Cat#MPK10096) to a total volume of 25 μL and incubated at room temperature for 15 minutes. The cells were dissociated using ACCUTASE®, subsequently resuspended in DMEM / F12 medium (Gibco, cat#11320033), counted using NC-200 (Chemometec), and centrifuged. A total of 1 × 10⁶ cells were obtained. 6The cells were resuspended with the RNP complex, and R-buffer was added to a total volume of 125 μL. This mixture was then electroporated at 1100 V for 30 seconds in 2 pulses. After electroporation, the cells were pipetted into Eppendorf tubes filled with StemFlex medium containing RevitaCell. This cell suspension was then seeded into tissue culture dishes pre-coated with BIOLAMININ 521 CTG at a 1:20 dilution. The cells were cultured for 48 hours in a normal oxygen pressure incubator (37°C, 8% CO2). After 48 hours, genomic DNA was collected from the cells using QuickExtract (Lucigen, Middleton, WI; Cat#QE09050).

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

[0467] [Table 2]

[0468] [Table 3]

[0469] The resulting amplification products were subjected to PCR purification and Sanger sequencing. The Sanger sequencing results, along with the guide sequences, were entered into Tsunami software. Indel percentages and identities were calculated by the software. Next, specific gRNAs were selected based on their indel frequencies in hPSCs. Figure 1 shows the cleavage efficiencies of B2M-1, B2M-2, and B2M-3 gRNAs.

[0470] Off-target effects of the selected gRNAs were evaluated in stem cell-derived DNA using hybrid capture analysis of predicted sequence-similarity sites. Neither the B2M-2 nor B2M-3 guides showed any detectable off-target activity. The B2M-2 gRNA was selected for further cloning due to its high on-target activity and undetectable off-target activity.

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

[0472] The B2M knockout status of the clones was confirmed via PCR and Sanger sequencing. The obtained DNA sequences of the target B2M region were aligned in Snapgene software to determine indel identity and juxtaposition status. Clones with the desired editing were grown and further validated via flow cytometry evaluation for B2M expression (see Table 4 for a list of antibodies used). Clones were evaluated with and without interferon-gamma treatment (25 ng / mL, R&D Systems, 285-IF). Figure 2A shows B2M expression in wild-type cells, and Figure 2B shows B2M expression in B2M knockout cells. The karyotype status of the clones was evaluated via Cell Line Genetics service (Madison, WI), and normal karyotypes were reported.

[0473] [Table 4]

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

[0475] Example 3: Production of B2M KO / PD-L1 knock-in (KI) human pluripotent stem cells (hPSCs) Design of the B2M-KO / PD-L1 KI strategy. Plasmid design for inserting PD-L1 (CD274) into the B2M locus was designed so that the B2M start codon is removed after homologous recombination repair (HDR) to insert PD-L1 and negate the opportunity for partial B2M expression. Figure 3 shows a schematic diagram of the plasmid, and Table 5 identifies its elements and locations. The donor plasmid contained CAGGS promoter-driven cDNA of PD-L1 adjacent to an 800 base pair homology arm with a sequence identical to the B2M locus around exon 1. The complete plasmid sequence includes SEQ ID NO: 33.

[0476] [Table 5]

[0477] B2M-2 gRNA was used to facilitate 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 B2M-targeted gRNA and Cas9 protein. 4 μg of plasmid DNA was delivered along with RNP per 1 million CyT49 cells (ViaCyte). Electroporation was performed as described in Example 2. Seven days after electroporation, cells were sorted for PD-L1 surface expression in 96-well plates coated with BIOLAMININ 521 CTG containing StemFlex along with Revitacell using a WOLF FACS sorter (Nanocellect). For FACS sorting, unedited cells served as a negative control. PD-L1-positive cells were selected for sorting and single-cell cloning.

[0478] Anti-PD-L1 fluorescent antibodies were used to detect PD-L1 surface expression (see Table 4). Seeded single cells were grown in an oxygen-pressure incubator (37°C, 8% CO2) with the medium changed every other day until the colonies grew large enough to be reseeded as single cells. At confluence, the samples were divided for maintenance and genomic DNA extraction.

[0479] Precisely targeted clones were identified via PCR, which allowed amplification of only the KI-integrating DNA for PD-L1 knock-in (KI) inserts, using primers that amplified the region from the outside of the plasmid homology arm to the PD-L1 cDNA insert. On-target inserts were tested for conjugation status by PCR to assess whether the KI occurred in a heterozygous or homozygous manner. If heterozygous clones were identified, the KI-negative allele was sent for Sanger sequencing to verify that it contained a B2M-disrupted indel in the non-KI allele. Precise KI clones with complete B2M disruption (either by KI insert or indel formation) were grown in an expanding tissue culture format until they reached a population size of 30 million cells. Approximately 10 clones were grown in this format and confirmed to be pluripotent by testing for OCT4 and SOX2 via intracellular flow cytometry (Figure 4). Clones that passed the above tests were then further tested for karyotyping (Cell Line Genetics) as described below. Furthermore, the clones were tested for their ability to differentiate into pancreatic endodermal progenitor cells (PECs) via the established protocol described below (Schulz et al. (2012) PLoS ONE 7(5):e37004). The absence of B2M was further confirmed by the lack of B2M expression with and without interferon-gamma treatment (25 ng / mL, R&D Systems, 285-IF) via flow cytometry. Figures 5A and 5B show PD-L1 expression in wild-type and B2M KO / PD-L1 KI cells, respectively.

[0480] Example 4: Karyotype analysis of edited clones G-banding karyotype analysis of edited embryonic stem (ES) cells. One million edited ES cells were passaged into T-25 culture flasks containing culture medium (DMEM / F12 + 10% Xeno-free KSR and 10 ng / mL activin and 10 ng / mL heregulin). After overnight culture, three T25 culture flasks were sent to the Cell Line Genetics, Inc. laboratory for karyotype analysis; FISH analysis for chromosomes 1, 12, 17, and 20; and array comparative genomic hybridization (aCGH) analysis using a standard 8 × 60K array. G-banding results of selected cells, electroporated with non-cleaved guides ("NCG"), B2M KO clones, and B2M KO / PD-L1 KI clones ("V1-A"), are shown in Table 6.

[0481] [Table 6]

[0482] Example 5: Differentiation of edited human embryonic stem cells into pancreatic endoderm cells (PECs) Maintenance of edited human embryonic stem cells (ES). Edited human embryonic stem cells of various passage numbers (P38-42) were passed at 33,000 cells / cm³ for 4 days in hESM medium (DMEM / F12 + 10% KSR + 10 ng / mL activin A and 10 ng / mL heregulin) and final 10% human AB serum. 2 Alternatively, 50,000 cells / cm³ for 3-day passaging. 2 It was sown there.

[0483] Aggregation of edited human embryonic stem cells for PEC differentiation. Edited ES cells were dissociated into single cells with ACCUTASE®, followed by centrifugation and resuspended at 1 million cells per ml in 2% StemPro (Cat#A1000701, Invitrogen, CA) in DMEM / F12 medium, resulting in a total of 3.5 to 4 million cells, which were rotated at a rotation speed of 8 RPM ± 0.5 RPM in a single 850 cm³ chamber. 2The cells were seeded in rotor bottles (Cat#431198, Corning, NY) for 18-20 hours prior to differentiation. Edited human embryonic stem cell aggregates were differentiated into pancreatic lineage cells using rotor bottles as described in Schulz et al. (2012) PLoS ONE 7(5):e37004.

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

[0485] [Table 7]

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

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

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

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

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

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

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

[0493] [Table 8]

[0494] TXNIP KO hPSC cloning and characterization. To evaluate the cleavage efficiency of these gRNAs in hPSCs, TC1133 hiPSC cells were electroporated using a Neon electroporator (Neon transfection system ThermoFisher Cat#MPK5000) with an RNP mixture of Cas9 protein (Biomay) and guide RNA (Synthego) in a 3:1 (gRNA:Cas9) molar ratio, with an absolute value of 125 pmol of Cas9 and 375 pmol of gRNA. To form the RNP complex, the gRNA and Cas9 were combined in a single container with R-buffer to a total volume of 25 μL and incubated at room temperature for 15 minutes. The cells were dissociated using ACCUTASE®, subsequently resuspended in DMEM / F12 medium (Gibco, cat#11320033), counted using NC-200 (Chemometec), and centrifuged. A total of 1 × 10⁶ cells were obtained. 6 The cells were resuspended with the RNP complex, and R-buffer was added to a total volume of 125 μL. This mixture was then electroporated using the parameters: 2 pulses, 30 ms, 1100 V. After electroporation, the cells were pipetted into Eppendorf tubes filled with StemFlex medium containing RevitaCell. This cell suspension was then seeded into tissue culture dishes pre-coated with BIOLAMININ 521 CTG. The cells were cultured for 48 hours in a normal oxygen pressure incubator (37°C, 8% CO2). After 48 hours, genomic DNA was collected from the cells using QuickExtract.

[0495] PCR was performed for the target TXNIP sequence, and the resulting amplified DNA was sequenced by Sanger sequencing. Output sequencing data for indel percentages using Tsunami software was analyzed using TIDE analysis. Figure 13 shows the cleavage efficiency for TXNIP gRNAs. Next, gRNAs were selected based on their indel frequencies in hPSCs.

[0496] The off-target effects of the gRNA with the highest cleavage efficiency were evaluated in stem cell-derived DNA using hybrid capture analysis at sites where sequence similarity was predicted. Further experiments with TXNIP gRNA T5 were conducted because it showed no detectable off-target effects and exhibited high on-target activity.

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

[0498] The TXNIP knockout status of the clones was confirmed via PCR and Sanger sequencing. The obtained DNA sequences of the target TXNIP region were aligned in Snapgene software to determine indel identity and juxtaposition status. Clones with the desired edits were grown and further validated via flow cytometry evaluation of TXNIP expression. The karyotype status of the clones was assessed via Cell Line Genetics services, and a normal karyotype was reported (Table 9).

[0499] [Table 9]

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

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

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

[0503] The plasmid design for inserting PD-L1 (CD274) into the B2M locus is shown in Example 3. The donor plasmid contains PD-L1 CAGGS promoter-driven cDNA flanked by an 800-base pair homology arm having a sequence identical to the B2M locus around exon 1. B2M-2 gRNA was used to facilitate insertion of the PD-L1 transgene at the targeted B2M locus. The PD-L1 donor plasmid was introduced with an RNP complex consisting of B2M-targeting gRNA and Cas9 protein. 4 μg of plasmid DNA was delivered with RNP per 1 million CyT49 cells (ViaCyte). Electroporation was performed as described in Example 2. Seven days after electroporation, cells were enriched for PD-L1-positive cells via magnetically assisted cell sorting (MACS) using Miltenyi reagent (anti-mouse IgG microbeads Cat#130-048-401, LS column Cat#130-042-401, and MidiMACS separator Cat#130-042-302) or Thermofisher reagent (DynaMag®-15 magnet Cat#12301D, CELLection® pan-mouse IgG kit Cat#11531D, Dynabeads® pan-mouse IgG Cat#11042).

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

[0505] [Table 10]

[0506] [Table 11]

[0507] TXNIP-T5 gRNA was used to facilitate the insertion of HLA-E transgenes at the targeted TXNIP locus. The HLA-E donor plasmid was introduced with an RNP complex consisting of TXNIP-T5 gRNA and Cas9 protein. 4 μg of HLA-E donor plasmid DNA (SEQ ID NO: 56) was delivered with RNP per 1 million PD-L1+ cells. Alternatively, HLA-E donor plasmid DNA (SEQ ID NO: 34) may be used. Electroporation was performed as described in Example 2. Seven days after electroporation, cells were enriched for HLA-E positive cells via MACS using Miltenyi or Thermofisher reagent. Following HLA-E enrichment, cells were sorted into single cells in 96-well plates coated with BIOLAMININ 521 CTG containing StemFlex and Revitacell using a WOLF FACS sorter (Nanocellect). Seeded single cells were grown in an oxygen-pressure incubator (37°C, 8% CO2) with the medium changed every other day until the colonies grew large enough to be reseeded as single cells. Upon confluence, the samples were divided for maintenance and genomic DNA extraction. Anti-PD-L1 and anti-HLA-E antibodies (Table 4) were used for FACS sorting in 96-well plates with MACS enrichment and gating settings for HLA-E and PD-L1 double-positive cells. For FACS sorting, unedited cells served as a negative control.

[0508] Precisely targeted clones were identified via PCR, enabling amplification of KI-integrating DNA only for PD-L1 KI inserts and HLA-E KI inserts, using primers that amplified the region from the outside of the plasmid homology arm to the PD-L1 cDNA insert or HLA-E cDNA insert, respectively. On-target inserts were tested for conjugation status by PCR to assess whether KI arose in a heterozygous or homozygous manner. If heterozygous clones were identified, the KI-negative alleles were sent for Sanger sequencing to verify that they contained either B2M-disrupted indels or TXNIP-disrupted indels, respectively. Precise KI clones with complete B2M and TXNIP disruption (either by KI insert or indel formation) were grown in an expanding tissue culture format until a population size of 30 million cells was reached. Approximately 10 clones were grown in this format and confirmed to be pluripotent by testing for OCT4 and SOX2 via intracellular flow cytometry (Figure 15).

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

[0510] [Table 12]

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

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

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

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

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

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

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

[0518] Following a standard procedure, pancreatic endoderm aggregates derived from designated clonal strains were loaded into a perforation device (PD) to generate test or control articles. The PD enables direct angiogenesis upon subcutaneous transplantation, and the encapsulated pancreatic progenitor cells mature in vivo into functional pancreatic endocrine cells, including glucose-responsive, insulin-producing cells.

[0519] As summarized in Table 13, five groups of thymus-deficient nude rats were each given approximately 7 × 10⁻¹⁰ clonal strains obtained from the differentiation of the four clonal strains mentioned above. 6 Two articles containing either individual pancreatic endoderm cells or wild-type CyT49 hES(ViaCyte) cells were subcutaneously transplanted.

[0520] [Table 13]

[0521] From week 12 onwards, all surviving animals were subjected to efficacy evaluation via glucose-stimulated insulin secretion (GSIS) testing. 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 via standard enzyme-linked immunosorbent assay.

[0522] The GSIS trial was conducted at weeks 12, 16, and 20. The results showed no substantial differences between the experimental groups, particularly after week 12. Compared to the C-peptide levels detected in the control group (Group 1, <40 pM~2.0 nM, mean 1.1 nM), C-peptide levels were elevated in 2 out of 6 animals from Group 3 (TXNIP KO, mean 1.5 nM). The other groups, Group 2 (NCG, mean 0.5 nM), Group 4 (B2M KO / PD-L1 KI, mean 0.5 nM), and Group 5 (B2M KO / HLA-E KI, mean 0.4 nM), showed similar ranges of C-peptide levels compared to the control group, but more animals were near the lower end of that range. However, these differences were not statistically significant. These results demonstrate that neither the introduced gene modifications nor the procedures required to create the cloned cell line affected the cell line's ability to differentiate into pancreatic endoderm cells in vitro and subsequently produce functional beta cells in vivo.

[0523] At 20 weeks, after the GSIS trial, the animals were euthanized, the transplanted test material was fixed in neutral buffered formalin, prepared as a slide, and stained by immunohistochemistry for H&E as well as insulin and glucagon.

[0524] In vivo efficacy evaluations in the GSIS trial showed no substantial difference between the unedited control product and the edited test products, each constructed from pancreatic endodermal cells derived from clonal cell lines, each containing a subset of genetically modified organisms. The results suggest that individual genetic modifications and the processes by which they are introduced are acceptable in vivo.

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

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

[0527] [Table 14]

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

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

[0530] Example 12. In vivo efficacy study of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI cell lines. B2M KO / PD-L1 KI, TXNIP KO / HLA-E KI: Aggregates of pancreatic endoderm cells (approximately 7 x 10⁻¹⁰) 6 The test material will be formulated with (including individual cells). Two test materials will be subcutaneously implanted into each of 46 thymus-deficient nude rats. The animals will be evaluated for GSIS, ITT, and non-fasting blood glucose (NFBG) during the study. Ten animals per group will be euthanized at the scheduled end times of weeks 13, 17, 26, and 39, while six additional animals will be tested in consideration of any possible unplanned early termination. From each animal, the two implanted test materials will be randomly assigned to either histological evaluation or total C-peptide content evaluation. Table 15 shows the study design.

[0531] [Table 15]

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

[0533] At weeks 25 and 33, surviving animals are subjected to an insulin challenge (insulin resistance test, ITT) to assess changes in serum human C-peptide in response to reduced blood glucose levels while the animals are not fed. Blood samples are obtained from fasted animals before and at multiple time points (15, 30, and 60 minutes) after intraperitoneal administration of 1 unit of insulin per kg of body weight. Serum concentrations of human C-peptide are determined via a standard enzyme-linked immunosorbent assay.

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

[0535] At the planned endpoints identified in Table 13, the animals will be euthanized. Euthanasia will be performed by bilateral thoracotomy after CO2 inhalation. Autopsies will be performed at all planned and unplanned endpoints, and any macroscopic abnormalities will be recorded.

[0536] After the designated grafts are frozen, the luminal contents are homogenized. The total C-peptide content of the homogenate is determined via a standard enzyme-linked immunosorbent assay. The total C-peptide content of the graft is used to predict clinical medication.

[0537] The designated transplanted test articles are fixed in neutral buffered formalin, prepared as slides, and stained by immunohistochemistry (IHC) for H&E as well as insulin and glucagon to identify human pancreatic endocrine cells. Additional IHC for the human-specific nuclear marker NuMA1 is performed to identify the potential location of cells originating from the transplant outside the lumen of the test article graft.

[0538] Example 13: Preparation of B2M KO / PD-L1 KI and TXNIP KO / HLA-E KI in human iPSCs Human iPSCs (iPSC 0025) with the PD-L1 coding sequence inserted into the B2M locus were generated. The RNP complex was formed by combining B2M-2 gRNA (SEQ ID NO: 2) and Cas9 in a molar ratio of 3:1 (gRNA:Cas9). To form the RNP complex, the gRNA and Cas9 were combined in a single container with R-buffer to a total volume of 25 μL and incubated at room temperature for 15 minutes. The cells were dissociated using ACCUTASE®, subsequently resuspended in DMEM / F12 medium (Gibco, cat#11320033), counted using NC-200 (Chemometec), and centrifuged. A total of 1 × 10⁶ cells were obtained. 6The cells were resuspended with the RNP complex. 4 μg of B2M-CAGGS-PD-L1 donor plasmid (SEQ ID NO: 33) and R-buffer were added for a total volume of 125 μL. This mixture was then electroporated using the parameters: 2 pulses, 30 ms, 1100 V. Seven days after electroporation, the cells were enriched for PD-L1-positive cells via MACS using Miltenyi reagent or Thermofisher reagent, essentially as described above in Example 8.

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

[0540] Precisely targeted clones were identified via PCR, enabling amplification of KI-integrating DNA only for PD-L1 KI inserts and HLA-E KI inserts, using primers that amplified the region from the outside of the plasmid homology arm to the PD-L1 cDNA insert or HLA-E cDNA insert, respectively. On-target inserts were tested for conjugation status by PCR to assess whether KI arose in a heterozygous or homozygous manner. If heterozygous clones were identified, the KI-negative alleles were sent for Sanger sequencing to verify that they contained either B2M-disrupted indels or TXNIP-disrupted indels, respectively. Precise KI clones with complete B2M and TXNIP disruption (either by KI insert or indel formation) were grown in an expanding tissue culture format until a population size of 30 million cells was reached. Selected clones were grown in this format and confirmed to be pluripotent by testing for OCT4 and SOX2 via intracellular flow cytometry.

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

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

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

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

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

[0546] PD-L1 and HLA-E double-positive cells were single-cell sorted. For this purpose, cells were supplied with StemFlex Complete along with Revitacell (final concentration of 1×Revitacell) 3-4 hours before dissociation with ACCUTASE®. After dissociation, single cells were sorted into single wells of a 96-well tissue culture plate coated with BIOLAMININ. Using a WOLF FACS sorter (Nanocellect), single cells were sorted into wells using the anti-PD-L1 and anti-HLA-E antibodies described above. The plates were pre-filled with 100-200 μL of StemFlex Complete along with Revitacell. Three days after cell seeding, cells were supplied with fresh StemFlex, and 100-200 μL of medium continued to be supplied every other day. Ten days after proliferation, cells were supplied with StemFlex daily until day 12-14. At this point, the plates were dissociated with ACCUTASE®, and the recovered cell suspension was divided 1:2 into two 96-well plates and cultured for approximately 4 days.

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

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

Claims

1. Genetically modified cells, wherein the genetically modified cells are A nucleotide sequence encoding a first immunotolerogenic factor, which is inserted into a gene encoding a survival factor, wherein the genetically modified cell expresses the first immunotolerogenic factor and has disrupted expression of the survival factor, and the survival factor is TXNIP, and the first immunotolerogenic factor is HLA-E, and A nucleotide sequence encoding a second immunotolerogenic factor, which is inserted into the gene encoding B2M, wherein the genetically modified cell expresses the second immunotolerogenic factor and has disrupted expression of B2M, and the second immunotolerogenic factor is PD-L1, the nucleotide sequence encoding the second immunotolerogenic factor. It includes, and, The genetically modified cells are genetically modified cells that have increased immune evasion and / or cell survival compared to cells without the inserted nucleotide sequence.

2. The genetically modified cell according to claim 1, wherein the cell without the inserted nucleotide sequence is a wild-type cell.

3. The genetically modified cell according to claim 1 or 2, wherein the disrupted expression of the survival factor includes reduced or absent expression.

4. The genetically modified cell according to claim 1, wherein the nucleotide sequence encoding HLA-E includes a sequence encoding an HLA-E trimer comprising a B2M signal peptide fused to an HLA-G presentation peptide fused to a B2M membrane protein fused to HLA-E without the signal peptide.

5. The genetically modified cell according to claim 4, wherein the sequence encoding the HLA-E trimer includes the nucleotide sequence shown in Sequence ID No.

55.

6. The genetically modified cell according to any one of claims 1 to 5, wherein the nucleotide sequence encoding PD-L1 includes the nucleotide sequence shown in Sequence ID No.

11.

7. The genetically modified cell according to any one of claims 1 to 6, wherein the nucleotide sequence encoding the first immunotolerogenic factor is operably linked to an exogenous promoter, and / or the nucleotide sequence encoding the second immunotolerogenic factor is operably linked to an exogenous promoter, and the exogenous promoter is a CMV, EF1α, PGK, CAG, or UBC promoter.

8. The aforementioned cells are stem cells including embryonic stem cells other than human embryonic stem cells, adult stem cells, induced pluripotent stem cells other than human induced pluripotent stem cells, or hematopoietic stem cells, or The aforementioned cells are differentiated cells or somatic cells. Genetically modified cells according to any one of claims 1 to 7.

9. The cells can differentiate into lineage-limited progenitor cells or fully differentiated somatic cells, and (a) the lineage-limited 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 / or (b) the fully differentiated somatic cells are pancreatic beta cells, epithelial cells, endodermal cells, macrophages, hepatocytes, adipocytes, renal cells, blood cells, cardiomyocytes or immune system cells, according to any one of claims 1 to 8.

10. Multiple genetically modified cells comprising two or more genetically modified cells according to any one of claims 1 to 9.

11. A population of lineage-limited progenitor cells or fully differentiated somatic cells derived from a plurality of genetically modified cells as described in claim 10.

12. A population of lineage-limited progenitor cells or fully differentiated somatic cells as described in claim 11, (i) The lineage-limited 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 beta cells, epithelial cells, endodermal cells, macrophages, hepatocytes, adipocytes, renal cells, blood cells, cardiomyocytes, or immune system cells, and at least 50% of the cells express the first immunotolerogenic factor, the second immunotolerogenic factor, or the first and second immunotolerogenic factors, or (ii) The lineage-limited progenitor cells are embryonic endoderm cells, primitive intestinal cells, posterior foregut cells, pancreatic endoderm progenitor cells, pancreatic endocrine progenitor cells, immature beta cells or mature beta cells, and the fully differentiated somatic cells are pancreatic beta cells, and at least 50% of the cells express the first immunotolerogenic factor, the second immunotolerogenic factor, or the first and second immunotolerogenic factors.

13. A composition comprising a plurality of genetically modified cells as described in claim 10, or a population of lineage-limited progenitor cells or fully differentiated somatic cells as described in claim 11 or 12.

14. The composition according to claim 13 for use as a pharmaceutical.

15. A composition for use in treating a genetically inherited disease, comprising a plurality of genetically modified cells as described in claim 10, or a population of lineage-limited progenitor cells or fully differentiated somatic cells as described in claim 11 or 12.

16. The composition according to claim 15, wherein the genetically inherited disease is diabetes mellitus.

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