Materials and methods for an iPSC population produced by biotechnology

Abstract

Provided herein is a method for producing pluripotent stem cells (iPSCs) generated by biotechnology and isolated populations thereof.

Description

Technical Field

[0001] (Cross - reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 357,922, filed on July 1, 2022, which is hereby incorporated by reference in its entirety.

[0002] (Field of the Invention) Provided herein are, among other things, materials and methods for producing induced pluripotent stem cells (iPSCs) made by biotechnology, and their use.

Background Art

[0003] Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs) have the proliferative and developmental potential to differentiate in vivo to generate multiple cell types. Thus, the scientific potential of these cells is, although uncertain, exceptional, especially since it has been shown that somatic cell gene expression profiles can be altered by research to epigenetically reprogram them into pluripotent stem cells (see, for example, Takahashi, K., & Yamanaka S, Nat. Rev. Mol. Cell Biol., 2016, 17(3):183 - 93).

[0004] Embryonic stem cells can be derived from the inner cell mass of mammalian blastocysts. See, for example, Human Genes and Genomes: Science, Health, Society (Rosenberg, L.E. & Rosenberg, D.D., 1st ed. 2012). Additionally, somatic cell nuclear transfer (SCNT)-mediated reprogramming has also been utilized to generate pluripotent ES cells, and in some instances, cloned animals have been used (Wilmut, I., et al., Nature, 1997, 385: 810-813; Wakayama, T., et al., Nature, 1998, 394: 369-374). Nevertheless, SCNT has been plagued by various technical (e.g., epigenetic) barriers because the destruction of embryos and the introduction of mammalian genetic information into unfertilized eggs are controversial (Matoba, S. & Zhang, Y., supra; Kastenberg, Z.J. & Odorico, J.S., Transplant Rev., 2008, 22(3): 215-22).

[0005] Alternative techniques for reprogramming somatic cells into pluripotent stem cells continue to be of interest. Induced pluripotent stem cell (iPSC) technology emerged as one such option when Yamanaka et al. reported that the transcription factors Oct3 / 4, Sox2, Klf4, and c-Myc could endow adult somatic cells with pluripotency and that pluripotency could be conferred to generate iPSCs (Takahashi, K., & Yamanaka, S, Cell, 2006, 126(4): 663-76; Wernig, M., et al., Nature, 2007, 448: 318-324; Maherali, N., et al., Cell Stem Cell, 2007, 1(1): 55-70). SUMMARY OF THE INVENTION

[0006] In one aspect, provided herein is a population of induced pluripotent stem cells (iPSCs), which are optionally generated by reprogramming γδ T cells, and the population comprises iPSCs that contain a disrupted beta-2-microglobulin (B2M) gene. In certain embodiments, the iPSCs are disrupted for both copies of the B2M gene (B2M - / - ).

[0007] In some aspects, the nucleotide sequence of the B2M gene encodes an amino acid sequence of any one of SEQ ID NOs: 12-16. In some aspects, the nucleotide sequence of the B2M gene encodes an amino acid sequence that is about, at least about, or up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 12-16. In some aspects, the amino acid sequence is about or at least about 50 contiguous amino acids in any one of SEQ ID NOs: 12-16 and is about, at least about, or up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some aspects, the nucleotide sequence encodes the amino acid sequence of B2M, a portion thereof, or an isoform thereof.

[0008] In some embodiments, the disrupted B2M gene comprises a deletion of at least a portion of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the deletion is about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleotide sequence of SEQ ID NO: 1. In some embodiments, about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the iPSCs do not express detectable levels of B2M.

[0009] In some embodiments, the disruption comprises a deletion of at least one nucleotide base pair. In some embodiments, the disruption comprises an insertion of at least one nucleotide base pair. In some embodiments, the disrupted B2M gene exhibits reduced B2M expression as compared to the non-disrupted B2M gene. In some embodiments, the reduced B2M expression is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the expression of B2M in reference iPSCs.

[0010] In some embodiments, iPSCs containing disrupted B2M genes exhibit reduced expression of HLA-A, HLA-B, and / or HLA-C compared to the expression of HLA-A, HLA-B, and / or HLA-C in reference iPSCs, e.g., reference iPSCs that do not contain disrupted B2M genes. In some embodiments, the reduced expression of HLA-A, HLA-B, and / or HLA-C is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the expression of HLA-A, HLA-B, and / or HLA-C in reference iPSCs. In some embodiments, the reference iPSCs are a population of iPSCs that do not contain disrupted B2M genes. In some embodiments, the reference iPSCs are a population of iPSCs in which the B2M gene has not been disrupted.

[0011] In some embodiments, the disrupted B2M gene is generated by contacting a population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease, and a guide RNA (gRNA), wherein the gRNA binds to a complementary sequence of a target motif of the B2M gene. In some embodiments, the RNA-guided endonuclease is selected from the group consisting of MAD7, MAD2, C2c1, C2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, and Cas13c. In some embodiments, the RNA-guided endonuclease is Cas12a (Cpf1). In some embodiments, the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1). In some embodiments, the RNA-guided endonuclease is MAD7.

[0012] In some embodiments, the gRNA binds to at least a portion of the complementary sequence of SEQ ID NO: 1. In some embodiments, the gRNA binds to a complementary sequence of any one of SEQ ID NOs: 2-6 or 17. In some embodiments, the complementary sequence includes any one of SEQ ID NOs: 19-24. In some embodiments, the gRNA includes any one of the sequences of SEQ ID NOs: 7-11 or 18. In some embodiments, the gRNA includes SEQ ID NO: 18. In some embodiments, the gRNA consists of any one of the sequences of SEQ ID NOs: 7-11 or 18. In some embodiments, the gRNA consists of SEQ ID NO: 18. In some embodiments, the complementary sequence includes any one of SEQ ID NOs: 19-24.

[0013] In one aspect, induced pluripotent stem cells (iPSCs) containing disrupted B2M gene, e.g., B2M - / - The iPSCs are produced by a method comprising: (a) contacting a population of isolated cells with an activation culture comprising IL-15 and zoledronic acid; (b) culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; (c) transducing the γδ T cells with a viral vector encoding one or more reprogramming factors; and (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state, thereby producing a population of iPSCs. In certain embodiments, the iPSCs containing disrupted B2M gene are generated by contacting the population of iPSCs produced thereby with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA), wherein the gRNA binds to a target motif of the beta-2-microglobulin (B2M) polynucleotide sequence in the population of iPSCs, and contacting results in cleavage of the B2M polynucleotide sequence, whereby the B2M gene is disrupted.

[0014] In one aspect, provided herein is a method of producing induced pluripotent stem cells (iPSCs), comprising: (a) contacting an isolated cell population with an activation culture comprising IL-15 and zoledronic acid; (b) culturing the isolated cell population in the activation culture to enrich and / or activate γδ T cells in the isolated cell population; (c) transducing the γδ T cells with a viral vector encoding one or more reprogramming factors; (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state, thereby producing a population of iPSCs; and (e) contacting the population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA), wherein the gRNA binds to a target motif of a beta-2-microglobulin (B2M) polynucleotide sequence in the population of iPSCs, and the contacting results in cleavage of the B2M polynucleotide sequence, whereby the B2M gene is disrupted.

[0015] In some embodiments, the RNA-guided endonuclease is selected from the group consisting of MAD7, MAD2, C2c1, C2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, and Cas13c. In some embodiments, the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1). In some embodiments, the RNA-guided endonuclease is Cas12a (Cpf1). In some embodiments, the RNA-guided endonuclease is MAD7.

[0016] In some embodiments, the target motif comprises a portion of SEQ ID NO: 1. In some embodiments, the target motif comprises any one of the sequences of SEQ ID NOs: 2-6 or 17. In some embodiments, the target motif consists of any one of the sequences of SEQ ID NOs: 2-6 or 17. In some embodiments, the gRNA comprises any one of the sequences of SEQ ID NOs: 7-11 or 18. In some embodiments, the gRNA consists of any one of the sequences of SEQ ID NOs: 7-11 or 18. In some embodiments, the gRNA comprises SEQ ID NO: 18. In some embodiments, the gRNA consists of SEQ ID NO: 18. In some embodiments, the gRNA binds to a complementary sequence to any one of SEQ ID NOs: 2-6 or 17. In some embodiments, the complementary sequence comprises any one of SEQ ID NOs: 19-24.

[0017] In some embodiments, cleavage of the B2M polynucleotide sequence results in reduced B2M expression in the iPSC as compared to the expression of B2M in the reference. In some embodiments, the reduced B2M expression is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the expression of B2M in the reference. In some embodiments, cleavage of the B2M polynucleotide sequence results in reduced HLA-A, HLA-B, and / or HLA-C expression as compared to the expression of HLA-A, HLA-B, and / or HLA-C in the reference. In some embodiments, the reduced HLA-A, HLA-B, and / or HLA-C expression is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the expression of HLA-A, HLA-B, and / or HLA-C in the reference. In some embodiments, the reference is an iPSC or a population of iPSCs without cleavage of the B2M polynucleotide sequence.

[0018] In some embodiments, the activated culture further comprises IL-2. In some embodiments, the viral vector is a Sendai virus (SeV) vector. In some embodiments, the method further comprises obtaining a population of cells isolated from a subject. In some embodiments, the population of isolated cells is a peripheral blood mononuclear cell (PBMC). In some embodiments, the population of isolated cells is a terminally differentiated cell. In some embodiments, the population of isolated cells is a mammalian cell. In some embodiments, the population of isolated cells is a human cell. In some embodiments, the population of isolated cells is cultured in the activated culture for up to 13 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, or up to 1 day. In some embodiments, the population of isolated cells is cultured in the activated culture for up to 3 days. In some embodiments, the population of isolated cells is cultured in the activated culture for 3 days. In some embodiments, after being cultured in the activated culture, the population of isolated cells comprises less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, or less than 30% γδ T cells. In some embodiments, after being cultured in the activated culture, the population of isolated cells comprises less than 35% γδ T cells. In some embodiments, the method further comprises, after step (b), enriching γδ T cells in the population of isolated cells. In some embodiments, the γδ T cells are enriched by cell-to-cell aggregate enrichment. In some embodiments, at least a portion of the γδ T cells are activated to Vγ9 + γδ T cells in step (b). In some embodiments, at least a portion of the γδ T cells are activated to Vγ9δ2 + γδ T cells in step (b).

[0019] In some embodiments, one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc. In some embodiments, in step (d), the transduced γδ T cells are cultured in the presence of one or more feeder cell layers. In some embodiments, in step (d), the transduced γδ T cells are cultured in the presence of a single layer of feeder cells. In some embodiments, the feeder cell layer comprises mouse embryonic fibroblasts (MEFs). In some embodiments, the method further comprises isolating and / or purifying the produced iPSCs. In some embodiments, the method further comprises differentiating the iPSCs into cells of a desired cell type ex vivo.

[0020] In some embodiments, the produced iPSCs are negative for Sendai virus (SeV) vectors. In some embodiments, the produced iPSCs are derived from γδ T cells. In some embodiments, the produced iPSCs have rearranged genes at the TRG locus and the TRD locus, and optionally, the produced iPSCs have a Vγ9 gene configuration and a Vδ2 gene configuration. In some embodiments, the produced iPSCs are not derived from αβ T cells. In some embodiments, the produced iPSCs do not produce or express TCRA and / or TCRB, or fragments thereof, such that there is no detectable or other surface expression of TCRA and TCRB.

[0021] In some embodiments, the produced iPSCs are genomically stable without chromosomal loss. In some embodiments, the genomic stability of the produced iPSCs is determined by karyotyping. In some embodiments, the produced iPSCs can grow in a medium without feeder cells after adaptation. In some embodiments, induced pluripotent stem cells (iPSCs) are produced according to the method. In some embodiments, iPSCs are produced according to the method. In some embodiments, the composition comprises iPSCs. In some embodiments, differentiated iPSCs are produced according to the method.

[0022] In one aspect, provided herein is a method of producing induced pluripotent stem cells (iPSCs), the method comprising: (a) a step for performing a function of enriching and / or activating γδ T cells in a population of isolated cells; (b) a step for performing a function of reprogramming γδ T cells into a pluripotent state, thereby producing iPSCs; and (c) a step for contacting the iPSCs with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), wherein the gRNA binds to a target motif of a beta-2-microglobulin (B2M) polynucleotide sequence in the iPSCs, and the contacting step results in cleavage of the B2M polynucleotide sequence. In some aspects, induced pluripotent stem cells (iPSCs) are produced according to the method.

[0023] In one aspect, provided herein is a population of isolated induced pluripotent stem cells (iPSCs) comprising pluripotent cells, wherein the pluripotent cells comprise means for expressing one or more reprogramming factors and / or the pluripotent cells comprise means for encoding rearrangement of the TRG gene and the TRD gene, and the pluripotent cells comprise means for cleaving the B2M gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0024]

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DETAILED DESCRIPTION OF THE INVENTION

[0025] The present disclosure provides, in part, β2m knockout iPSCs and methods for producing β2m knockout iPSCs, which are derived from T cells, particularly γδ T cells. In some embodiments, the present disclosure provides a population of induced pluripotent stem cells (iPSCs), wherein at least a portion of the iPSCs comprise a disrupted beta-2-microglobulin (B2M) gene and the iPSCs are generated by reprogramming γδ T cells.

[0026] γδ T cells are a subset of T lymphocytes that express a TCR different from that expressed by αβ T cells, which are the major subset of T lymphocytes in human peripheral blood (Kalyan, S. & Kabelitz, D., Cell Mol. Immunol., 2013, 10(1):21-29). Vγ9Vδ2 T cells are the major subset of γδ T cells and exhibit significant effector functions against tumor cells (Tyler, C.J., et al., Cellular Immunology, 2015, 296(1):10-21, Silva-Santos, B., Nat Rev Immunol., 2015, 15:683-91).

[0027] 5.1. Definitions As used herein, the terms "about" or "approximately" refer to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% with respect to the amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. Ranges of amounts, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths can be ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% with respect to the amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. The term "about" associated with a reference numerical value includes the numerical value itself and a range of values, for example, plus or minus 10% from that numerical value. In some embodiments, an amount of "about 10" includes 10 and any amount from 9 to 11. In some cases, numerical values disclosed throughout may be the numerical value "about" even if the term "about" is not specifically stated.

[0028] Unless otherwise specified, the terms "at least", "up to", or "about" preceding a series of elements should be understood to refer to all elements of the series.

[0029] As used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural references.

[0030] As used herein, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or". For example, the condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0031] As used herein, the conjunctive term "and / or" between a plurality of recited elements is understood to cover both the individual and combined choices. For example, when two elements are connected by "and / or", the first choice refers to the applicability of the first element without the second element. The second choice refers to the applicability of the second element without the first element. The third choice refers to the applicability of the first and second elements together. Any one of these choices is meant to be included, and thus, as used herein, is understood to meet the requirements of the term "and / or". The simultaneous applicability of two or more of the choices is also meant to be included, and thus, is understood to meet the requirements of the term "and / or".

[0032] As used herein, the terms "beta-2 microglobulin", "B2M", or "β2m" refer to the beta chain component of MHC class I molecules. Human beta-2 microglobulin is encoded by the B2M gene (e.g., NCBI Gene ID 567). The expression of beta-2 microglobulin is required for the assembly and function of MHC class I molecules on the cell surface.

[0033] As used herein, the term "MHC class I molecule" refers to the major histocompatibility complex (MHC) found on the cell surface that presents peptide fragments of non-self proteins. MHC class I molecules consist of two polypeptide chains. The alpha chain consists of three polypeptides designated as alpha-1, alpha-2, and alpha-3 domains. The alpha chain is non-covalently linked via the alpha-3 domain to a beta chain consisting of beta-2 microglobulin (B2M). The alpha chain is polymorphic and is encoded by the HLA genes (i.e., HLA-A, HLA-B, and HLA-C), while beta-2 microglobulin is not polymorphic and is encoded by the B2M gene.

[0034] As used herein, the terms "deletion" or "knockout" refer to a genetic modification in which a site or region of genomic DNA is removed by any molecular biology method, such as the methods described herein, e.g., by delivering an endonuclease and at least one gRNA to a site of genomic DNA. The terms "deletion" or "knockout" include deleting all or a portion of a target polynucleotide sequence so as to interfere with the function of the target polynucleotide sequence. In some embodiments, a "deletion" or "knockout" can result in a complete or partial loss of expression of the target gene. Any number of nucleotides can be deleted. In some embodiments, the deletion involves the removal of at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, or more than 25 nucleotides. In some embodiments, the deletion involves the removal of 10-50, 25-75, 50-100, 50-200, or more than 100 nucleotides. In some embodiments, the deletion involves the removal of an entire target gene, e.g., the B2M gene. In some embodiments, the deletion involves the removal of all or a portion of a part of the target gene, e.g., the promoter and / or coding sequence of the B2M gene. In some embodiments, the deletion involves the removal of a transcriptional regulatory factor of the target gene, e.g., the promoter region. In some embodiments, the deletion involves the removal of all or a portion of the coding region such that the product normally expressed by the coding region is no longer expressed, is expressed as a truncated form, or is expressed at reduced levels. In some embodiments, the deletion results in a decrease in gene expression as compared to an unmodified cell. In some embodiments, a knockout can be achieved by modifying a target polynucleotide sequence by inducing an indel in the target polynucleotide sequence within a functional domain (e.g., a DNA binding domain) of the target polynucleotide sequence. The terms "disruption" or "disrupted" refer to a modification that results in a gene product that does not exhibit wild-type function and / or activity levels. In some aspects, disruption refers to a modification of a gene such that the disrupted gene results in the production of such a non-wild-type gene product.In certain embodiments, disruption truncates a gene, such as the beta-2 microglobulin (B2M) gene. In certain embodiments, disruption deletes a gene, such as the β-2 microglobulin (B2M) gene. In certain embodiments, disruption results in a gene that produces an inactive protein. In certain embodiments, disruption results in disruption of the B2M reading frame by multiple out-of-frame deletions. In certain embodiments, disruption results in disruption of the B2M reading frame by a single out-of-frame deletion. In certain embodiments, disruption results from an insertion of about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleotides or nucleotide base pairs (e.g., an insertion that changes the reading frame of a gene, such as B2M). In certain embodiments, disruption results in disruption of the B2M reading frame. In certain embodiments, the gene is the B2M gene and disruption results in a B2M gene that produces an inactive B2M protein. In certain embodiments, disruption results in a gene that expresses a reduced amount of gene product, such as a reduced amount of B2M polypeptide. In certain embodiments, the gene is the B2M gene and disruption results in a B2M gene that expresses a reduced amount of B2M polypeptide. In certain embodiments, disruption results in a gene that does not express a detectable amount of gene product, e.g., a gene that does not express a detectable amount of B2M polypeptide. In certain embodiments, the gene is the B2M gene and disruption results in a B2M gene that does not express a detectable amount of B2M polypeptide. A disrupted gene, such as a disrupted B2M gene, can refer to a gene that contains an insertion, deletion, or substitution compared to the corresponding wild-type gene such that the disrupted gene does not express a reduced, e.g., detectable, amount of functional protein compared to the expression of the wild-type gene. A gene can be disrupted via a method of inserting, deleting, or substituting at least one nucleotide / nucleic acid in the endogenous gene such that expression of the functional protein from the endogenous gene is reduced or inhibited.In some embodiments, the terms “disruption,” “disrupted,” “knockout,” or “deletion” are used interchangeably in this disclosure.

[0035] As used herein, the term "endonuclease" generally refers to an enzyme that cleaves phosphodiester bonds within a polynucleotide. In some embodiments, the endonuclease specifically cleaves phosphodiester bonds within a DNA polynucleotide. In some embodiments, the endonuclease is a zinc finger nuclease (ZFN), a transcription activator like effector nuclease (TALEN), a homing endonuclease (HE), a meganuclease, a MegaTAL, or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated endonuclease. A CRISPR cluster includes spacers, sequences complementary to a preceding mobile element, and a target invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In some embodiments, the endonuclease is an RNA-guided endonuclease. In certain embodiments, the RNA-guided endonuclease is a CRISPR nuclease, such as a type II CRISPR Cas9 endonuclease or a type V CRISPR Cpf1 (or Cas12a) endonuclease. In some embodiments, the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1). The CRISPR-Cas system can be characterized as a class 1 or class 2 system. Class 1 systems are characterized by multi-subunit effectors, i.e., they contain multiple Cas proteins. Class 1 systems can be further characterized as types I, III, and IV. Class 2 systems are characterized by a single effector protein having multiple domains. Class 2 systems can be further characterized as types II, V, and VI. For example, the class 2 type II system includes Cas9, and the class 2 type V system includes Cpf1 (Cas12a).Further examples of Cas proteins include, but are not limited to, Cas9 protein, Cas9-like proteins encoded by Cas9 orthologs, Cas9-like synthetic proteins, Cpf1 protein, proteins encoded by Cpf1 orthologs, Cpf1-like synthetic proteins, C2c1 protein, C2c2 protein, C2c3 protein, and variants and modifications thereof. In some embodiments, the endonuclease is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1 (also known as Cas12a), MAD7, MAD2 endonuclease, or homologs thereof, recombinants of their naturally occurring molecules, codon-optimized versions thereof, or modified versions thereof, or combinations thereof. Examples of Cas proteins include, but are not limited to, MAD7, MAD2, Cpf1, C2c1, C2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, and Cas13c.In some embodiments, the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1). In some embodiments, the endonuclease can introduce one or more single-stranded breaks (SSBs) and / or one or more double-stranded breaks (DSBs).

[0036] As used herein, the terms "Cas12" or "Cas12 protein" refer to any Cas12 protein including, but not limited to, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e. In some embodiments, the Cas12 protein has an amino acid sequence that is at least 85% (or at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of a functional Cas12 protein. In some embodiments, the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1). In some embodiments, the Cas12 protein can be a Cas12 polypeptide that is substantially identical to a naturally-occurring protein or has at least about 85% sequence identity (or at least about 90% sequence identity, or at least about 95% sequence identity, or at least about 96% sequence identity, or at least about 97% sequence identity, or at least about 98% sequence identity, or at least about 99% sequence identity) to a naturally-occurring Cas12 protein and has substantially the same biological activity. Examples of Cas12a proteins include, but are not limited to, FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, or Lb4Cas12a. Examples of Cas12b proteins include, but are not limited to, AacCas12b, Aac2Cas12b, AkCas12b, AmCas12b, AhCas12b, and AcCas12b.

[0037] In some embodiments, the term "Cpf endonuclease" refers to an RNA-guided DNA endonuclease associated with CRISPR that cleaves a target DNA sequence when bound to a guide RNA. The Cpf endonuclease is guided by the guide RNA to recognize and cleave a specific target site in double-stranded DNA in the genome of a cell. In some embodiments, the CRISPR-Cpf system uses an Acidaminococcus genus Cpf1 endonuclease, a Lachnospiraceae genus Cpf1 endonuclease, or a Francisella novicide Cpf1 endonuclease, or variants thereof. The Cpf1-crRNA is cleaved by the specification of the protospacer adjacent motif (PAM) 5'-TTTN of the Acidaminococcus genus Cpf1 endonuclease and the Lachnospiraceae genus Cpf1 endonuclease, as well as the PAM sequence 5'-TTN of Francisella novicide Cpf1. After the specification of the PAM, Cpf1 introduces a sticky-end DNA double-strand break with an overhang of 4 to 5 nucleotides distal from the 3' end of the targeted PAM, which is then repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). The term "Cpf1 endonuclease" is understood to include variants thereof.

[0038] As is known to those skilled in the art, the term "Mad endonuclease" refers to an RNA-guided DNA endonuclease associated with CRISPR that cleaves a target DNA sequence when bound to a guide RNA. The Mad endonuclease is induced by the guide RNA to recognize and cleave a specific target site in double-stranded DNA in the genome of a cell. The CRISPR-Mad system is closely related to the type V (Cpf1-like) of the class 2 family of CAS enzymes. In some embodiments, the CRISPR-Mad system uses the Eubacterium rectale MAD7 endonuclease or a variant thereof. In some embodiments, MAD7 is a class 2 type V-A CRISPR family identified in Eubacterium rectale. The MAD7-crRNA complex cleaves the target by the specification of the protospacer adjacent motif (PAM) 5'-YTTN. After the specification of the PAM, MAD7 introduces a sticky-ended DNA double-strand break with an overhang of 4 to 5 nucleotides at the 3' end of the targeted PAM, which is then repaired by either non-homologous end joining (NHEJ) or homologous recombination repair (HDR). The term "Mad endonuclease" is understood to encompass variants thereof. In some embodiments, the B2M target motif specified or used in the CRISPR-Cpf1 (Cas12a) system is the same B2M target motif as when using MAD7. In some embodiments, the same guide nucleic acid or guide RNA can be used with Cpf1 (or Cas12a) and the MAD7 nuclease. In some aspects, the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1).

[0039] As used herein, the terms “guide RNA” or “gRNA” generally refer to short ribonucleic acids that interact with, e.g., can bind to, and can bind or hybridize to a target genomic site or region. In some embodiments, the gRNA is a single-molecule guide RNA (sgRNA). In some embodiments, the gRNA can include a spacer extension region. In some embodiments, the gRNA can include a tracrRNA extension region. In some embodiments, the gRNA is single-stranded. In some embodiments, the gRNA includes naturally occurring nucleotides. In some embodiments, the gRNA is a chemically modified gRNA. In some embodiments, the chemically modified gRNA is a gRNA that includes at least one nucleotide having a chemical modification, e.g., a 2’-O-methyl sugar modification. In some embodiments, the chemically modified gRNA includes a modified nucleic acid backbone. In some embodiments, the chemically modified gRNA includes 2’-O-methyl-phosphorothioate residues. In some embodiments, the gRNA may be pre-complexed with a DNA endonuclease. In some embodiments, the gRNA sequence includes modifications. In some embodiments, the modifications increase the stability of the gRNA. In some embodiments, the gRNA sequence includes AltR1 and / or AltR2. In some embodiments, AltR1 and AltR2 are proprietary (IDT) modifications used to increase the stability of short RNAs (e.g., gRNAs).

[0040] As used herein, the term "gene modification" generally refers to a site of genomically edited or engineered DNA, using any molecular biological method, such as the methods described herein, for example, by delivering an endonuclease and at least one gRNA to a site in genomic DNA. Examples of gene modifications include insertions, deletions, duplications, inversions, and translocations, and combinations thereof. In some embodiments, the gene modification is a deletion. In some embodiments, the gene modification is an insertion. In other embodiments, the gene modification is an insertion-deletion mutation (or indel), resulting in a shift in the reading frame of the target gene, either resulting in a modified gene product or no gene product.

[0041] In some embodiments, "modification," "alteration," or "at least partial deletion" of a gene (e.g., the B2M gene) results in reduced expression of the target polynucleotide sequence (e.g., as compared to a cell or population of cells without B2M modification, alteration, or at least partial deletion). The terms "decrease," "reduction," and "lower" are all used interchangeably herein and mean a statistically significant amount (e.g., below two standard deviations (2SD) from normal) of reduction. In some embodiments, "decrease," "reduction," or "lower" means at least about a 5% reduction as compared to a reference level, e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduction as compared to a reference level. In some embodiments, "decrease," "reduction," or "lower" is any reduction from 10 to 100% as compared to a reference level. In some embodiments, "decrease," "reduction," or "lower" means at least about a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold reduction as compared to a reference level. In some embodiments, the decrease or reduction in expression results in undetectable levels of the target gene or target polynucleotide sequence in a cell or population of cells as determined by methods used by those of skill in the art or methods disclosed in the present disclosure (e.g., FACS). In some embodiments, the decrease in B2M expression is reduced as compared to a reference. In some embodiments, the reference is an iPSC or population of iPSCs without cleavage of the B2M polynucleotide sequence.

[0042] In some embodiments, the terms "increase", "enhancement", and "elevation" are all used interchangeably herein and mean an increase of at least about 5%, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a reference level. In some embodiments, "increase", "enhancement", or "elevation" is any increase from 10 to 100% compared to a reference level. In some embodiments, "increase", "enhancement", or "elevation" means an increase of at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a reference level.

[0043] As used herein, the term "polynucleotide", which may be used interchangeably with the term "nucleic acid", generally refers to a biomolecule comprising two or more nucleotides. Typically, the polynucleotides of the present disclosure are composed of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) naturally found in DNA or RNA, linked by phosphodiester bonds. In some embodiments, the polynucleotide is a hybrid DNA / RNA molecule. In some embodiments, the term encompasses molecules containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When the present application refers to a polynucleotide, it is understood that both DNA and RNA, and both single-stranded and double-stranded forms in each case (and the complement of each single-stranded molecule) are provided. As used herein, the term "polynucleotide sequence" can refer to the polynucleotide substance itself and / or the sequence information (i.e., the sequence of letters used as abbreviations for bases) that biochemically characterizes a particular nucleic acid. The polynucleotide sequences presented herein are presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, the polynucleotide comprises at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 250, at least 500, or any number of nucleotides. In some embodiments, the polynucleotide is a site or region of genomic DNA. In some embodiments, the polynucleotide is an endogenous gene contained within the genome of a cell. In some embodiments, the polynucleotide is an exogenous polynucleotide not integrated into genomic DNA. In some embodiments, the polynucleotide is an exogenous polynucleotide that is integrated into genomic DNA.In some embodiments, the polynucleotide is a plasmid or an adeno-associated virus vector. In some embodiments, the polynucleotide is a circular or linear molecule.

[0044] As used herein, "cell culture medium" (also referred to herein as "culture medium" or "culture" or "medium") is a medium for culturing cells containing nutrients that maintain cell viability and support growth. The cell culture medium can contain any of salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum substitutes, and other components such as peptide growth factors in appropriate combinations. Cell culture media commonly used for specific cell types are known to those skilled in the art. Some non-limiting examples are provided herein.

[0045] As used herein, "cell line" typically refers to a population of cells that are derived from a single ancestral cell or from a defined and / or substantially identical population of ancestral cells, and are mostly or substantially identical. A cell line may be maintained in culture over a long period of time (e.g., for months, years, an unlimited period) or may be capable of being maintained. It may have undergone an autologous or induced process of transformation that confers an unlimited culture lifespan on the cells. A cell line includes all cell lines so recognized in the art. It will be understood that cells can acquire mutations and, in some cases, epigenetic changes over time such that at least some of the properties of the individual cells of the cell line can differ from one another.

[0046] As used herein, the terms "differentiate", "differentiation", etc. refer to the process by which unspecialized (or undetermined) or relatively unspecialized cells acquire the characteristics of specialized cells such as blood cells or muscle cells. Differentiated cells or cells induced to differentiate are cells that are in a more specialized (or determined) position within a cell lineage. A cell is determined when it has proceeded along a differentiation pathway to a point where, under normal circumstances, it continues to differentiate into a particular cell type or subset of cell types and, under normal circumstances, cannot differentiate into a different cell type or revert to a relatively undifferentiated cell type.

[0047] As used herein, the term "encode" refers to the unique property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA that functions as a template for the synthesis of other polymers and macromolecules in a biological process, having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, as well as the biological properties resulting therefrom. Thus, in a cell or other biological system, when transcription and translation of the mRNA corresponding to a gene produce a protein, that gene encodes the protein. Both a nucleotide sequence that is identical to the mRNA sequence and the coding strand, which is normally provided in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0048] As used herein, the term "exogenous" is intended to mean that the referenced molecule or activity is introduced into the host cell. This molecule can be introduced, for example, by introduction of the coding nucleic acid into the host genetic material, such as by integration into the host chromosome, or as extrachromosomal genetic material, such as a plasmid. Thus, when used with respect to the expression of a coding nucleic acid, the term refers to the introduction of the coding nucleic acid in an expressible form into a cell. The term "endogenous" refers to a reference molecule or activity that is present in the host cell. Similarly, when used with respect to the expression of a coding nucleic acid, the term refers to the expression of a coding nucleic acid that is contained within the cell and is not exogenous.

[0049] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a stem cell produced from a differentiated adult cell that has been induced or altered (i.e., reprogrammed) to be capable of differentiating into cells of all three germ layers or cortical tissues, the mesoderm, endoderm, and ectoderm.

[0050] As used herein, terms such as "isolated," when used with respect to a cell, are intended to mean a cell that is substantially free of at least one component as it is found in nature. The term includes a cell that has been removed from some or all of the components as it is found in its natural environment. The term also includes a cell that has been removed from at least one, some, or all of the components such that it is found in an environment where it does not occur naturally. Thus, an isolated cell is partially or completely separated from other substances as it is found in nature or as it grows, stores, or exists in an environment where it does not occur naturally. Specific examples of isolated cells include cells that are partially pure, substantially pure, and cells cultured in a non-naturally occurring medium.

[0051] As used herein, terms such as "purify" refer to increasing purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% (e.g., as compared to a reference).

[0052] As used herein, the term "pluripotency" refers to the ability of a cell to form all lineages of the body or cell mass (i.e., the embryo proper). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: the ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential from less primitive and less pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot give rise to a complete organism to more primitive and more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism.

[0053] As used herein, the term "population," when used with respect to T lymphocytes, refers to a group of cells containing two or more T lymphocytes. A population of isolated T lymphocytes can have only one type of T lymphocyte or two or more types of T lymphocytes. A population of isolated T lymphocytes can be a homogeneous population of one type of T lymphocyte or a heterogeneous population of two or more types of T lymphocytes. A population of isolated T lymphocytes can also be a heterogeneous population having T lymphocytes and at least cells other than T lymphocytes, such as B cells, macrophages, neutrophils, red blood cells, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. The heterogeneous population can have from 0.01% to about 100% T lymphocytes. Thus, a population of isolated T lymphocytes can have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T lymphocytes. A population of isolated T lymphocytes can contain only one type of T lymphocyte or a mixture of two or more types of T lymphocytes. A population of isolated T lymphocytes can contain one or two or more, or all, of the different types of T lymphocytes, including but not limited to those disclosed herein. A population of isolated T lymphocytes can contain all known types of T lymphocytes. In a population of isolated T lymphocytes containing two or more types of T lymphocytes, the ratio of each type of T lymphocyte can range from 0.01% to 99.99%. An isolated population can also be a clonal population of T lymphocytes, where all T lymphocytes in the population are clones of a single T lymphocyte.

[0054] A "recombinant" polynucleotide is a polynucleotide that is not in its natural state. For example, the polynucleotide contains a nucleotide sequence not found in nature, or the polynucleotide is in a context other than its natural context. For example, the polynucleotide is separated from nucleotide sequences that are normally adjacent in nature, or is adjacent (or contiguous) to nucleotide sequences that are not normally adjacent. For example, the sequence in question can be cloned into a vector or, alternatively, recombined with one or more additional nucleic acids.

[0055] As used herein, "reprogramming" refers to the process of changing or reversing the differentiated state of a somatic cell. The cell can be partially or terminally differentiated prior to reprogramming. Reprogramming encompasses the complete reversion of the differentiated state of a somatic cell (e.g., a T cell) to a pluripotent state. Reprogramming also encompasses the partial reversal of the differentiated state of a somatic cell to a state that makes the cell more sensitive to complete reprogramming to a pluripotent state when subjected to further manipulations as described herein. Such exposure can result in the expression of specific genes by the cell, and this expression contributes to reprogramming. In certain embodiments of the present disclosure, reprogramming of a somatic cell results in a somatic cell that is pluripotent and in an ES-like state. The resulting cells are referred to herein as reprogrammed pluripotent somatic cells or induced pluripotent stem cells (iPSCs). In some embodiments, reprogramming also encompasses the partial reversal of the differentiated state of a somatic cell to a multipotent state.

[0056] Reprogramming is different from simply maintaining the existing undifferentiated state of cells that are already pluripotent, or maintaining the existing not fully differentiated state of cells that are already multipotent (e.g., hematopoietic stem cells). Reprogramming is also different from promoting self-renewal or proliferation of cells that are already pluripotent or multipotent. In some embodiments, the methods described herein contribute to establishing a pluripotent state by reprogramming. In some embodiments, the methods described herein may be performed on fully differentiated cells and / or certain types of cells (e.g., γδ T cells) rather than cells that are already multipotent or pluripotent.

[0057] As used herein, "reprogramming factor" refers to a gene, RNA, or protein that promotes or contributes to cell reprogramming, e.g., in vitro. Examples of reprogramming factors for reprogramming somatic cells to pluripotency in vitro include Oct3 / 4, Klf4, c-Myc, Nanog, Sox2, and Lin28, and any gene / protein that can substitute for one or more of these in a method of reprogramming somatic cells, e.g., in vitro.

[0058] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably and refer to the major types of white blood cells that mature in the thymus and have various roles in the immune system, including the identification of specific foreign antigens in the body and the activation and inactivation of other immune cells. T lymphocytes can be any T lymphocytes, such as cultured T lymphocytes, e.g., primary T lymphocytes, or T lymphocytes from cultured T cell lines, e.g., T lymphocytes from Jurkat, SupT1, etc., or T lymphocytes obtained from mammals. T lymphocytes can be CD3+ cells. T lymphocytes can be any type of T lymphocyte, including, but not limited to, CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMC), peripheral blood leukocytes (PBL), tumor infiltrating lymphocytes (TIL), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδ T cells), etc., at any stage of development. T lymphocytes can be regulatory T cells, including nTreg (natural Treg), iTreg (induced Treg), CD8 + Treg, regulatory Tr1 cells, and Th3 cells. Further types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Further types of memory T cells include cells such as central memory T cells (T CM cells), effector memory T cells (T EM cells and T EMRA cells), etc. T lymphocytes can also refer to genetically engineered T lymphocytes, such as T lymphocytes modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). Additionally, T lymphocytes can be differentiated from stem cells, definitive angioblasts, CD34+ cells, HSC (hematopoietic stem cells and progenitor cells), hematopoietic multipotent progenitor cells, or T cell progenitor cells.

[0059] As used herein, the term "γδ T cell" refers to a T cell having a T cell receptor that includes a γ chain and a δ chain on its surface.

[0060] As used herein, the term "selectable marker" refers to a gene, RNA, or protein that, when expressed, confers a selectable phenotype on a cell, such as resistance to a cytotoxic or cytostatic agent (e.g., antibiotic resistance), nutritional prototrophy, or the expression of a specific protein that can be used as a basis for distinguishing cells expressing the protein from cells that do not. Proteins that can be easily detected for expression ( "detectable markers"), such as fluorescent or luminescent proteins, or enzymes that act on a substrate to produce a colored, fluorescent, or luminescent substance, constitute a subset of selectable markers. The presence of a selectable marker linked to native expression control elements for genes that are normally expressed selectively or exclusively in pluripotent cells enables the identification and selection of somatic cells that have been reprogrammed to a pluripotent state. A variety of selectable marker genes can be used, such as the neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyl transferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and the hisD gene. Detectable markers include green fluorescent protein (GFP), blue, sapphire, yellow, red, orange, and cyan fluorescent proteins, as well as variants of any of these.Luminescent proteins such as luciferase (e.g., firefly or Renilla luciferase) are also useful. As will be apparent to those skilled in the art, as used herein, the term "selection marker" can refer to a gene or the expression product of a gene, e.g., the encoded protein.

[0061] In some embodiments, a selection marker confers a growth and / or survival benefit to cells that express it compared to cells that do not express it or express it at a significantly lower level. Such growth and / or survival benefits typically occur when the cells are maintained under certain conditions, i.e., "selective conditions." To ensure effective selection, a population of cells can be maintained under conditions and for a sufficient period of time such that cells that do not express the marker do not grow and / or do not survive and are excluded from the population or their numbers are reduced to only a very small percentage of the population. By maintaining a population of cells under selective conditions such that cells that do not express the marker are largely or completely excluded, the process of selecting cells that express a marker that confers a growth and / or survival benefit is referred to herein as "positive selection," and the marker is said to be "useful for positive selection." Negative selection and markers useful for negative selection are also of interest in certain methods described herein. Expression of such a marker confers a growth and / or survival disadvantage to cells that express it compared to cells that do not express it or express it at a significantly lower level (or, alternatively viewed, cells that do not express the marker have a growth and / or survival benefit compared to cells that express the marker). Thus, cells that express the marker can be largely or completely excluded from a population of cells when maintained under selective conditions for a sufficient period of time.

[0062] As used herein, "feeder cell (feeder)" is a type of cell that is co-cultured with a second type of cell, and by providing stimuli, growth factors, and nutrients for the support of the second type of cell, an environment is provided in which the second type of cell can grow, expand, or differentiate. Feeder cells are optionally derived from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by a primary culture of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, cells derived from peripheral blood or transformed leukemia cells support the proliferation and maturation of natural killer cells. Feeder cells can typically be inactivated by treatment with mitotic inhibitors such as radiation or mitomycin to prevent overgrowth of the cells they support when co-cultured with other cells. Feeder cells can include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limitation, one particular cell type of feeder cell can be a human feeder cell such as human dermal fibroblasts. Another cell type of feeder cell can be mouse embryonic fibroblasts (MEFs). In general, various feeder cells can be used in part to direct differentiation toward a particular lineage, enhance proliferative capacity, and promote maturation into specialized cell types such as effector cells.

[0063] As used herein, a "feeder-free" (FF) environment refers to an environment such as a culture condition, cell culture, or culture medium that is essentially free of feeder cells or stromal cells and / or has not been pre-conditioned by the culture of feeder cells. In some embodiments, a feeder-free medium containing a recombinant protein surface is provided. One of ordinary skill in the art will understand the use of media such as iMatrix / laminin for use in a feeder-free environment, for example.

[0064] The term "pre-conditioned" medium refers to a medium that is harvested after the feeder cells have been cultured for a defined period, such as at least one day, in the medium. The pre-conditioned medium contains many mediator substances, including growth factors and cytokines secreted by the feeder cells cultured in the medium. In some embodiments, an environment free of feeder cells does not contain both feeder cells and stromal cells and is not pre-conditioned by culturing of feeder cells. In some embodiments, an additional step of filtering the medium to remove non-human elements is performed.

[0065] The term "pluripotency-related gene" refers to a gene whose expression occurs in pluripotent stem cells under normal conditions (e.g., in the absence of genetic or other manipulations designed to alter gene expression), is typically restricted to pluripotent stem cells, and is important for the functional identity of the gene itself. It is understood that polypeptides encoded by genes that are functionally related to pluripotency may be present as maternal factors in the oocyte. This gene may be expressed by at least some cells of the embryo, for example, over at least a portion of the pre-implantation period and / or in adult germ cell precursors.

[0066] The term "pluripotency factor" is used to refer to the expression product of a pluripotency-related gene, e.g., a polypeptide encoded by a gene. In some embodiments, the pluripotency factor is not normally substantially expressed in somatic cell types (excluding germ cells or their precursors) that make up the body of an adult animal. For example, the average level of the pluripotency factor in ES cells can be at least 50-fold or 100-fold greater than its average level in terminally differentiated cell types present in the body of an adult mammal. In some embodiments, the pluripotency factor is essential for maintaining the viability or pluripotent state of ES cells and / or ES cells induced using conventional methods in vivo. Thus, if the gene encoding the factor is knocked out or inhibited (i.e., its expression is eliminated or substantially reduced), ES cells are not formed and die or, in some embodiments, differentiate. In some embodiments, inhibiting the expression of a gene having a function associated with pluripotency in ES cells (e.g., resulting in at least 50%, 60%, 70%, 80%, 90%, 95%, or more reduction in the average steady-state level of the RNA transcript and / or protein encoded by the gene) results in cells that are viable but no longer pluripotent. In some embodiments, the gene is characterized in that its expression in ES cells decreases (e.g., resulting in at least 50%, 60%, 70%, 80%, 90%, 95%, or more reduction in the average steady-state level of the RNA transcript and / or protein encoded by the gene) when the cell differentiates into a terminally differentiated cell.

[0067] As used herein, the term "pluripotency-inducing gene" refers to a gene whose expression contributes to reprogramming somatic cells into a pluripotent state. The term "pluripotency-inducing factor" refers to the expression product of a pluripotency-inducing gene. A pluripotency-inducing factor may or may not be a pluripotency factor. Expression of an exogenously introduced pluripotency-inducing factor may be transient, i.e., required during at least a portion of the reprogramming process to induce pluripotency and / or establish a stable pluripotent state, but not required thereafter to maintain pluripotency. For example, this factor may induce the expression of endogenous genes having functions associated with pluripotency. These genes may then maintain the reprogrammed cells in a pluripotent state.

[0068] "Polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically about 2 to 60 amino acids in length. Polypeptides used herein typically contain amino acids such as the 20 L-amino acids most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. One or more of the amino acids in a polypeptide may be modified, for example, by the addition of chemical moieties such as carbohydrate groups, phosphate groups, fatty acid groups, linkers for conjugation, functionalization, etc. A polypeptide is still considered a "polypeptide" even if a non-polypeptide moiety is associated with it covalently or non-covalently. Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, or synthesized through chemical means such as conventional solid-phase peptide synthesis. As used herein, the terms "polypeptide sequence" or "amino acid sequence" can refer to the polypeptide material itself and / or the sequence information that biochemically characterizes the polypeptide (i.e., the sequence of letters or three-letter codes used as abbreviations for amino acid names). Polypeptide sequences presented herein are presented in the N-terminal to C-terminal direction unless otherwise indicated.

[0069] 5.2. Abbreviations A list of abbreviations used in this disclosure is provided in Table 1 below.

[0070] [Table 1]

[0071] 5.3. Target Genes In one aspect, provided herein is a method of modifying a target polynucleotide sequence (e.g., B2M) in a cell (e.g., an iPSC derived from a γδ T cell (Sections 5.6 and 5.7)), which includes contacting the target polynucleotide sequence with the genome editing method of the present disclosure (Section 5.4). In some embodiments, the iPSCs used in the present disclosure and methods of making the same (e.g., to knockout B2M using a gene editing system) are provided in Sections 5.6 and 5.7 of the present disclosure.

[0072] In one aspect, provided herein is a method of modifying a target polynucleotide sequence in a cell (e.g., an iPSC derived from a γδ T cell) using a CRISPR / Cas system (see Section 5.4). Any CRISPR / Cas system capable of modifying the target polynucleotide sequence in the cell can be used in the present disclosure (e.g., to modify B2M in an iPSC derived from a γδ T cell). In one aspect, provided herein is a method of modifying a target polynucleotide sequence in a cell (e.g., an iPSC derived from a γδ T cell) using a TALEN system (see Section 5.4). In one aspect, provided herein is a method of modifying a target polynucleotide sequence in a cell (e.g., an iPSC derived from a γδ T cell) using a zinc finger system (see Section 5.4). Examples of methods utilizing CRISPR / Cas (e.g., cpf1) are described in detail herein, but it should be understood that the present disclosure is not limited to the use of these methods / systems. Other targeting methods known to those skilled in the art for reducing or eliminating expression in a target cell, e.g., B2M, can be utilized herein.

[0073] In one aspect, provided herein is a method of modifying a target B2M polynucleotide sequence in a cell, comprising contacting at least a portion of the B2M polynucleotide sequence with a clustered regularly interspaced short palindromic repeat (Cas) protein or endonuclease of the disclosure and at least one ribonucleic acid (e.g., one or two ribonucleic acids), wherein the at least one ribonucleic acid directs the Cas protein or endonuclease to a target motif of the target B2M polynucleotide sequence and hybridizes therewith. In some embodiments, the target B2M polynucleotide sequence is cleaved.

[0074] In some embodiments, modifying the target polynucleotide sequence results in knockout or reduced expression of the target polynucleotide sequence (e.g., B2M) or a portion thereof (e.g., as compared to a reference). In some embodiments, the reference is an iPSC or a population of iPSCs without cleavage of the B2M polynucleotide sequence. In some embodiments, modifying or knocking out the target polynucleotide sequence is performed in vitro. In some embodiments, modification or knockout of the target polynucleotide sequence is performed ex vivo.

[0075] In some embodiments, the target polynucleotide sequence is beta-2-microglobulin (also referred to as B2M or β2m) or a portion thereof. The terms "beta-2-microglobulin", "B2M", and "β2m" are used interchangeably in the present disclosure. In some embodiments, the target polynucleotide sequence is a variant of B2M. In some embodiments, the target polynucleotide sequence is an isoform of B2M. In some embodiments, the target polynucleotide sequence is a homolog of B2M. In some embodiments, the target polynucleotide sequence is an ortholog of B2M. In some embodiments, the target polynucleotide sequence is beta-2-microglobulin (B2M; Gene ID: 567). In some embodiments, the target polynucleotide sequence is NCBI reference sequence: NG_012920.2 or a portion thereof. In some embodiments, the target polynucleotide sequence is SEQ ID NO: 1 or a portion thereof. In some embodiments, the target polynucleotide sequence is B2M from any species (e.g., mammalian, human, mouse, rat, pig, or any other species).

[0076] In some embodiments, disruption or B2M knockout of B2M results from deletion of at least a portion of the B2M target polynucleotide sequence (e.g., SEQ ID NO: 1). In some embodiments, the deletion is about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% deletion of the target polynucleotide sequence.

[0077] In some embodiments, disruption or B2M knockout of B2M results from the insertion of about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleotides or nucleotide base pairs (bp) in the B2M target polynucleotide sequence. In some embodiments, disruption or B2M knockout of B2M results from the insertion of about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 nucleotides or nucleotide base pairs in the target polynucleotide sequence, such insertion changing the reading frame of B2M. In some embodiments, disruption or B2M knockout of B2M results from an insertion of about 1 to about 10 bp, about 1 to about 50 bp, about 1 to about 100 bp, about 1 to about 200 bp, about 1 to about 300 bp, about 1 to about 400 bp, about 1 to about 500 bp, about 1 to about 600 bp, about 1 to about 700 bp, about 1 to about 800 bp, about 1 to about 900 bp, about 1 to about 1000 bp, about 1 to about 5000 bp, about 1 to about 10,000 bp, or more than 10,000 bp.

[0078] In some embodiments, the present disclosure provides a method of disrupting a gene or polynucleotide sequence encoding the amino acid sequence of B2M. In some embodiments, the amino acid sequence of B2M is any one of SEQ ID NOs: 12-16. In some embodiments, the amino acid sequence of B2M is any one of UniProtKB P61769, UniProtKB F5H6I0, UniProtKB H0YLF3, UniProtKB J3KNU0, and / or UniProtKB Q16446. In some embodiments, the amino acid sequence is about, at least about, or up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 12-16. In some embodiments, the amino acid sequence is about or at least about 50 contiguous amino acids in any one of SEQ ID NOs: 12-16 and about, at least about, or up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical. In some embodiments, the amino acid sequence is about 50 to about 120 amino acids, about 50 to about 200 amino acids, or about 30 to about 300 amino acids. In some embodiments, the amino acid sequence is about, at least about, or up to about 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 95, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or more than 120 amino acids (e.g., of any one of SEQ ID NOs: 12-16). In some embodiments, the nucleotide sequence encodes the amino acid sequence of B2M, a portion thereof, or an isoform thereof.

[0079] In some embodiments, modifying a target polynucleotide sequence comprises contacting at least a portion of the polynucleotide sequence (e.g., B2M) with an endonuclease of the present disclosure and at least one ribonucleic acid (e.g., gRNA), wherein the at least one ribonucleic acid directs the endonuclease to and hybridizes with a target motif of the target B2M polynucleotide sequence. In some embodiments, the target motif is a portion of a target polypeptide. In some embodiments, the target motif is a portion of the NCBI reference sequence: NG_012920.2. In some embodiments, the target motif is a portion of a target polypeptide. In some embodiments, the target motif is a portion of B2M. In some embodiments, the target motif is a portion of SEQ ID NO: 1. In some embodiments, the target motif is about, at least about, or up to about 5 nucleotides (nt), 10 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, or more than 60 nt. In some embodiments, the target motif is a sequence of about 15 nt to about 20 nt, about 15 nt to about 25 nt, about 15 nt to about 30 nt, about 15 nt to about 35 nt, about 15 nt to about 40 nt, about 15 nt to about 45 nt, about 15 nt to about 50 nt, about 20 nt to about 25 nt, about 20 nt to about 30 nt, about 20 nt to about 35 nt, about 20 nt to about 40 nt, about 20 nt to about 45 nt, or about 20 nt to about 50 nt. In some embodiments, the target motif is a sequence of about 20 nt to about 24 nt. In some embodiments, the target motif is about, at least about, or up to about 20 nt.In some embodiments, the target motif is about 21 NTs. In some embodiments, the target motif is about 22 NTs. In some embodiments, the target motif is about 23 NTs. In some embodiments, the target motif is about 24 NTs. In some embodiments, the target motif is about 25 NTs. In some embodiments, the target motif is about 26 NTs. In some embodiments, the target motif is about 27 NTs. In some embodiments, the target motif is about 28 NTs. In some embodiments, the target motif is about 29 NTs. In some embodiments, the target motif is about 30 NTs.

[0080] In some embodiments, the target motif comprises at least one mutation, substitution, addition, and / or deletion relative to a portion of the target polypeptide. In some embodiments, the target motif is about, at least about, or up to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of the target polypeptide. In some embodiments, the target motif comprises or consists of one or more nucleotide modifications (e.g., nucleotide substitutions, additions, deletions, and / or insertions) relative to a portion of the target polypeptide. In some embodiments, the target motif comprises or consists of about 1, 2, 3, 4, 5, or more than 5 nucleotide modifications relative to a portion of the target polypeptide.

[0081] In some embodiments, the target motif is selected to minimize the off-target effects of the CRISPR / Cas systems or any gene or genome editing of the present disclosure. One of ordinary skill in the art will understand that various techniques (e.g., bioinformatics analysis) can be used to select a suitable target motif for minimizing off-target effects. In some embodiments, the target motif is selected to include at least one mismatch (e.g., at least two, three, four, or more mismatches) when compared to the genomic nucleotide sequence in a cell.

[0082] In some embodiments, the target motif includes any one of SEQ ID NOs: 2-6 or 17. In some embodiments, the target motif consists of any one of SEQ ID NOs: 2-6 or 17. In some embodiments, the target motif includes or consists of at least one nucleotide modification relative to any one of SEQ ID NOs: 2-6 or 17. In some embodiments, the target motif includes or consists of about 1, 2, 3, 4, 5, or more than 5 nucleotide modifications relative to any one of SEQ ID NOs: 2-6 or 17. In some embodiments, the target motif is about, at least about, or up to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 2-6 or 17. In some embodiments, the target motif includes a sequence that includes at least 1, 2, 3, 4, 5, or more than 5 nucleotide mismatches compared to any one of SEQ ID NOs: 2-6 or 17. In some embodiments, the complementary sequence includes any one of SEQ ID NOs: 19-24.

[0083] 5.4. Gene or Genome Editing In some embodiments, the genome editing methods described herein, for example, using a CRISPR-endonuclease system, can be used to genetically modify the cells described herein to form, for example, B2M knockout cells (B2M knockout iPSC cells derived from γδ T cells). In some embodiments, the genome editing methods described herein, for example, using a CRISPR-endonuclease system, can be used to genetically modify the cells described herein to introduce at least one genetic modification within or near at least one gene (e.g., B2M).

[0084] Some embodiments can generate the B2M knockout cells of the present disclosure using any known method of genome editing known to those of skill in the art. In some embodiments, the methods of genome editing described herein use site-specific nucleases to cleave deoxyribonucleic acid (DNA) at precise target locations in the genome, thereby forming single-stranded or double-stranded DNA breaks at specific locations within the genome. Such breaks can be repaired by natural endogenous cellular processes such as homologous recombination repair (HDR) and non-homologous end joining (NHEJ), and are typically repaired. NHEJ directly ligates the DNA ends resulting from double-stranded breaks and sometimes involves the loss or addition of nucleotide sequences that can disrupt or enhance gene expression. HDR utilizes a homologous sequence or donor sequence as a template for inserting a defined DNA sequence at the cleavage site. The homologous sequence may be in the endogenous genome such as a sister chromatid. Alternatively, the donor sequence has regions of high homology to the nuclease cleavage locus (e.g., left and right homology arms), but may also contain additional sequences or sequence changes including deletions that can be incorporated into the cleaved target locus, and can be an exogenous polynucleotide such as a plasmid, single-stranded oligonucleotide, double-stranded oligonucleotide, double-stranded oligonucleotide, or virus. A third repair mechanism can be microhomology-mediated end joining (MMEJ), also referred to as "alternative NHEJ", where the genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ can utilize homologous sequences of several base pairs adjacent to the DNA cleavage site to drive more favorable DNA end ligation repair outcomes (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).

[0085] Using each of these genome editing mechanisms, a desired gene modification can be formed. The steps in the genome editing process can form one or two DNA breaks, the latter as a double-strand break or as two single-strand breaks, at the target locus near the site of the intended mutation or modification. This can be achieved via the use of endonucleases, as described herein.

[0086] In some embodiments, the target gene or target polypeptide (e.g., B2M) of the present disclosure is disrupted or at least partially deleted via the CRISPR-Cas system. In some embodiments, the CRISPR / Cas system used to modify the target polynucleotide sequence in a cell comprises an RNA-binding protein, an endonuclease and exonuclease, a helicase, and / or a polymerase. In some embodiments, the CRISPR-endonuclease system comprises an endonuclease and at least one guide nucleic acid that hybridizes with a recognition site in genomic DNA (or a target motif of the target polynucleotide) to direct the DNA cleavage of the endonuclease. In some embodiments, the gRNA of the embodiments herein binds to the complementary strand of the target gene. In some aspects, the complementary sequence comprises any one of SEQ ID NOs: 19-24. In some embodiments, the CRISPR-endonuclease system comprises an endonuclease and at least one ribonucleic acid (e.g., guide RNA (gRNA)) that hybridizes with a recognition site in genomic DNA (or a target motif of the target polynucleotide) to direct the DNA cleavage of the endonuclease. Those skilled in the art will understand that the gRNA is directed to the target motif and binds to its complementary sequence in the target motif. In some aspects, the complementary sequence comprises any one of SEQ ID NOs: 19-24. In some embodiments herein, the disrupted B2M gene is generated by contacting a population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease, and a guide RNA (gRNA), and the gRNA binds to the target motif of the B2M gene. The target motif comprises a double-stranded nucleic acid region to which the gRNA binds to one of the strands, i.e., the complementary strand to the gRNA. In some embodiments, the CRISPR system is a type I, type II, type III, type IV, type V, and / or type VI system. In some embodiments, the CRISPR system is a type II CRISPR / Cas9 system. In some embodiments, the CRISPR system is a type V CRISPR / Cpf1 (or Cas12a) system.In some embodiments, the CRISPR system is the CRISPR-MAD7 system. In some embodiments, the CRISPR system includes an endonuclease, such as Cas9, Cpf1, or MAD7, and one or two non-coding RNAs for targeting DNA cleavage - a crisprRNA (crRNA) and a trans-activating RNA (tracrRNA).

[0087] In some embodiments, the genome editing method of the present disclosure uses at least one and / or any ribonucleic acid (e.g., gRNA) that can direct an endonuclease (Cas protein) to a target motif of a target polynucleotide sequence and hybridize with it. In some embodiments, at least one of the ribonucleic acids includes a tracrRNA. In some embodiments, at least one of the ribonucleic acids includes a CRISPR RNA (crRNA). In some embodiments, the CRISPR RNA (crRNA) is or includes a nucleotide sequence of about 17 to 20 nucleotides complementary to the target DNA (the target motif of the target polynucleotide). In some embodiments, the tracrRNA functions as a binding scaffold for an endonuclease (e.g., Cas9, Cpf1, MAD7, or any other endonuclease of the present disclosure). In some embodiments, a single ribonucleic acid includes a guide RNA that directs an endonuclease or Cas protein to a target motif of a target polynucleotide sequence in a cell and hybridizes with it. In some embodiments, at least one of the ribonucleic acids includes a guide RNA that directs an endonuclease or Cas protein to a target motif of a target polynucleotide sequence in a cell and hybridizes with it. In some embodiments, both of the one or two ribonucleic acids include a guide RNA that directs an endonuclease or Cas protein to a target motif of a target polynucleotide sequence in a cell and hybridizes with it. In some embodiments, at least one ribonucleic acid of the present disclosure can be selected to hybridize with various different target motifs, e.g., different target motifs within a target polynucleotide. In some embodiments, at least one ribonucleic acid of the present disclosure can be selected to hybridize with various different target motifs depending on the specific CRISPR / Cas system used and the sequence of the target polynucleotide, as understood by those skilled in the art. In some embodiments, at least one ribonucleic acid (e.g., one or two ribonucleic acids) can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence.In some embodiments, at least one ribonucleic acid (e.g., 1 to 2 ribonucleic acids) hybridizes to a target motif that contains at least two mismatches when compared to all other genomic nucleotide sequences in the cell. In some embodiments, at least one ribonucleic acid (e.g., 1 to 2 ribonucleic acids) hybridizes to a target motif that contains at least one mismatch when compared to all other genomic nucleotide sequences in the cell. In some embodiments, at least one ribonucleic acid (e.g., 1 to 2 ribonucleic acids) is designed to hybridize to a target motif that is directly adjacent to a deoxyribonucleic acid motif recognized by an endonuclease or a Cas protein. In some embodiments, at least one ribonucleic acid (e.g., 1 to 2 ribonucleic acids) is designed to hybridize to a target motif that is directly adjacent to a deoxyribonucleic acid motif recognized by an endonuclease or a Cas protein that is adjacent to a variant allele located between the target motifs.

[0088] In some embodiments, at least one ribonucleic acid (e.g., guide RNA) is complementary to and / or hybridizes with a sequence on the same strand of a target polynucleotide sequence (e.g., B2M). In some embodiments, at least one ribonucleic acid (e.g., guide RNA) is complementary to and / or hybridizes with a sequence on the opposite strand of a target polynucleotide sequence. In some embodiments, at least one ribonucleic acid (e.g., guide RNA) is not complementary to and / or does not hybridize with a sequence on the opposite strand of a target polynucleotide sequence. In some embodiments, at least one ribonucleic acid (e.g., guide RNA) is complementary to and / or hybridizes with an overlapping target motif of a target polynucleotide sequence. In some embodiments herein, the gRNA of the present application can bind to a complementary sequence in a target motif. The target motif includes a double-stranded nucleic acid, and the gRNA binds to one of the strands to which it is complementary. In some embodiments, at least one ribonucleic acid (e.g., guide RNA) is complementary to and / or hybridizes with an offset target motif of a target polynucleotide sequence. In some aspects, the complementary sequence includes any one of SEQ ID NOs: 19 to 24.

[0089] In some embodiments, the CRISPR endonuclease is Cas9 and / or Cpf1, e.g., L. bacteria ND2006 Cpf1 and / or Acidaminococcus sp. BV3L6 Cpf1 (Example 1, Section 7), and / or MAD7. In various embodiments, CRISPR / MAD7 is used (especially in Example 1 of the present disclosure, e.g., the same B2M target motif and gRNA sequence (Section 7 of the present disclosure) is used with other type V CRISPR enzymes in MAD7). In some embodiments, since MAD7 is a Cas12a-like endonuclease, the B2M target motif and / or guide nucleic acid (e.g., gRNA) used or specified for Cpf1 or Cas-12a is the same as the B2M target motif and / or guide nucleic acid (e.g., gRNA) used for MAD7. In some embodiments, the B2M target motif specified or used for the CRISPR-Cpf1 system is the same B2M target motif used for the CRISPR-MAD7 system. In some embodiments, the guide nucleic acid (e.g., gRNA) specified or used for the CRISPR-Cpf1 system is the same guide nucleic acid (e.g., gRNA) used for the CRISPR-MAD7 system. In some embodiments, the B2M target motif and guide nucleic acid (e.g., gRNA) specified or used for the CRISPR-Cpf1 system are the same B2M target motif and the same guide nucleic acid (e.g., gRNA) used for the CRISPR-MAD7 system. In some embodiments, the CRISPR endonuclease is MAD7. In some embodiments, the nuclease used in the methods of the present disclosure is Inscripta's MAD7™ nuclease. In some embodiments, the nuclease used in the methods of the present disclosure is Inscripta's nuclease. In some embodiments, the CRISPR endonuclease is Cas9 (CRISPR-associated protein 9). In some embodiments, the Cas9 endonuclease is derived from Streptococcus pyogenes.In some embodiments, other Cas9 homologs are used, such as S. aureus Cas9, N. meningitidis Cas9, S. thermophilus CRISPR 1 Cas9, S. thermophilus CRISPR 3 Cas9, or T. denticola Cas9. In some embodiments, the endonuclease is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and / or Cpf1 endonuclease. In some embodiments, wild-type variants can be used. In some embodiments, modified versions of the endonuclease (e.g., homologs thereof, recombinants of its naturally occurring molecules, its codon optimization, or modified versions thereof) can be used. In some embodiments, the endonuclease is any one or more of the endonucleases of the present disclosure. In some embodiments, the endonuclease is any one or more endonucleases known to those skilled in the art. In some embodiments, the exogenous Cas protein can be introduced into the cell in polypeptide form. In certain embodiments, the Cas protein can be conjugated or fused to a cell-permeable polypeptide or a cell-permeable peptide. As used herein, "cell-permeable polypeptide" and "cell-permeable peptide" refer to a polypeptide or a peptide that facilitates the uptake of a molecule into a cell, respectively. In some embodiments, the cell-permeable polypeptide can include a detectable label.

[0090] In some embodiments, the endonuclease or Cas protein can be conjugated or fused to a charged protein (e.g., having a positive charge, a negative charge, or an overall neutral charge). Such linkages can be covalent. In some embodiments, the endonuclease or Cas protein can be fused to GFP that is overly positively charged to greatly increase the ability of the Cas protein to enter cells (Cronican et al. ACS Chem Biol. 2010;5(8):747-52). In some embodiments, the endonuclease or Cas protein can be fused to a protein transduction domain (PTD) to facilitate its entry into cells. Exemplary PTDs include Tat, oligoarginine, and penetratin. In some embodiments, the endonuclease or Cas protein comprises a Cas polypeptide fused to a cell-penetrating peptide.

[0091] In some embodiments, the endonuclease is linked to at least one nuclear localization signal (NLS). The at least one NLS can be located at the amino terminus of the endonuclease or within 50 amino acids thereof, and / or the at least one NLS can be located at the carboxy terminus of the endonuclease or within 50 amino acids thereof.

[0092] In some embodiments, the CRISPR-endonuclease system includes an RNA-guided endonuclease. In some embodiments, the RNA-guided endonuclease comprises an amino acid sequence having at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% amino acid sequence identity with a wild-type endonuclease, such as Cpf1, MAD7, Cas9, and / or any other endonuclease of the present disclosure. In some embodiments, the endonuclease comprises about or at least about 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with a wild-type endonuclease (e.g., Cpf1, MAD7, Cas9, and / or any other endonuclease of the present disclosure) over about or at least about 10 contiguous amino acids. In some embodiments, the endonuclease comprises about or at least about 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with a wild-type endonuclease (e.g., Cpf1, MAD7, Cas9, and / or any other endonuclease of the present disclosure) over about or at least about 10 contiguous amino acids, with a maximum of about 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity. In some embodiments, the endonuclease comprises at least about 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with a wild-type endonuclease (e.g., Cpf1, MAD7, Cas9, and / or any other endonuclease of the present disclosure) over about or at least about 10 contiguous amino acids in the HNH nuclease domain of the endonuclease. In some embodiments, the endonuclease comprises about or at least about 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with a wild-type endonuclease (e.g., Cpf1, MAD7, Cas9, and / or any other endonuclease of the present disclosure) over about or at least about 10 contiguous amino acids in the HNH nuclease domain of the endonuclease, with a maximum of about 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity.In some embodiments, the endonuclease comprises at least about 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with a wild-type endonuclease (e.g., Cpf1, MAD7, Cas9, and / or any other endonuclease of the present disclosure) over about or at least about 10 contiguous amino acids in the RuvC nuclease domain of the endonuclease. In some embodiments, the endonuclease comprises up to about 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity with a wild-type endonuclease (e.g., Cpf1, MAD7, Cas9, and / or any other endonuclease of the present disclosure) over about or at least about 10 contiguous amino acids in the RuvC nuclease domain of the endonuclease. The present invention provides a guide RNA (gRNA) that can direct the activity of a related endonuclease to a specific target site within a polynucleotide. In some embodiments, the gRNA comprises a spacer sequence that hybridizes to a target nucleic acid sequence of interest and a CRISPR repeat sequence. In some embodiments, for example, in a CRISPR type II system, the gRNA also comprises a second RNA called a tracrRNA sequence. In some embodiments, in a CRISPR type II guide RNA (gRNA), the CRISPR repeat sequence and the tracrRNA sequence hybridize to each other to form a double strand. In some embodiments, in a CRISPR type V system, the gRNA comprises a crRNA that forms a double strand. In some embodiments, the gRNA is capable of binding to the endonuclease such that the gRNA and the endonuclease form a complex. The gRNA can provide target specificity to the complex by virtue of its association with the endonuclease.

[0093] In some embodiments, the tracrRNA sequence comprises nucleotides that hybridize to the CRISPR repeat sequence within the cell. The tracrRNA sequence and the CRISPR repeat sequence can form a double-stranded, i.e., base-paired double-stranded structure. At the same time, the tracrRNA sequence and the CRISPR repeat can bind to an RNA-guided endonuclease. In some embodiments, at least a portion of the tracrRNA sequence can hybridize to the CRISPR repeat sequence. In some embodiments, the tracrRNA sequence can be at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementary to the CRISPR repeat sequence. In some embodiments, the tracrRNA sequence can have a length of from about 7 nucleotides to about 100 nucleotides. For example, the tracrRNA sequence can be from about 7 nucleotides (NTs) to about 50 NTs, from about 7 NTs to about 40 NTs, from about 7 NTs to about 30 NTs, from about 7 NTs to about 25 NTs, from about 7 NTs to about 20 NTs, from about 7 NTs to about 15 NTs, from about 8 NTs to about 40 NTs, from about 8 NTs to about 30 NTs, from about 8 NTs to about 25 NTs, from about 8 NTs to about 20 NTs, from about 8 NTs to about 15 NTs, from about 15 NTs to about 100 NTs, from about 15 NTs to about 80 NTs, from about 15 NTs to about 50 NTs, from about 15 NTs to about 40 NTs, from about 15 NTs to about 30 NTs, or from about 15 NTs to about 25 NTs in length. In some embodiments, the tracrRNA sequence can be approximately 9 nucleotides in length. In some embodiments, the tracrRNA sequence can be about 12 nucleotides.

[0094] In some embodiments, the tracrRNA sequence can be at least about 60% identical to a reference tracrRNA (e.g., a wild-type tracrRNA from S. pyogenes) sequence over an interval of at least 6, 7, or 8 consecutive nucleotides. For example, the tracrRNA sequence can 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 the reference tracrRNA sequence over an interval of at least 6, 7, or 8 consecutive nucleotides.

[0095] In some embodiments, the Cas protein or endonuclease can be introduced into a cell containing a target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein or endonuclease (e.g., Cas9, Cpf1, MAD7, or any endonuclease or Cas protein of the present disclosure). In some embodiments, the method includes techniques for introducing the nucleic acid into γδ iPSC cells. The process of introducing the nucleic acid into the cell can be accomplished by any suitable technique. Suitable techniques include, but are not limited to, transfection (e.g., neon transfection, calcium phosphate or lipid-mediated transfection), electroporation, and transduction or infection using viral vectors. In some embodiments, the transfection is performed using Neon transfection. In some embodiments, the transfection is performed using Lonza nucleofection. In some embodiments, the nucleic acid is introduced into the cell using a non-viral system (e.g., neon transfection). In some embodiments, the nucleic acid is introduced into the cell using a viral system (e.g., adeno-associated virus). In some embodiments, the method includes, for example, electroporation of γδ iPSC cells for introducing genetic material including RNA and / or mRNA. In some embodiments, the techniques for introducing the protein or nucleic acid can include introducing the protein or nucleic acid via electroporation, microinjection, viral delivery, exosomes, liposomes, particle guns, jet injection, hydrodynamic injection, ultrasound, magnetic field-mediated gene transfer, electrical pulse-mediated gene transfer, use of nanoparticles including, for example, lipid-based nanoparticles, incubation with endosome-lysing agents, use of cell-penetrating peptides, or any other suitable technique. In some embodiments, the method includes, for example, electroporation of γδ iPSC cells including using a Neon transfection system (Thermo Fisher Scientific Inc.).

[0096] In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises modified DNA. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises modified mRNA.

[0097] In some embodiments, the Cas protein or endonuclease is complexed with at least one ribonucleic acid (e.g., 1 to 2 ribonucleic acids). In some embodiments, the Cas protein or endonuclease is complexed with two ribonucleic acids. In some embodiments, the Cas protein or endonuclease is complexed with one ribonucleic acid. In some embodiments, the Cas protein or endonuclease is encoded by a modified nucleic acid.

[0098] In some embodiments, the endonuclease and gRNA can each be administered to the cell separately. In some embodiments, the endonuclease can be pre-complexed with one or more guide RNAs, or one or more crRNAs together with a tracrRNA. The pre-complexed material can then be administered to the cell. Such pre-complexed materials are known as ribonucleoprotein particles (RNPs). The endonuclease in the RNP can be, for example, a Cpf1 endonuclease, a MAD7 endonuclease, a Cas9 endonuclease, or any endonuclease of the present disclosure. In some aspects, the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1). In some embodiments, the endonuclease can be flanked at the N-terminus, C-terminus, or both the N-terminus and C-terminus by one or more nuclear localization signals (NLSs). In some embodiments, the weight ratio of the genomic targeting nucleic acid to the endonuclease in the RNP can be 1:1, 2:1, 1:2, or any suitable ratio.

[0099] In some embodiments, the gRNA can be a bimolecular guide RNA. In some embodiments, the gRNA can be a single-molecule guide RNA (sgRNA).

[0100] In some embodiments, the gRNA comprises a sequence that hybridizes with a sequence in the target polynucleotide. In some embodiments, the nucleotide sequence of the gRNA can vary according to the sequence of the target nucleic acid of interest. In some embodiments, the gRNA comprises a variable-length sequence having 17 to 30 nucleotides, at least a portion of which hybridizes with a sequence in the target polynucleotide. In some embodiments, the gRNA sequence can be designed to hybridize with a target polynucleotide located 5' of the PAM of the endonuclease used in the system.

[0101] In some embodiments, the gRNA comprises another moiety (e.g., a stability control sequence, an endonuclease binding sequence, or a ribozyme). This moiety can decrease or increase the stability of the nucleic acid targeting nucleic acid. In some embodiments, the moiety can be a transcription termination segment (i.e., a transcription termination sequence). In some embodiments, the moiety can function in eukaryotic cells. The moiety can function in prokaryotic cells. In some embodiments, the moiety can function in both eukaryotic and prokaryotic cells. Non-limiting examples of suitable moieties include a 5’ cap (e.g., a 7-methylguanylic acid cap (m7G)), a riboswitch sequence (e.g., for enabling stability and / or regulated accessibility modulated by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the RNA to an intracellular location (e.g., the nucleus, mitochondria, chloroplast, etc.), a modification or sequence that provides tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescence detection, a sequence that enables fluorescence detection, etc.), and / or a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including transcription activators, transcription repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.).

[0102] In some embodiments, a portion of the gRNA that hybridizes to a sequence or target motif in the target polynucleotide is referred to as a spacer. In some embodiments, a portion of the gRNA that hybridizes to a sequence or target motif in the target polynucleotide (spacer) comprises about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than about 25 nucleotides. In some embodiments, a portion of the gRNA that hybridizes to a sequence or target motif in the target polynucleotide comprises less than about 25 nucleotides. In some embodiments, a portion of the gRNA that hybridizes to a sequence or target motif in the target polynucleotide, or the gRNA, comprises more than about 20 nucleotides. In some embodiments, a portion of the gRNA that hybridizes to a sequence or target motif in the target polynucleotide, or the gRNA, comprises about or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides. In some embodiments, a portion of the gRNA that hybridizes to a sequence or target motif in the target polynucleotide, or the gRNA, comprises up to about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides. In some embodiments, a sequence or target motif in the target polynucleotide sequence comprises about, at least about, or up to about 20 bases immediately 5' of the first nucleotide of the PAM.

[0103] In some embodiments, a portion of the gRNA that hybridizes to a sequence or target motif in the target polynucleotide has a length of at least about 6 nucleotides (NTs). In some embodiments, a portion of the gRNA that hybridizes to a sequence or target motif in the target polynucleotide, or the gRNA, is about or at least about 6 NTs, about or at least about 10 NTs, about or at least about 15 NTs, about or at least about 18 NTs, about or at least about 19 NTs, about or at least about 20 NTs, about or at least about 21 NTs, about or at least about 22 NTs, about or at least about 23 NTs, about or at least about 24 NTs, about or at least about 25 NTs, about or at least about 30 NTs, about or at least about 35 NTs, about or at least about 40 NTs, about or at least about 45 NTs, about or at least about 50 NTs, or more than about 50 NTs.In some embodiments, a portion of the gRNA that hybridizes to a sequence or target motif in the target polynucleotide, or the gRNA, is from about 6 NT to about 40 NT, from about 6 NT to about 35 NT, from about 6 NT to about 30 NT, from about 6 NT to about 29 NT, from about 6 NT to about 28 NT, from about 6 NT to about 27 NT, from about 6 NT to about 26 NT, from about 6 NT to about 25 NT, from about 6 NT to about 24 NT, from about 6 NT to about 23 NT, from about 6 NT to about 22 NT, from about 6 NT to about 21 NT, from about 6 NT to about 20 NT, from about 10 NT to about 50 NT, from about 10 NT to about 40 NT, from about 10 NT to about 35 NT, from about 10 NT to about 30 NT, from about 10 NT to about 30 NT, from about 10 NT to about 29 NT, from about 10 NT to about 28 NT, from about 10 NT to about 27 NT, from about 10 NT to about 26 NT, from about 10 NT to about 25 NT, from about 10 NT to about 24 NT, from about 10 NT to about 23 NT, from about 10 NT to about 22 NT, from about 10 NT to about 21 NT, from about 10 NT to about 20 NT, from about 19 NT to about 23 NT, from about 19 NT to about 24 NT, from about 19 NT to about 25 NT, from about 19 NT to about 30 NT, from about 19 NT to about 35 NT, from about 19 NT to about 40 NT, from about 19 NT to about 45 NT, from about 19 NT to about 50 NT, from about 19 NT to about 60 NT, from about 20 NT to about 25 NT, from about 20 NT to about 30 NT, from about 20 NT to about 35 NT, from about 20 NT to about 40 NT, from about 20 NT to about 45 NT, from about 20 NT to about 50 NT, or from about 20 NT to about 60 NT.

[0104] In some embodiments, the percent complementarity between the gRNA or a portion of the gRNA (e.g., the spacer or crRNA) and the target polynucleotide is about or at least about 30%, about or at least about 40%, about or at least about 50%, about or at least about 60%, about or at least about 65%, about or at least about 70%, about or at least about 75%, about or at least about 80%, about or at least about 85%, about or at least about 90%, about or at least about 95%, about or at least about 97%, about or at least about 98%, about or at least about 99%, or 100%. In some embodiments, the percent complementarity between the gRNA or a portion of the gRNA and the target polynucleotide is at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 97%, at most about 98%, at most about 99%, or 100%. In some embodiments, the lengths of the gRNA and a portion of the target nucleic acid can differ by 1 to 6 nucleotides, which can be considered a bulge(s).

[0105] In some embodiments, the gRNA or a portion of the gRNA (e.g., the spacer or crRNA) comprises any one of SEQ ID NOs: 7-11 or 18, or a portion or fragment thereof. In some embodiments, the gRNA or a portion of the gRNA (e.g., the spacer or crRNA) consists of any one of SEQ ID NOs: 7-11 or 18, or a portion or fragment thereof. In some embodiments, the gRNA or a portion of the gRNA (e.g., the spacer or crRNA) comprises or consists of at least one nucleotide modification relative to any one of SEQ ID NOs: 7-11 or 18. In some embodiments, the gRNA or a portion of the gRNA (e.g., the spacer or crRNA) comprises or consists of about 1, 2, 3, 4, 5, or more than 5 nucleotide modifications relative to any one of SEQ ID NOs: 7-11 or 18. In some embodiments, the gRNA or a portion of the gRNA (e.g., the spacer or crRNA) is about, at least about, or up to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 7-11 or 18, or a portion or fragment thereof. In some embodiments, the gRNA or a portion of the gRNA (e.g., the spacer or crRNA) is about, at least about, or up to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to about or at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of any one of SEQ ID NOs: 7-11 or 18. In some embodiments, the gRNA or a portion of the gRNA (e.g., the spacer or crRNA) comprises a sequence that contains at least 1, 2, 3, 4, 5, or more than 5 nucleotide mismatches compared to any one of SEQ ID NOs: 7-11 or 18.

[0106] In some embodiments, the gRNA is modified or chemically modified. In some embodiments, the chemically modified gRNA is a gRNA comprising at least one nucleotide having a chemical modification, such as a 2'-O-methyl sugar modification. In some embodiments, the chemically modified gRNA comprises a modified nucleic acid backbone. In some embodiments, the chemically modified gRNA comprises 2'-O-methyl-phosphorothioate residues. In some embodiments, the chemical modification enhances stability, reduces the likelihood or extent of the innate immune response, and / or enhances other properties, as described in the art.

[0107] In some embodiments, the modified gRNA comprises a modified backbone, such as a phosphorothioate, phosphotriester, morpholino, methylphosphonate, short-chain alkyl or cycloalkyl sugar linkage, or short-chain heteroatom or heterocyclic sugar linkage.

[0108] In some embodiments, the modified gRNA has one or more substituted sugar moieties, such as at the 2'-position, OH, SH, SCH3, F, OCN, OCH3, OCH3O(CH2)nCH3, O(CH2)nNH2, or O(CH2)nCH3 (where n is from 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; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; intercalator; 2'-O-(2-methoxyethyl); 2'-methoxy (2'-O-CH3); 2'-propoxy (2'-OCH2CH2CH3); and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the gRNA, such as the 3'-position of the sugar on the 3'-terminal nucleotide and / or the 5'-position of the 5'-terminal nucleotide. In some examples, both the sugar of the nucleotide unit and the internucleoside linkage, i.e., the backbone, can be replaced with different groups.

[0109] In some embodiments, the gRNA additionally or alternatively includes nucleic acid base (or "base") 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 natural nucleic acids such as hypoxanthine, 6-methyladenine, 5-Me pyrimidine, 5-methylcytosine (also referred to as 5-methyl-2'deoxycytosine or 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, and synthetic nucleic acid bases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine, or other hetero-substituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6-diaminopurine, which are rarely or transiently found in nucleic acid bases.

[0110] In some embodiments, the modified nucleic acid bases can include other synthetic nucleic acid bases and natural nucleic acid bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azauracil, cytosine, and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 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-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0111] In some embodiments, the method of modifying a gene of the present disclosure or the method of genome editing can be performed using zinc finger nucleases (ZFNs). Zinc finger nucleases (ZFNs) are modular proteins composed of engineered zinc finger DNA-binding domains linked to the catalytic domain of the type II endonuclease FokI. Since FokI functions as a dimer, a pair of ZFNs are engineered to bind to cognate target "half-site" sequences on opposite DNA strands, with an exact spacing between them, allowing the formation of a catalytically active FokI dimer. Upon dimerization of the FokI domains, a DNA double-strand break is generated between the ZFN half-sites as an initial step in genome editing.

[0112] In some embodiments, the DNA-binding domain of each ZFN is composed of 3 to 6 zinc fingers with a rich Cys2-His2 structure, and each finger mainly recognizes a triplet of nucleotides on one strand of the target DNA sequence, but cross-strand interactions with the fourth nucleotide can also occur. Modification of the amino acids of the finger at positions that make important contacts with the DNA modifies the sequence specificity of a given finger. Thus, a four-finger zinc finger protein selectively recognizes a 12-bp target sequence, which is a complex of triplet preferences contributed by each finger, although the triplet preferences can be affected to varying degrees by adjacent fingers. ZFNs can be easily re-targeted to almost any genomic address simply by modifying the individual fingers. In some embodiments, proteins of 4 to 6 fingers that recognize 12 to 18 bp each are used. Thus, a pair of ZFNs will typically recognize a combined target sequence of 24 to 36 bp that does not include a typical 5 to 7 bp spacer between the half-sites. The binding sites can be further separated by larger spacers that include 15 to 17 bp.

[0113] A variety of ZFN-based systems have been described in the art, and modified forms thereof have been reported regularly, and numerous references describe the rules and parameters used to guide the design of ZFNs. See, for example, Segal et al., Proc Natl Acad Sci, 1999 96(6):2758-63, Dreier B et al., J Mol Biol., 2000, 303(4):489-502, Liu Q et al., J Biol Chem., 2002, 277(6):3850-6, Dreier et al., J Biol Chem., 2005, 280(42):35588-97, and Dreier et al., J Biol Chem. 2001, 276(31):29466-78.

[0114] In some embodiments, the method of modifying a gene of the present disclosure or the method of genome editing can be carried out using transcription activator-like effector nucleases (TALENs). TALENs, like ZFNs, represent another form of modular nuclease in which the engineered DNA-binding domain is linked to the FokI nuclease domain, and a pair of TALENs operate tandemly to achieve targeted DNA cleavage. The main difference from ZFNs is the nature of the DNA-binding domain and the associated target DNA sequence recognition characteristics. The TALEN DNA-binding domain is derived from the TALE protein originally described in the plant bacterial pathogen Xanthomonas. TALEs are composed of tandem arrays of 33 - 35 amino acid repeats, each repeat recognizing a single base pair in a target DNA sequence typically 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 the repeat variable diresidue (RVD), which contains exactly two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine, and thymine are recognized mainly by four RVDs: Asn-Asn, Asn-Ile, His-Asp, and Asn-Gly, respectively. Various TALEN-based systems have been described in the art and its modified forms 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 multiple 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.

[0115] In some embodiments, the methods for modifying genes or genome editing methods of the present disclosure can be performed using a homing endonuclease (HE). A homing endonuclease (HE) is a sequence-specific endonuclease that has a long recognition sequence (14 to 44 base pairs), high specificity, and often cleaves DNA at unique sites in the genome. There are at least six known HE families classified by their structures, including GIY-YIG, His-Cis box, H-N-H, PD-(D / E)xK, and Vsr-like, derived from a wide range of hosts, including eukaryotes, protists, bacteria, archaea, cyanobacteria, and phages. Similar to ZFN and TALEN, HE can be used to form DSBs at target gene loci as the first step of genome editing. In addition, some natural and engineered HE cleave only one strand of DNA, thereby functioning as site-specific nickases. Various HE-based systems have been described in the art, and modified forms of them are reported regularly. See, for example, reviews by Steentoft et al., Glycobiology, 2014, 24(8):663-80, Belfort and Bonocora, Methods Mol Biol., 2014, 1123:1-26, and Hafez and Hausner, Genome, 2012, 55(8):553-69.

[0116] In some embodiments, the methods of modifying the genes of the present disclosure or the methods of genome editing can be performed using the MegaTAL or Tev-mTALEN platforms. The MegaTAL platform and the Tev-mTALEN platform utilize both the adjustable DNA binding and specificity of TALE and the cleavage sequence specificity of HE to use a fusion of the TALE DNA binding domain and the catalytically active 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.

[0117] In some embodiments, the MegaTev construct is a fusion of a meganuclease (Mega) and a nuclease domain derived from the GIY-YIG homing endonuclease I-TeeI (Tev). The two active sites are located approximately 30 bp apart on the DNA substrate and generate two DSBs with incompatible sticky ends. See, for example, Wolfs et al., Nucleic Acids Res., 2014, 42, 8816-29. Other combinations of existing nuclease-based approaches have evolved and are expected to be useful for achieving the target genome modifications described herein.

[0118] 5.5. γδ T cell-derived B2M knockout iPSC γδ T cell-derived iPSCs express HLA class I, one of the most polymorphic human antigens, and are frequently recognized by allogeneic T cells, resulting in allogeneic rejection. As a result, such iPSCs (and immunotherapeutic agents based thereon) have limited applicability in clinical settings without additional intervention (e.g., host immune system suppression / depletion, etc.). B2M is required for surface expression of HLA-I molecules. Thus, disruption of B2M results in reduced / eliminated surface expression of HLA-I molecules. Therefore, the resulting iPSCs are improved for use in allogeneic settings (e.g., for developing so-called "off-the-shelf" therapies).

[0119] In some embodiments, the genetically modified cells of the present disclosure (e.g., B2M knockout derived from γδ T cells, and / or HLA-A, HLA-B, and / or HLA-C knockout iPSCs) result in reduced or modified expression of B2M and / or one or more MHC-I human leukocyte antigens (e.g., HLA-A, HLA-B, and / or HLA-C) compared to unmodified cells, and include the introduction of at least one gene modification into or near at least one gene (e.g., B2M). In some embodiments, the genetically modified cells or a population thereof of the present disclosure have reduced immunogenicity or a reduced immune response compared to unmodified cells or a population of unmodified cells (e.g., compared to iPSCs derived from γδ T cells and not including B2M knockout and / or HLA-A, HLA-B, and / or HLA-C knockout). In some embodiments, the population of genetically modified cells of the present disclosure has reduced immunogenicity or a reduced immune response that is about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100% (lower) compared to the population of unmodified cells. In some embodiments, the cells are evaluated for immunogenicity using any suitable method known to those skilled in the art. In some embodiments, the cells are analyzed for the presence of antibodies on the cell surface, for example, by staining with anti-IgM antibodies. In some embodiments, immunogenicity is evaluated by a PBMC cytotoxicity assay. In some embodiments, a population of cells is incubated with peripheral blood mononuclear cells (PBMCs), and then the lysis of the cells by PBMCs is evaluated. In some embodiments, immunogenicity is evaluated by a natural killer (NK) cell cytotoxicity assay. In some embodiments, a population of cells is incubated with NK cells, and then the lysis of the cells by NK cells is evaluated. In some embodiments, immunogenicity is evaluated by a CD8+ T cell cytotoxicity assay.In some embodiments, the population of cells is incubated with CD8+ T cells and then evaluated for cell lysis by the CD8+ T cells. In some embodiments, the genetically modified cells or population thereof of the present disclosure have an increased survival rate or increased residual rate compared to unmodified cells or a population of unmodified cells (e.g., compared to iPSCs derived from γδ T cells and not including B2M knockout and / or HLA-A, HLA-B, and / or HLA-C knockout). In some embodiments, the population of genetically modified cells of the present disclosure has an increased survival rate or increased residual rate by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100% (higher) compared to the population of unmodified cells. In some embodiments, the cells are evaluated for increased survival rate or increased residual rate using any suitable method known to those skilled in the art. In some embodiments, cell survival rate or residual rate is determined using flow cytometry, high-content imaging, tetrazolium reduction (MTT) assay, resazurin reduction assay, protease survival marker assay, and / or ATP detection assay.

[0120] In some embodiments, the genetically modified cells of the present disclosure include the introduction of at least one genetic modification within or in the vicinity of at least one gene that reduces the expression of one or more MHC-I human leukocyte antigens compared to unmodified cells. In some embodiments, the genetically modified cells of the present disclosure include at least one deletion of a gene or at least partial inactivation of a gene (e.g., B2M). In some embodiments, the genetically modified cells of the present disclosure include at least one deletion within or in the vicinity of at least one gene that modifies the expression of one or more MHC-I human leukocyte antigens compared to unmodified cells.

[0121] In some embodiments, the genome of the cell is modified to reduce the expression of β-2-microglobulin (B2M). HLA-I proteins are closely associated with B2M in the endoplasmic reticulum, which is essential for forming functional cell surface-expressed HLA-I molecules. In some embodiments, the gRNA targets sites within the B2M gene described in Sections 5.3 and 5.4. In some embodiments, B2M expression is not detected in the population of genetically modified cells of the present disclosure (e.g., not detected by conventional methods (e.g., FACS)). In some embodiments, the population of genetically modified cells of the present disclosure has a complete or at least partial reduction in B2M expression compared to the population of unmodified cells. In some embodiments, the expression of B2M in the population of genetically modified cells is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% (lower) compared to the expression of B2M in the population of unmodified cells.

[0122] In some embodiments, the genetically modified cells of the present disclosure do not have a detectable level of B2M. In some embodiments, the genetically modified cells do not have a detectable RNA transcript encoding B2M. In some embodiments, the genetically modified cells do not have a detectable level of B2M protein. In some embodiments, the iPSCs do not express a detectable level of B2M protein when analyzed by flow cytometry.

[0123] In some embodiments, the genetically modified cells of the present disclosure comprise genomic modification of one or more MHC-I genes. In some embodiments, the genetically modified cells of the present disclosure comprise genomic modification of one or more polynucleotide sequences that regulate the expression of MHC-I. In some embodiments, the genetic modification of the present disclosure is carried out using any gene editing method including, but not limited to, those methods described herein (see Section 5.4). The genes encoding the major histocompatibility complex (MHC) are located on human Chr. 6p21. The MHC-I genes (HLA-A, HLA-B, and HLA-C) are expressed in almost all tissue cell types, presenting "non-self" antigen-processing peptides to CD8+ T cells, thereby promoting their activation against cytolytic CD8+ T cells. The MHC-I protein is closely associated with beta-2-microglobulin (B2M) in the endoplasmic reticulum, which is essential for forming functional MHC-I molecules on the cell surface.

[0124] In some embodiments, reducing the expression of one or more MHC-I human leukocyte antigens as compared to unmodified cells is achieved, for example, for gene deletion, by directly targeting at least one base pair in the MHC-I gene. In some embodiments, reducing the expression of one or more MHC-I human leukocyte antigens as compared to unmodified cells is achieved, for example, for gene deletion, by targeting the B2M gene. In some embodiments, reducing the expression of one or more MHC-I human leukocyte antigens as compared to unmodified cells is achieved, for example, for gene deletion, by targeting at least one transcriptional regulator of MHC-I. In some embodiments, the genome of the cell is modified to delete all or part of the HLA-A, HLA-B, and / or HLA-C genes. In some embodiments, the genome of the cell 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 genome of the cell is modified to delete all or part of the gene encoding the transcriptional regulator of MHC-I. In some embodiments, the genome of the cell is modified to delete all or part of the promoter region of the gene encoding the transcriptional regulator of MHC-I.

[0125] In some embodiments, the expression of HLA-A, HLA-B, and / or HLA-C is not detected in the population of genetically modified cells of the present disclosure (e.g., not detected by conventional methods (e.g., FACS)). In some embodiments, the population of genetically modified cells of the present disclosure has a complete or at least partial reduction in the expression of HLA-A, HLA-B, and / or HLA-C as compared to the population of unmodified cells. In some embodiments, the expression of HLA-A in the population of genetically modified cells is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% (lower) as compared to the expression of HLA-A in the population of unmodified cells. In some embodiments, the expression of HLA-B in the population of genetically modified cells is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% (lower) as compared to the expression of HLA-B in the population of unmodified cells. In some embodiments, the expression of HLA-C in the population of genetically modified cells is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% (lower) as compared to the expression of HLA-C in the population of unmodified cells. In some embodiments, the expression of B2M, HLA-A, HLA-B, and / or HLA-C in the population of genetically modified cells is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% (lower) as compared to the expression of B2M, HLA-A, HLA-B, and / or HLA-C in the population of unmodified cells.In some embodiments, the expression of B2M, HLA-A, HLA-B, and HLA-C in a population of genetically modified cells is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% (lower) compared to the expression of B2M, HLA-A, HLA-B, and HLA-C in a population of unmodified cells. In some embodiments, the expression of B2M, HLA-A, HLA-B, and HLA-C is detected by FACS.

[0126] 5.6. Method for Producing Induced Pluripotent Stem Cells (iPSCs) In some embodiments, the iPSCs of the present disclosure and methods of making the same are disclosed in International Publication No. 2021 / 257679 (International Application PCT / US2021 / 037594), the entire contents of which are incorporated herein by reference. In one aspect, methods are provided herein for reprogramming somatic cells (e.g., T cells) to a less differentiated state. The resulting cells are referred to herein as reprogrammed somatic cells. The reprogrammed somatic cells may be reprogrammed somatic cells in various differentiated states. In some embodiments, the reprogrammed somatic cells are induced pluripotent stem cells. The present disclosure is based in part on the surprising discovery that combinations of various factors, such as the combination of zoledronic acid and Interleukin-15 (IL-15), can activate γδ T cells and thus improve the induction efficiency of pluripotency in non-pluripotent mammalian T cells transformed with transcription factors. Accordingly, in one aspect, the present disclosure provides a method for inducing pluripotency in non-pluripotent mammalian γδ T cells (e.g., Vγ9 + γδ T cells), the method comprising contacting peripheral blood mononuclear cells (PBMCs) with an activation culture comprising IL-15 and zoledronic acid.

[0127] In some embodiments, the iPSCs of the present disclosure and methods of making the same are disclosed in International Publication No. 2021 / 176373 (International Application PCT / IB2021 / 051779), which is hereby incorporated by reference in its entirety. In some embodiments, provided herein is a method of producing induced pluripotent stem cells (iPSCs), comprising: (a) contacting a population of isolated cells with an activation culture comprising IL-15 and zoledronic acid; (b) culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; (c) transducing the γδ T cells with a viral vector encoding one or more reprogramming factors; (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state, thereby producing a population of iPSCs; and (e) contacting the population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA). In some embodiments, the gRNA binds to a target motif of the beta-2-microglobulin (B2M) polynucleotide sequence in the population of iPSCs. In some embodiments, the contacting results in cleavage of the B2M polynucleotide sequence.

[0128] In some embodiments, provided herein is a method of reprogramming somatic cells, comprising: (a) contacting a population of isolated cells with an activation culture comprising IL-15 and zoledronic acid; (b) culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; (c) transducing the γδ T cells with one or more viral vectors encoding one or more reprogramming factors; and (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a less differentiated state. In some embodiments, the less differentiated state is a pluripotent state. In some embodiments, the less differentiated state is a multipotent state.

[0129] In some more specific embodiments, provided herein is a method of generating induced pluripotent stem cells (iPSCs), comprising: (a) contacting a population of isolated cells with an activation culture comprising IL-15 and zoledronic acid; (b) culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; (c) transducing the γδ T cells with one or more viral vectors encoding one or more reprogramming factors; and (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0130] In certain embodiments, the activation culture further comprises one or more additional agents or compounds, for example, to improve the efficiency of activation or induction. In one embodiment, the activation culture further comprises Interleukin-2 (IL-2).

[0131] Accordingly, in some embodiments, provided herein is a method of reprogramming somatic cells, comprising: (a) contacting a population of isolated cells with an activation culture comprising IL-15, zoledronic acid, and IL-2; (b) culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; (c) transducing the γδ T cells with one or more viral vectors encoding one or more reprogramming factors; and (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a less differentiated state.

[0132] In some more specific embodiments, provided herein is a method of generating induced pluripotent stem cells (iPSCs), comprising: (a) contacting a population of isolated cells with an activation culture comprising IL-15, zoledronic acid, and IL-2; (b) culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; (c) transducing the γδ T cells with one or more viral vectors encoding one or more reprogramming factors; and (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0133] In some embodiments, the method further comprises delivery of a donor template comprising a knock-in cassette comprising a cargo sequence for insertion into a target gene. In some embodiments, an endonuclease is introduced to edit the B2M gene to insert the knock-in gene. In some embodiments, the endonuclease comprises a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or an endonuclease of the CRISPR / CAS system.

[0134] In some embodiments, a transposase, recombinase, or integrase is introduced to insert the knock-in gene into the B2M gene. In some embodiments, the transposase, recombinase, or integrase is conjugated to a targeting moiety such as a zinc finger, transcription activator-like effector (TALE), or nuclease-deficient CRISPR / CAS molecule. Some embodiments include the introduction of vector DNA and transposase for the introduction of a construct encoding a cargo. In some embodiments, zinc finger nucleases are used as gene editing factors in any one of the embodiments described herein. Certain embodiments can use transcription activator-like nucleases (TALENs) as gene editing factors. In some embodiments, a targeted gene manipulation approach such as a CRISPR nuclease system can be used.

[0135] One of ordinary skill in the art will understand that a transposon system can be used to insert a cassette encoding a cargo into the B2M gene. In some embodiments, a Sleeping Beauty transposon system is used to insert an expression cassette into the B2M gene, and the expression cassette encodes a transgene. In some embodiments, a MOS1 transposase system is used to insert an expression cassette into the B2M gene, and the expression cassette encodes a transgene. In some embodiments herein, a PiggyBac transposase is used to insert an expression cassette into the B2M gene, and the expression cassette encodes a transgene. In some embodiments, an ISY100 transposase is used to insert an expression cassette into the B2M gene, and the expression cassette encodes a transgene. In some embodiments, the expression cassette further includes transcriptional regulatory elements. In some embodiments, the transposases described herein further include a fusion protein comprising a nuclease-inactive Cas protein, TALE, or zinc finger protein. In some embodiments, the transgene encodes a therapeutic cargo, protein, TCR, or chimeric antigen receptor.

[0136] In some embodiments, the method further comprises forming a DSB within the B2M gene and inserting a nucleic acid construct within or near the target gene. In some embodiments, the double-strand break is effected by an endonuclease and the polynucleotide is inserted via HDR. In some embodiments, the method further comprises providing a nucleic acid for insertion into the target gene. In some embodiments, the nucleic acid is a vector encoding a cargo. In some embodiments, the cargo comprises a therapeutic protein. In some embodiments, the nucleic acid comprises a gene encoding a therapeutic protein. In some embodiments, the nucleic acid comprises a gene encoding a TCR. In some embodiments, the nucleic acid encodes a CAR construct (CAR) for insertion into the target gene. In some embodiments, the target is within the B2M gene. In some embodiments, the target is within SEQ ID NO: 1. In some embodiments, the CAR comprises an ectodomain comprising an antigen recognition region, a transmembrane domain, and an endodomain comprising at least one co-stimulatory domain. In some embodiments, the nucleic acid further comprises a promoter, at least one gene regulatory element, or a combination thereof.

[0137] In certain embodiments, the method further comprises obtaining a population of cells isolated from a subject. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0138] In certain embodiments, the population of isolated cells is peripheral blood cells, umbilical cord blood cells, or bone marrow cells. In one embodiment, the population of isolated cells is peripheral blood mononuclear cells (PBMC).

[0139] Methods for identifying reprogrammed mammalian somatic cells having a low differentiation state or a pluripotent state are known in the art. For example, in some embodiments, reprogrammed somatic cells are identified by selecting cells that express an appropriate selectable marker. In some embodiments, reprogrammed somatic cells are further evaluated for pluripotency characteristics. The presence of pluripotency characteristics indicates that the somatic cells have been reprogrammed to a pluripotent state.

[0140] The differentiation state of a cell is a continuous spectrum, with a fully differentiated state at one end of the spectrum and a dedifferentiated state (pluripotent state) at the other end. Reprogramming, as used herein, refers to the process of changing or reversing the differentiation state of a somatic cell that can partially or terminally differentiate. Reprogramming includes both a complete reversal and a partial reversal of the differentiation state of a somatic cell. In other words, the term "reprogramming," as used herein, encompasses any transition to a less differentiated state along the spectrum of a cell's differentiation state. For example, reprogramming includes returning a multipotent cell to a pluripotent cell, or returning a terminally differentiated cell to either a multipotent or a pluripotent cell. In one embodiment, reprogramming of a somatic cell returns the somatic cell to a pluripotent state. In another embodiment, reprogramming of a somatic cell returns the somatic cell to a multipotent state. Thus, as used herein, the term "low differentiation state" is a relative term and includes both a fully dedifferentiated state and a partially differentiated state.

[0141] The term "pluripotency characteristics" refers to many characteristics associated with pluripotency, including, for example, the ability to differentiate into all types of cells, as well as the expression of pluripotency genes, the expression of other ES cell markers, and a characteristic expression profile known as "stem cell molecular signature" or "stemness" at the global level, which includes an expression pattern characteristic of pluripotent cells.

[0142] Thus, to evaluate the reprogrammed somatic cells for pluripotent properties, such cells may be analyzed for various growth characteristics and ES cell-like morphology. In some embodiments, the cells may be subcutaneously injected into immunodeficient SCID mice to induce teratomas (a standard assay for ES cells). ES-like cells can differentiate into embryoid bodies (another ES-specific feature). Additionally, ES-like cells can be differentiated in vitro by adding certain growth factors known to drive differentiation into specific cell types. Self-renewal ability, marked by the induction of telomerase activity, is another pluripotent property that can be monitored.

[0143] In some embodiments, a functional assay of the reprogrammed somatic cells may be performed by introducing the reprogrammed somatic cells into a blastocyst to determine whether the cells can give rise to all cell types. If the reprogrammed cells can form some of the body's cell types, they are multipotent, and if the reprogrammed cells can form all of the body's cell types, including germ cells, they are pluripotent.

[0144] In other embodiments, the expression of individual pluripotency genes in the reprogrammed somatic cells may be examined to evaluate their pluripotent properties.

[0145] In addition, the expression of other ES cell markers may be evaluated. Stage-specific embryonic antigen-1, -3, and -4 (SSEA-1, SSEA-3, SSEA-4) are glycoproteins that are specifically expressed during early embryonic development and are markers for ES cells (Solter and Knowles, 1978, Proc. Natl. Acad. Sci. USA 75:5565-5569, Kannagi et al., 1983, EMBO J 2:2355-2361).

[0146] High expression of the enzyme alkaline phosphatase (AP) is another marker associated with undifferentiated embryonic stem cells (Wobus et al., 1984, Exp. Cell 152:212-219; Pease et al., 1990, Dev. Biol. 141:322-352). Other stem / precursor cell markers include intermediate neurofilament nestin (Lendahl et al., 1990, Cell 60:585-595; Dah-Istrand et al., 1992, J. Cell Sci. 103:589-597), the membrane glycoprotein prominin / AC133 (Weigmann et al., 1997, Proc. Natl. Acad. USA 94:12425-12430; Corbeil et al., 1998, Blood 91:2625-22626), the transcription factor Tcf-4 (Korinek et al., 1998, Nat. Genet. 19:379-383; Lee et al., 1999, J. Biol. Chem. 274:1566-1572), and the transcription factor Cdx1 (Duprey et al., 1988, Genes Dev. 2:1647-1654; Subramania’n et al., 1998, Differentiation 64:11-18).

[0147] In some embodiments, expression profiling of reprogrammed somatic cells may be used to evaluate their pluripotent properties. Pluripotent cells, such as embryonic stem cells, and multipotent cells, such as adult stem cells, are known to have characteristic patterns of overall gene expression profiles. This characteristic pattern is referred to as the “stem cell molecular signature” or “stemness.” See, for example, Ramalho-Santos et al., Science 298:597-600 (2002); Ivanova et al., Science 298:601-604.

[0148] Somatic cells can be reprogrammed to obtain any complete set of pluripotency characteristics and are thus pluripotent. Alternatively, somatic cells can be reprogrammed to obtain only a subset of pluripotency characteristics. In another option, somatic cells can be reprogrammed to be multipotent.

[0149] Activation culture In certain embodiments, the population of isolated cells is cultured in an activation culture for a first period. In certain embodiments, the first period is from 1 to 20 days. In certain embodiments, the first period is from 1 to 17 days. In certain embodiments, the first period is from 1 to 15 days. In certain embodiments, the first period is from 1 to 13 days. In certain embodiments, the first period is from 1 to 11 days. In certain embodiments, the first period is from 1 to 9 days. In certain embodiments, the first period is from 1 to 7 days. In certain embodiments, the first period is from 1 to 5 days. In certain embodiments, the first period is from 1 to 3 days. In certain embodiments, the first period is from 12 to 72 hours. In certain embodiments, the first period is from 12 to 60 hours. In certain embodiments, the first period is from 12 to 48 hours. In certain embodiments, the first period is from 12 to 36 hours. In certain embodiments, the first period is from 12 to 24 hours. In certain embodiments, the first period is from 8 to 16 hours. In certain embodiments, the first period is from 4 to 8 hours. In certain embodiments, the first period is from 2 to 4 hours. In certain embodiments, the first period is from 4 to 8 hours. In certain embodiments, the first period is from 50 to 80 hours. In certain embodiments, the first period is from 4 to 8 hours. In certain embodiments, the first period is from 55 to 75 hours. In certain embodiments, the first period is from 4 to 8 hours. In certain embodiments, the first period is from 60 to 75 hours. In certain embodiments, the first period is from 4 to 8 hours. In certain embodiments, the first period is from 70 to 75 hours. In certain embodiments, the first period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0150] In certain embodiments, a population of isolated cells is cultured in an activation culture for a period of time that is less than or equal to a certain period. For example, the population of isolated cells is cultured in the activation culture for up to 13 days, up to 12 days, up to 11 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, or up to 1 day. In certain embodiments, the population of isolated cells is cultured in the activation culture for up to 5 days. In certain preferred embodiments, the population of isolated cells is cultured in the activation culture for up to 3 days. In certain preferred embodiments, the population of isolated cells is cultured in the activation culture for about 3 days.

[0151] In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 100% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 95% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 90% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 85% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 80% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 75% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 70% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 65% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 60% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 55% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 50% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 45% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 40% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 35% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 15% to 35% γδ T cells.In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 25% to 35% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 30% to 35% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 30% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 25% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 20% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises 5% to 15% γδ T cells.

[0152] In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% γδ T cells. In certain embodiments, after culturing in an activation culture over a first period, a population of isolated cells comprises less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, or less than about 30% γδ T cells.

[0153] In one embodiment, after culturing in an activation culture over a first period, a population of isolated cells comprises less than about 60% γδ T cells. In another embodiment, after culturing in an activation culture over a first period, a population of isolated cells comprises less than about 55% γδ T cells. In yet another embodiment, after culturing in an activation culture over a first period, a population of isolated cells comprises less than about 50% γδ T cells. In yet another embodiment, after culturing in an activation culture over a first period, a population of isolated cells comprises less than about 45% γδ T cells. In yet another embodiment, after culturing in an activation culture over a first period, a population of isolated cells comprises less than about 40% γδ T cells. In yet another embodiment, after culturing in an activation culture over a first period, a population of isolated cells comprises less than about 35% γδ T cells.

[0154] In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 100% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 95% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 90% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 85% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 80% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 75% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 70% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 65% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 60% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 55% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 50% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 45% TCRVγ9+ T cells.In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 40% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 35% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 30% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 25% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 20% TCRVγ9+ T cells. In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population comprise 5% to 15% TCRVγ9+ T cells.

[0155] In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population are about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% TCRVγ9 + T cells (also known as Vγ9 + T cells). In certain embodiments, after culturing in an activation culture for a first period, the γδ T cells in the isolated cell population are less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, or less than about 30% TCRVγ9 + T cells.

[0156] In one embodiment, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population comprise less than about 60% TCRVγ9+ T cells. In one embodiment, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population comprise less than about 55% TCRVγ9+ T cells. In yet another embodiment, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population comprise less than about 50% TCRVγ9+ T cells. In yet another embodiment, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population comprise less than about 45% TCRVγ9+ T cells. In yet another embodiment, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population comprise less than about 40% TCRVγ9+ T cells. In yet another embodiment, after culturing in an activation culture over a first period, the γδ T cells in the isolated cell population comprise less than about 35% TCRVγ9+ T cells.

[0157] In certain embodiments, the method further comprises enriching the γδ T cells in the isolated cell population. In certain embodiments, the γδ T cells are enriched by cell - cell aggregate enrichment.

[0158] In certain embodiments, at least a portion of the activated γδ T cells in step (b) are Vγ9 + γδ T cells.

[0159] In certain embodiments, at least a portion of the activated γδ T cells in step (b) are Vγ9δ2 + γδ T cells.

[0160] cells The present disclosure is based on the discovery that isolated cell populations, such as isolated γδ T cell populations, can be activated (e.g., in the presence of zoledronic acid and IL - 15) and reprogrammed to pluripotency by introduction of transcription factors (e.g., by Sendai virus vectors).

[0161] The isolated cell population of the present disclosure includes any T cells of the body that are not stem cells, germ cells, or iPSCs. Non-limiting examples of non-iPSCs are T cells derived from any tissue of the body, including visceral organs, skin, bone, blood, nerve tissue, and connective tissue.

[0162] In certain embodiments, the isolated cell population is blood cells. In certain embodiments, the blood cells are preferably peripheral blood mononuclear cells (PMBCs) and can include all types of blood cells present in the entire differentiation process from hematopoietic stem cells to their final differentiation into peripheral blood. In one embodiment, the blood cells include, for example, hematopoietic stem cells, lymphoid stem cells, lymphoid dendritic cell precursors, lymphoid dendritic cells, T lymphocyte precursors, T cells, B lymphocyte precursors, B cells, plasma cells, NK precursors, NK cells, monocytes, and macrophages.

[0163] In some embodiments, the isolated cell population can be peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), or combinations thereof. In some embodiments, the isolated cell population is peripheral blood mononuclear (PBMC) cells.

[0164] In certain embodiments, the isolated cell population is T cells. In some embodiments, the isolated population of T cells can be selected from the group consisting of CD4+ / CD8+ double-positive T cells, cytotoxic T cells, Th3 (Treg) cells, Th9 cells, Thαβ helper cells, Tfh cells, stem memory TSCM cells, central memory TCM cells, effector memory TEM cells, effector memory TEMRA cells, gamma delta T cells, and any combination thereof.

[0165] In some embodiments, the isolated cell population is derived from cell types that are easily accessible and require minimal invasion, such as fibroblasts, skin cells, cord blood cells, peripheral blood cells, and renal epithelial cells.

[0166] In certain embodiments, the isolated cell population is a terminally differentiated cell. In certain embodiments, the isolated cell population is a terminally differentiated T cell. In certain embodiments, the isolated cell population is a terminally differentiated PBMC cell. In certain embodiments, the isolated cell population is a terminally differentiated γδ T cell.

[0167] The isolated cell populations of the present disclosure can be derived from mammals, preferably humans, but include, without limitation, non-human primates, rodents (i.e., mice and rats), dogs, cats, horses, cows, sheep, pigs, goats, etc.

[0168] In certain embodiments, the isolated cell population is a mammalian cell.

[0169] In certain embodiments, the isolated cell population is a human cell.

[0170] In some embodiments, the isolated cell population is a human PBMC cell.

[0171] Introduction of pluripotency-related genes The present disclosure also relates to introducing an endogenous locus, which is a pluripotency-related gene, into a population of activated cells. In some embodiments, such pluripotency-related genes can be introduced using an expression vector. In some embodiments, such pluripotency-related genes can be introduced using a CRISPR activation system having at least one sgRNA targeting a desired locus. In some embodiments, such pluripotency-related genes can be introduced by expression from a recombinant expression cassette introduced into the target cell. In some embodiments, such pluripotency-related genes can be introduced by incubating the cells in the presence of an exogenous reprogramming transcription factor polypeptide.

[0172] In certain embodiments, the expression vectors used to introduce pluripotency-related genes include modified viral polynucleotides derived from, for example, adenovirus, Sendai virus, herpes virus, or retrovirus (such as lentiviral vectors). The expression vectors are not limited to recombinant viruses and include non-viral vectors such as DNA plasmids and in vitro-transcribed mRNA. In a preferred embodiment, a Sendai virus vector is used.

[0173] To address safety issues arising from the target cell genome carrying the integrated exogenous sequences, several modified gene protocols have been developed and can be used in the production methods described herein. These protocols produce potentially less risky iPS cells and include non-integrating adenoviruses (Stadtfeld, M., et al., Science, 2008, 322:945 - 949) for delivering reprogramming genes, transient transfection of reprogramming plasmids (Okita, K., et al., Science, 2008, 322:949 - 953), the piggyBac transposition system (Woltjen, K., et al., Nature, 2009, 458:766 - 770, Yusa, et al., Nat. Methods, 2009, 6:363 - 369, Kaji, K., et al. (2009)), Cre-excisable viruses (Soldner, F., et al., Cell, 2009, 136:964 - 977), and the oriP / EBNA1-based episomal expression system (Yu, J., et al., Science, 2009, 324(5928):797 - 801).

[0174] Non-limiting examples of pluripotency-related genes (genes encoding reprogramming transcription factors) are Oct3 / 4, Sox2, Nanog, Klf4, c-Myc, Nanog, Lin28, Nr5a2, Glis1, Cebpa, Esrrb, and Rex1. In some embodiments, the endogenous locus is Oct4 or Sox2.

[0175] In certain embodiments, a population of isolated cells endogenously expresses at least one or more proteins from the group consisting of Oct3 / 4 polypeptide, Klf4 polypeptide, c-Myc polypeptide, Sox2 polypeptide, Nanog polypeptide, Lin28 polypeptide, Nr5a2 polypeptide, Glis1 polypeptide, Cebpa polypeptide, Esrrb polypeptide, and Rex1 polypeptide. In certain embodiments, a population of isolated cells does not endogenously express any reprogramming transcription factors.

[0176] In certain embodiments, the reprogramming factors include Oct3 / 4, Sox2, Klf4, and c-Myc.

[0177] In certain embodiments, the reprogramming factors are Oct3 / 4, Sox2, KLF4, c-Myc, and Lin28.

[0178] In certain embodiments, the reprogramming factors are Oct3 / 4, Sox2, Klf4, and c-Myc.

[0179] Exogenous introduction of pluripotency genes can be done in several ways. In one embodiment, the exogenously introduced pluripotency genes can be expressed from chromosomal loci different from the endogenous chromosomal loci of the pluripotency genes. Such chromosomal loci may be loci having an open chromatin structure and may contain genes that are not essential for somatic cells. In other words, the desired chromosomal loci contain genes whose disruption does not cause cell death. Exemplary chromosomal loci include, for example, the mouse ROSA26 locus and the type II collagen (Col2a1) locus (see Zambrowicz et al., 1997).

[0180] Exogenously introduced pluripotency genes can be expressed from inducible promoters such that their expression can be regulated as desired.

[0181] In an alternative embodiment, the exogenously introduced pluripotency genes can be transiently transfected into cells, either individually or as part of a cDNA expression library prepared from pluripotent cells. Such pluripotent cells can be embryonic stem cells, oocytes, blastomeres, inner cell mass cells, embryonic germ cells, embryoid (embryonic) cells, morula-derived cells, teratoma (teratocarcinoma) cells, and multipotent partially differentiated embryonic stem cells taken at later stages of the embryonic development process.

[0182] The cDNA library is prepared by conventional techniques. Briefly, mRNA is isolated from the organism of interest. Reverse transcriptase is used for the first-strand synthesis using the mRNA as a template. The second-strand synthesis is carried out using DNA-dependent DNA polymerase to yield the cDNA product. Following conventional procedures to facilitate cloning of the cDNA, the cDNA is inserted into an expression vector such that the cDNA is operably linked to at least one regulatory sequence. The choice of expression vector for use in connection with the cDNA library is not limited to a particular vector. Any expression vector suitable for use in mouse cells is appropriate. In one embodiment, the promoter driving expression from the cDNA expression construct is an inducible promoter. The term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). For example, any of a variety of expression control sequences that control the expression of a DNA sequence when operably linked can be used in these vectors to express the cDNA. Such useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, the adenovirus or cytomegalovirus immediate early promoter, the lac, trp, TAC or TRC systems, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage λ, the control region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatase, e.g., Pho5, the promoter of yeast α mating factor, the polyhedron promoter of the baculovirus system, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof.It should be understood that the design of the expression vector can depend on factors such as the choice of host cell to be transformed and / or the type of protein desired to be expressed. Further, the copy number of the vector, the ability to control that copy number, and the expression of any other protein encoded by the vector, such as an antibiotic marker, should also be considered.

[0183] Exogenously introduced pluripotency genes can be expressed from inducible promoters. As used herein, the term "inducible promoter" refers to a promoter that does not direct, or directs low levels of, expression of an operably linked gene (including cDNA) in the absence of an inducer (such as a chemical agent and / or a biological agent), and whose ability to direct expression is enhanced in response to the inducer. Exemplary inducible promoters include, for example, promoters that respond to heavy metals (CRC Boca Raton, Fla. (1991), 167 - 220, Brinster et al. Nature (1982), 296, 39 - 42), heat shock, hormones (Lee et al. P.N.A.S. USA (1988), 85, 1204 - 1208; (1981), 294, 228 - 232, Klock et al. Nature (1987), 329, 734 - 736, Israel and Kaufman, Nucleic Acids Res. (1989), 17, 2589 - 2604), and promoters that respond to chemical agents such as glucose, lactose, galactose, or antibiotics.

[0184] The tetracycline-inducible promoter is an example of an inducible promoter that responds to antibiotics. See Gossen et al., 2003. The tetracycline-inducible promoter comprises a minimal promoter operably linked to one or more tetracycline operators. In the presence of one of tetracycline or its analogs, a transcriptional activator binds to the tetracycline operator sequence, which activates the minimal promoter and thus activates the transcription of the relevant cDNA. Tetracycline analogs include any compound that exhibits structural homology to tetracycline and is capable of activating the tetracycline-inducible promoter. Exemplary tetracycline analogs include, for example, doxycycline, chlorotetracycline, and anhydrotetracycline.

[0185] Accordingly, in one embodiment, the present disclosure provides a somatic cell carrying at least one pluripotency gene expressed as a transgene under an inducible promoter. Somatic cells having such inducible pluripotency transgenes may be more likely to be reprogrammed.

[0186] Any of the engineered somatic cells of the present disclosure can be used in a method. In one embodiment, the somatic cell used in the method contains only one endogenous pluripotency gene linked to a first selection marker, and the selection step is performed to select for the expression of the first selection marker. In an alternative embodiment, the somatic cell used in the method contains any number of endogenous pluripotency genes, each of which is linked to a separate selection marker, and the selection step is performed to select for at least a subset of the selection markers. For example, the selection step may be performed to select for all selection markers linked to various endogenous pluripotency genes.

[0187] In an alternative embodiment, the somatic cells used in the method comprise a selectable marker linked to an endogenous pluripotency gene and a further pluripotency gene expressed as a transgene under an inducible promoter. For these cells, the reprogramming method may include inducing the expression of the pluripotency transgene and selecting for the expression of the selectable marker.

[0188] In certain embodiments, in step (d) as described in the above method, the transduced γδ T cells are cultured in the presence of one or more feeder cell layers. In certain embodiments, in step (d), the transduced γδ T cells are cultured in the presence of a monolayer of feeder cells. In certain embodiments, the feeder cell layer comprises mouse embryonic fibroblasts (MEFs). In certain embodiments, in step (d), the transduced γδ T cells are cultured in the presence of one or more feeder cell layers. In certain embodiments, in step (d), the transduced γδ T cells are cultured in the presence of mitotically inactivated mouse embryonic fibroblasts (MEFs). In certain embodiments, in step (d), the transduced γδ T cells are cultured under conditions that do not include feeder cells. In certain embodiments, in step (d), the transduced γδ T cells are cultured on plates coated with iMatrix-511. In certain embodiments, after step (d), the method further comprises step (e) of isolating and / or purifying the produced iPSCs.

[0189] In certain more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), the method comprising contacting a population of isolated cells with an activation culture comprising IL-15, zoledronic acid, and IL-2; culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; transducing the γδ T cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors; and culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0190] In certain more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), the method comprising obtaining a population of cells isolated from a subject (e.g., a human) (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)); contacting the population of isolated cells with an activation culture comprising IL-15, zoledronic acid, and IL-2; culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; transducing the γδ T cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors; and culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0191] In other more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising obtaining a population of cells isolated from a subject (e.g., a human) (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)), contacting the isolated population of cells with an activation culture comprising IL-15, zoledronic acid, and IL-2, culturing the isolated population of cells in the activation culture for about 3 days to enrich and / or activate γδ T cells in the isolated population of cells, transducing the γδ T cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors, and culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state.

[0192] In other more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising obtaining a population of cells isolated from a subject (e.g., a human) (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)), contacting the isolated population of cells with an activation culture comprising IL-15, zoledronic acid, and IL-2, culturing the isolated population of cells in the activation culture for about 3 days such that after culturing in the activation culture, the isolated population of cells comprises less than 35% γδ T cells, transducing the γδ T cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors, and culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state.

[0193] In other more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising obtaining a population of cells isolated from a subject (e.g., a human) (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)), contacting the isolated population of cells with an activation culture comprising IL-15, zoledronic acid, and IL-2, culturing the isolated population of cells in the activation culture for about 3 days such that after culturing in the activation culture, the isolated population of cells comprises less than 35% γδ T cells, further enriching γδ T cells by cell-cell aggregate enrichment, transducing the γδ T cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors, and culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0194] In other more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising obtaining a population of cells isolated from a subject (e.g., a human) (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)), contacting the isolated population of cells with an activation culture comprising IL-15, zoledronic acid, and IL-2, culturing the isolated population of cells in the activation culture for about 3 days such that after culturing in the activation culture, the isolated population of cells comprises less than 35% γδ T cells, optionally, further enriching γδ T cells by cell-cell aggregate enrichment, transducing the γδ T cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc, and culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0195] In other more specific embodiments, provided herein is a method of producing induced pluripotent stem cells (iPSCs), comprising obtaining a population of cells isolated from a subject (e.g., a human) (e.g., terminally differentiated cells such as peripheral blood mononuclear cells (PBMCs)), contacting the isolated population of cells with an activation culture comprising IL-15, zoledronic acid, and IL-2, culturing the isolated population of cells in the activation culture for about 3 days such that after culturing in the activation culture, the isolated population of cells comprises less than 35% γδ T cells, optionally further enriching for γδ T cells by cell aggregate enrichment, transducing the γδ T cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc, and culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state in the presence of a monolayer of a feeder cell layer.

[0196] In other more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising obtaining a population of cells isolated from a subject (e.g., a human), such as a population of terminally differentiated cells (e.g., peripheral blood mononuclear cells (PBMCs)), contacting the isolated population of cells with an activation culture comprising IL-15, zoledronic acid, and IL-2, culturing the isolated population of cells in the activation culture for about 3 days such that after culturing in the activation culture, the isolated population of cells contains less than 35% γδ T cells, optionally further enriching γδ T cells by cell clump concentration, transducing the γδ T cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc, and culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state in the presence of a monolayer of feeder cells comprising mouse embryonic fibroblasts (MEFs).

[0197] iPSCs derived from γδ T cells In certain embodiments, the produced iPSCs are derived from γδ T cells. In certain embodiments, the produced iPSCs have rearranged genes at the TRG locus and the TRD locus. In certain embodiments, the produced iPSCs do not produce or express TCRA and / or TCRB, or fragments thereof, such that there is no detectable or otherwise surface expression of TCRA and TCRB.

[0198] In certain embodiments, the produced iPSCs are not derived from αβ T cells.

[0199] In certain embodiments, the produced iPSCs are negative for the Sendai virus (SeV) vector.

[0200] In certain embodiments, the generated iPSCs are genomically stable without chromosomal loss. In one embodiment, the genomic stability of the generated iPSCs is determined by karyotype analysis.

[0201] In certain embodiments, the generated iPSCs can be grown and maintained in a medium without feeder cells after domestication.

[0202] In certain embodiments, the method further comprises differentiating the generated iPSCs into a desired cell type in vitro or ex vivo. In certain embodiments, the method further comprises differentiating the generated iPSCs into a desired cell type in vitro. In certain embodiments, the method further comprises differentiating the generated iPSCs into a desired cell type ex vivo.

[0203] 5.7 T cell-derived induced pluripotent stem cells (iPSCs) Also provided herein is a population of isolated induced pluripotent stem cells (iPSCs) having novel properties. In some embodiments, the population of isolated iPSCs comprises pluripotent cells that express one or more reprogramming factors and comprises a nucleotide sequence encoding rearrangement of the TRG and TRD genes.

[0204] In certain embodiments, the population of isolated iPSCs is produced according to the methods described herein (e.g., in Section 5.6).

[0205] In certain embodiments, the reprogramming factor is selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28.

[0206] In certain embodiments, the reprogramming factors include Oct3 / 4, Sox2, Klf4, and c-Myc.

[0207] In certain embodiments, the reprogramming factors are Oct3 / 4, Sox2, KLF4, c-Myc, and Lin28.

[0208] In certain embodiments, the reprogramming factors are Oct3 / 4, Sox2, Klf4, and c-Myc.

[0209] In certain embodiments, the isolated population of iPSCs is derived from γδ T cells. In certain embodiments, the isolated population of iPSCs has rearranged genes of the TRG locus and the TRD locus. In certain embodiments, the isolated population of iPSCs does not produce PCR products from the TCRG locus and the TCRD locus.

[0210] In certain embodiments, the isolated population of iPSCs is not derived from αβ T cells. In certain embodiments, the isolated population of iPSCs does not have rearranged genes of the TRA locus and the TRB locus. In certain embodiments, the isolated population of iPSCs does not produce PCR products from the TCRA locus and the TCRB locus.

[0211] In certain embodiments, the isolated population of iPSCs is negative for Sendai virus (SeV) vectors.

[0212] In certain embodiments, the isolated population of iPSCs is genomically stable without chromosomal loss. In one embodiment, the genomic stability of the isolated population of iPSCs is determined by karyotype analysis.

[0213] In certain embodiments, the isolated population of iPSCs can be grown and maintained in a medium without feeder cells after adaptation.

[0214] In some embodiments, provided herein is a population of isolated induced pluripotent stem cells (iPSCs) comprising pluripotent cells that express one or more reprogramming factors, wherein (i) the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, or have rearrangement genes at the TRG locus and the TRD locus, (ii) the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28, (iii) the population of isolated iPSCs is negative for Sendai virus (SeV) vectors, (iv) the population of isolated iPSCs is derived from γδ T cells but not from αβ T cells, (v) the population of isolated iPSCs does not produce PCR products from the TCRA locus and the TCRB locus, (vi) the population of isolated iPSCs is genomically stable without chromosomal loss, as determined, for example, by karyotyping, and / or (vii) the population of isolated iPSCs can be grown and maintained in a feeder cell-free medium after domestication, a population of isolated induced pluripotent stem cells (iPSCs).

[0215] In a specific embodiment, provided herein is a population of isolated induced pluripotent stem cells (iPSCs) comprising pluripotent cells that express one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, and the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28, a population of isolated induced pluripotent stem cells (iPSCs).

[0216] In another specific embodiment, provided herein is a population of isolated induced pluripotent stem cells (iPSCs) comprising pluripotent cells that express one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, and the reprogramming factors are Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28, a population of isolated induced pluripotent stem cells (iPSCs).

[0217] In yet another specific embodiment, provided herein is a population of isolated induced pluripotent stem cells (iPSCs) comprising pluripotent cells that express one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, and the reprogramming factors are Oct3 / 4, Sox2, Klf4, and c-Myc, which is a population of isolated induced pluripotent stem cells (iPSCs).

[0218] In yet another specific embodiment, provided herein is a population of isolated induced pluripotent stem cells (iPSCs) comprising pluripotent cells that express one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, and the isolated population of iPSCs is negative for Sendai virus (SeV) vectors, which is a population of isolated induced pluripotent stem cells (iPSCs).

[0219] In yet another specific embodiment, provided herein is a population of isolated induced pluripotent stem cells (iPSCs) comprising pluripotent cells that express one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28, and the isolated population of iPSCs is negative for Sendai virus (SeV) vectors, which is a population of isolated induced pluripotent stem cells (iPSCs).

[0220] In yet another specific embodiment, provided herein is a population of isolated induced pluripotent stem cells (iPSCs) comprising pluripotent cells that express one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, and the isolated population of iPSCs is derived from γδ T cells but not from αβ T cells, which is a population of isolated induced pluripotent stem cells (iPSCs).

[0221] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells that express one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, and the isolated population of iPSCs is a population of isolated induced pluripotent stem cells (iPSCs) that do not produce PCR products from the TCRA locus and the TCRB locus.

[0222] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells that express one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene or have rearrangement genes of the TRG locus and the TRD locus, and the isolated population of iPSCs is a population of isolated induced pluripotent stem cells (iPSCs) that is genomically stable without chromosomal loss, as determined, for example, by karyotyping.

[0223] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells that express one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene or have rearrangement genes of the TRG locus and the TRD locus, and the isolated population of iPSCs is a population of isolated induced pluripotent stem cells (iPSCs) that can be grown and maintained in a medium free of feeder cells after domestication.

[0224] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells that express one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, or have rearrangement genes at the TRG locus and the TRD locus, the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28, the isolated population of iPSCs is negative for Sendai virus (SeV) vectors, the isolated population of iPSCs is derived from γδ T cells but not from αβ T cells, the isolated population of iPSCs does not produce PCR products from the TCRA locus and the TCRB locus, the isolated population of iPSCs is genomically stable without chromosomal loss as determined, for example, by karyotype analysis, and the isolated population of iPSCs can be grown and maintained in a feeder cell-free medium after adaptation, an isolated induced pluripotent stem cell (iPSC).

[0225] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells that express one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, or have rearrangement genes at the TRG locus and the TRD locus, the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin28, the isolated population of iPSCs is negative for Sendai virus (SeV) vectors, the isolated population of iPSCs is derived from γδ T cells but not from αβ T cells, the isolated population of iPSCs does not produce PCR products from the TCRA locus and the TCRB locus, the isolated population of iPSCs is genomically stable without chromosomal loss as determined, for example, by karyotype analysis, and the isolated population of iPSCs can be grown and maintained in a feeder cell-free medium after adaptation, an isolated induced pluripotent stem cell (iPSC).

[0226] In yet another specific embodiment, provided herein is a population of isolated induced pluripotent stem cells (iPSCs) comprising pluripotent cells, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, or have rearrangement genes of the TRG locus and the TRD locus, the population of isolated iPSCs is negative for Sendai virus (SeV) vectors, the population of isolated iPSCs is derived from γδ T cells but not from αβ T cells, the population of isolated iPSCs does not produce PCR products from the TCRA locus and the TCRB locus, the population of isolated iPSCs is genomically stable without chromosomal loss as determined, for example, by karyotype analysis, and the population of isolated iPSCs can be grown and maintained in a medium without feeder cells after domestication, which is an isolated induced pluripotent stem cell (iPSC).

[0227] In yet another specific embodiment, provided herein is a population of isolated induced pluripotent stem cells (iPSCs) comprising pluripotent cells, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, or have rearrangement genes of the TRG locus and the TRD locus, the population of isolated iPSCs is negative for Sendai virus (SeV) vectors, the population of isolated iPSCs is derived from γδ T cells but not from αβ T cells, the population of isolated iPSCs does not produce PCR products from the TCRA locus and the TCRB locus, the population of isolated iPSCs is genomically stable without chromosomal loss as determined, for example, by karyotype analysis, and the population of isolated iPSCs can be grown and maintained in a medium without feeder cells after domestication, which is an isolated induced pluripotent stem cell (iPSC).

[0228] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, or have rearrangement genes at the TRG locus and the TRD locus, the isolated population of iPSCs does not produce PCR products from the TCRA locus and the TCRB locus, the isolated population of iPSCs is genomically stable without chromosomal loss, as determined, for example, by karyotyping, and the isolated population of iPSCs can be grown and maintained in a feeder cell-free medium after domestication, and is an isolated induced pluripotent stem cell (iPSC).

[0229] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells, wherein the pluripotent cells comprise a nucleotide sequence encoding rearrangement of the TRG gene and the TRD gene, or have rearrangement genes at the TRG locus and the TRD locus, the isolated population of iPSCs does not produce PCR products from the TCRA locus and the TCRB locus, and the isolated population of iPSCs is genomically stable without chromosomal loss, as determined, for example, by karyotyping, and is an isolated population of induced pluripotent stem cells (iPSCs).

[0230] Also provided herein are reprogrammed somatic cells comprising reprogrammed pluripotent somatic cells such as iPSCs produced by the methods of the present disclosure.

[0231] In some embodiments, the reprogrammed somatic cells of the present invention are ES-like cells and can thus be induced to differentiate to obtain a desired cell type according to known methods for differentiating ES cells. For example, iPSCs can be induced to differentiate into hematopoietic stem cells, muscle cells, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, urinary tract cells, etc. by culturing such cells in a differentiation medium under conditions that result in cell differentiation. Suitable culture conditions, media, and methods for differentiating embryonic stem cells are known in the art.

[0232] In some specific embodiments, iPSCs are induced to differentiate into hematopoietic stem cells as described, for example, in Palacios et al., Proc. Natl. Acad. Sci., USA, 92:7530-37 (1995), which teaches the production of hematopoietic stem cells from embryonic cell lines by subjecting the stem cells to a certain induction procedure. This procedure involves first culturing aggregates of such cells in a suspension culture medium lacking retinoic acid, then culturing in the same medium containing retinoic acid, and subsequently transplanting the cell aggregates onto a substrate that provides cell attachment.

[0233] In other specific embodiments, iPSCs are induced to differentiate according to methods as described in Pedersen, J. Reprod. Fertil. Dev., 6:543-52 (1994), which refers to a number of papers disclosing methods for in vitro differentiation of embryonic stem cells to produce various differentiated cell types, including, inter alia, hematopoietic cells, muscle, cardiomyocytes, and nerve cells.

[0234] In other specific embodiments, iPSCs are induced to differentiate according to Bain et al., Dev. Biol., 168:342-357 (1995), which teaches the in vitro differentiation of embryonic stem cells to produce nerve cells with nerve characteristics.

[0235] These references are reported exemplary methods for obtaining differentiated cells from germ cells or stem-like cells. The disclosures therein regarding these references and in particular methods for differentiating embryonic stem cells are incorporated herein by reference in their entirety.

[0236] Thus, using known methods and culture media, one of ordinary skill in the art can culture the subject germ cells or stem-like cells to obtain the desired differentiated cell type, such as neurons, muscle cells, hematopoietic cells, etc. In addition, the use of inducible Bcl-2 or Bcl-x1 may be useful for enhancing the in vitro development of certain cell lineages.

[0237] The iPSCs provided herein can be used to obtain any desired differentiated cell type.

[0238] The iPSCs produced according to the present disclosure can be used to produce genetically engineered differentiated cells or transgenic differentiated cells. In essence, this is achieved by introducing the desired gene or genes, or removing all or part of one or more endogenous genes of the iPSCs produced according to the present invention, and differentiating such cells into the desired cell type. A preferred method for achieving such modifications is by homologous recombination, because such techniques can be used to insert, delete, or modify genes or genes at specific sites or sites in the stem-like cell genome.

[0239] The iPSCs of the present disclosure can be used, for example, as an in vitro model of differentiation, particularly for the study of genes involved in the regulation of early development. Differentiated cell tissues and organs using iPSCs can be used in drug research.

[0240] Compositions comprising the iPSCs of the present disclosure are also provided herein. In some embodiments, the composition comprises a B2M knockout iPSC or a population of B2M knockout iPSCs. In some embodiments, the iPSC is generated by reprogramming γδ T cells. In some embodiments, provided herein are compositions comprising an isolated population or subpopulation of functionally enhanced derived immune cells differentiated from iPSCs produced according to the methods provided herein.

[0241] 5.8. Method for reprogramming somatic cells or identifying an agent contributing to reprogramming In another aspect, provided herein is a method for identifying an agent that, alone or in combination with one or more other agents, reprograms a somatic cell (e.g., a T cell) to a less differentiated state. The present disclosure further provides an agent identified according to the methods provided herein.

[0242] In one embodiment, the method comprises contacting the somatic cell with an activation culture comprising IL-15, zoledronic acid, and / or IL-2, contacting the somatic cell with a candidate agent, and then determining whether the presence of the candidate agent results in enhanced reprogramming (e.g., increased reprogramming rate and / or efficiency) compared to the reprogramming that would occur if the cell was not contacted with the candidate agent.

[0243] In some embodiments, provided herein is a method for identifying an agent that reprograms somatic cells (e.g., T cells) to a less differentiated state, either alone or in combination with one or more other agents, the method comprising: (a) contacting a population of isolated cells with an activation culture comprising IL-15 and zoledronic acid; (b) culturing the population of isolated cells in the activation culture to enrich for and / or activate γδ T cells in the population of isolated cells; (c) contacting the population of isolated cells with a candidate agent; (d) transducing the γδ T cells with one or more viral vectors encoding one or more reprogramming factors; (e) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a less differentiated state; and (f) determining whether at least a portion of the somatic cells have been reprogrammed to a less differentiated state. In some embodiments, the less differentiated state is a pluripotent state. In some embodiments, the less differentiated state is a multipotent state. In certain embodiments, the activation culture further comprises one or more additional agents or compounds, for example, to improve the efficiency of activation or induction. In one embodiment, the activation culture further comprises interleukin-2 (IL-2).

[0244] In some embodiments, IL-15, zoledronic acid, and / or IL-2 and the candidate agent are present together in the cell culture medium, while in other embodiments, IL-15, zoledronic acid, and / or IL-2 and the candidate agent are not present together (e.g., the cells are sequentially exposed to the agents). In certain embodiments, the cells are maintained in culture for 1 to 20 days. In certain embodiments, the cells are maintained in culture for 1 to 17 days. In certain embodiments, the cells are maintained in culture for 1 to 15 days. In certain embodiments, the cells are maintained in culture for 1 to 13 days. In certain embodiments, the cells are maintained in culture for 1 to 11 days. In certain embodiments, the cells are maintained in culture for 1 to 9 days. In certain embodiments, the cells are maintained in culture for 1 to 7 days. In certain embodiments, the cells are maintained in culture for 1 to 5 days. In certain embodiments, the cells are maintained in culture for 1 to 3 days. In certain embodiments, the cells are maintained in culture for 12 to 72 hours. In certain embodiments, the cells are maintained in culture for 12 to 60 hours. In certain embodiments, the cells are maintained in culture for 12 to 48 hours. In certain embodiments, the cells are maintained in culture for 12 to 36 hours. In certain embodiments, the cells are maintained in culture for 12 to 24 hours. In certain embodiments, the cells are maintained in culture for 8 to 16 hours. In certain embodiments, the cells are maintained in culture for 4 to 8 hours. In certain embodiments, the cells are maintained in culture for 2 to 4 hours. The cells can be maintained in culture, for example, for up to 13 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, or up to 1 day, during which they are contacted with IL-15, zoledronic acid, and / or IL-2 and the candidate agent for all or part of the time. In some embodiments, an agent is identified as an agent that reprograms the cells if there are at least 2, 5, or 10 times more reprogrammed cells or colonies predominantly containing reprogrammed cells after that period than when the cells are not in contact with the agent.

[0245] Candidate agents can be any molecule or supramolecular complex, such as peptides, small organic or inorganic molecules, polysaccharides, polynucleotides, etc., which are tested for their ability to reprogram cells, or to facilitate or enhance reprogramming. Candidate agents may be obtained from a variety of sources, including libraries of synthetic or natural compounds, as will be understood by those skilled in the art. In some embodiments, the candidate agent is a synthetic compound. Numerous techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules. In some embodiments, the candidate modulator is provided as a mixture of natural compounds in the form of bacterial, fungal, plant and animal extracts, fermentation broths, conditioned media, etc., which are available or can be readily prepared.

[0246] In some embodiments, a library of compounds is screened. A library is typically a collection of compounds that can be presented or displayed such that the compounds can be identified in a screening assay. In some embodiments, the compounds in the library are housed in individual wells (e.g., of a microtiter plate), containers, tubes, etc., to facilitate easy transfer to individual wells or containers for contact with cells, performance of cell-free assays, etc. The library may be composed of molecules having a common structural feature that differs in the number or type of groups attached to the main structure, or may be completely random. Examples of libraries include, but are not limited to, phage display libraries, peptide libraries, polysome libraries, aptamer libraries, synthetic small molecule libraries, natural compound libraries, and chemical libraries. Methods for preparing libraries of molecules are well known in the art, and many libraries are available from commercial or non-commercial sources. An example of a library of interest is a synthetic organic combinatorial library. Libraries such as synthetic small molecule libraries and chemical libraries can contain a structurally diverse collection of chemical molecules. Small molecules often include organic molecules having multiple carbon-carbon bonds. The library can include cyclic carbon or heterocyclic structures, and / or aromatic or polyaromatic structures substituted with one or more functional groups. In some embodiments, the small molecule has 5 to 50 carbon atoms, for example, 7 to 30 carbons. In some embodiments, the compound is macrocyclic. The library of interest can also include peptide libraries, randomized oligonucleotide libraries, etc. The library can be synthesized from peptidomimetic and non-peptidic synthetic moieties. Such libraries containing non-peptidic synthetic moieties that are less susceptible to enzymatic degradation compared to their natural origin counterparts can be further synthesized. Small molecule combinatorial libraries can also be generated. Combinatorial libraries of organic small compounds can differ from one another in one or more aspects of diversity and can include collections of closely related analogs synthesized by organic techniques using multi-step processes.Combinatorial libraries can contain an enormous number of small organic compounds. As used herein, a "compound array" is a collection of compounds that can be identified by their spatial addresses in Cartesian coordinates and are arranged such that each compound has a common molecular core and one or more variable structural diversity elements. Compounds in such a compound array are made in parallel in separate reaction vessels, and each compound is identified and tracked by its spatial address. In some embodiments, mixtures containing two or more compounds, extracts obtained from natural sources or other preparations (which may contain dozens or more compounds), and / or inorganic compounds, etc. are screened.

[0247] In one embodiment, the method of the present disclosure is used to screen "approved drugs". An "approved drug" is any compound (this term includes biomolecules such as proteins and nucleic acids) that has been approved for use in humans by the FDA or a similar government agency in another country for any purpose. This represents a set of compounds that are safe and, at least in the case of FDA-approved drugs, are thought to have a therapeutic effect for at least one purpose, and thus can be a particularly useful class of compounds to screen. Therefore, these drugs are likely to be safe for at least other purposes.

[0248] Representative examples of libraries that can be screened include DIVERSet™ available from ChemBridge Corporation, 16981 Via Tazon, San Diego, Calif. 92127. DIVERSet contains 10,000 - 50,000 drug-like, manually synthesized small molecules. The compounds are preselected to form a "universal" library that covers maximum pharmacophore diversity with a minimum number of compounds and is suitable for either high-throughput or lower throughput screening. For further library descriptions, see, for example, Tan, et al., Am. Chem Soc. 120, 8565 - 8566, 1998, Floyd C D, Leblanc C, Whittaker M, Prog Med Chem 36:91 - 168, 1999. Numerous libraries are commercially available from, for example, AnalytiCon USA Inc., P.O. Box 5926, Kingwood, Tex. 77325, 3-Dimensional Pharmaceuticals, Inc., 665 Stockton Drive, Suite 104, Exton, PA. 19341 - 1151, Tripos, Inc., 1699 Hanley Rd., St. Louis, Mo., 63144 - 2913, etc. For example, libraries based on quinic acid and shikimic acid, hydroxyproline, santonin, dianhydro-D-glucitol, hydroxypipecolic acid, andrographolide, piperazine-2-carboxylic acid-based libraries, cytosine, etc. are commercially available.

[0249] In some embodiments, the candidate agent is a cDNA from a cDNA expression library prepared from cells, e.g., pluripotent cells. Such cells can be embryonic stem cells, oocytes, blastomeres, teratocarcinoma, embryonic germ cells, inner cell mass cells, etc.

[0250] The candidate reprogramming agents to be tested are typically understood to be those that are not present in the standard culture medium or, if present, are present in a lesser amount than when used in the present invention. It is also understood that useful reprogramming agents or other forms of reprogramming treatment need not be capable of reprogramming all types of somatic cells, nor all somatic cells of a given cell type. By way of non-limiting example, candidate agents that result in a population enriched about 2-fold, 5-fold, 10-fold, 50-fold, 100-fold or more for reprogrammed cells (i.e., the percentage of reprogrammed cells in the population is 2, 5, 10, 50, or 100-fold greater than that present in the starting population of cells treated in the same manner but not contacted with the candidate agent) are useful.

[0251] In some embodiments, the screening methods provided herein are used to identify agents or combinations of agents that replace Klf4 when reprogramming cells to a pluripotent state. In some embodiments, the method is used to identify an agent that replaces Sox2 when reprogramming cells to a pluripotent state. In some embodiments, the method is used to identify an agent that replaces Oct3 / 4 when reprogramming cells to a pluripotent state. In some embodiments, the method is used to identify an agent that replaces c-Myc when reprogramming cells to a pluripotent state. In some embodiments, the method is used to identify an agent that replaces Lin28 when reprogramming cells to a pluripotent state. In some embodiments, the method is performed using human cells. In some embodiments, the method is performed using mouse cells. In some embodiments, the method is performed using non-human primate cells.

[0252] In another aspect, methods are provided herein for identifying genes that activate the expression of endogenous pluripotency genes in somatic cells (e.g., T cells).

[0253] In some embodiments, the method comprises: (a) contacting a population of isolated cells with an activation culture comprising IL-15 and zoledronic acid; (b) culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; (c) transducing the γδ T cells with one or more viral vectors encoding one or more candidate reprogramming factors; (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a less differentiated state; and (e) determining whether at least a portion of the somatic cells have been reprogrammed to a less differentiated state. In some embodiments, the less differentiated state is a pluripotent state. In some embodiments, the less differentiated state is a multipotent state.

[0254] In some more specific embodiments, the method comprises: (a) contacting a population of isolated cells with an activation culture comprising IL-15 and zoledronic acid; (b) culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; (c) transducing the γδ T cells with one or more viral vectors encoding one or more candidate reprogramming factors; (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state; and (e) determining whether at least a portion of the somatic cells have been reprogrammed to a pluripotent state.

[0255] In certain embodiments, the activation culture further comprises one or more additional agents or compounds, for example, to improve the efficiency of activation or induction. In one embodiment, the activation culture further comprises interleukin-2 (IL-2).

[0256] Thus, in some embodiments, the methods provided herein include: (a) contacting a population of isolated cells with an activation culture comprising IL-15, zoledronic acid, and IL-2; (b) culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; (c) transducing the γδ T cells with one or more viral vectors encoding one or more candidate reprogramming factors; (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a less differentiated state; and (e) determining whether at least a portion of the somatic cells have been reprogrammed to a less differentiated state.

[0257] In some more specific embodiments, the methods provided herein include: (a) contacting a population of isolated cells with an activation culture comprising IL-15, zoledronic acid, and IL-2; (b) culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; (c) transducing the γδ T cells with one or more viral vectors encoding one or more candidate reprogramming factors; (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state; and (e) determining whether at least a portion of the somatic cells have been reprogrammed to a pluripotent state.

[0258] In other embodiments, the method comprises culturing somatic cells as provided herein in the presence of, for example, IL-15, zoledronic acid, and / or IL-2, then transfecting the somatic cells of the disclosure with a cDNA library prepared from ES cells or oocytes, selecting cells that express a first selectable marker, and assessing the expression of a first endogenous pluripotency gene in the transfected cells that express the first selectable marker. The expression of the first endogenous pluripotency gene indicates that the cDNA encodes a gene that activates the expression of the endogenous pluripotency gene in the somatic cells.

[0259] The method is applicable for identifying genes that activate the expression of at least two endogenous pluripotency genes in somatic cells. The somatic cells used in the method further comprise a second endogenous pluripotency gene linked to a second selectable marker. The method can be modified to select transfected cells that express both selectable markers, among which the expression of the first and second endogenous pluripotency genes is assessed. The expression of both the first and second endogenous pluripotency genes indicates that the cDNA encodes a gene that activates the expression of at least two pluripotency genes in the somatic cells.

[0260] The method is further applicable for identifying genes that activate the expression of at least three endogenous pluripotency genes in somatic cells. The somatic cells used in the method further comprise a third endogenous pluripotency gene linked to a third selectable marker. The method is modified to select transfected cells that express all three selectable markers, among which the expression of all three endogenous pluripotency genes is assessed. The expression of all three endogenous pluripotency genes indicates that the cDNA encodes a gene that activates the expression of at least three pluripotency genes in the somatic cells.

[0261] The practice of the present invention, unless otherwise indicated, uses conventional techniques of mouse genetics, developmental biology, cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the scope of the art. Such techniques are described in the literature. See, for example, Current Protocols in Cell Biology, ed. by Bonifacino, Dasso, Lippincott-Schwartz, Harford, and Yamada, John Wiley and Sons, Inc., New York, 1999; Manipulating the Mouse Embryos, A Laboratory Manual, 3rd Ed., by Hogan et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2003; Gene Targeting: A Practical Approach, IRL Press at Oxford University Press, Oxford, 1993; and Gene Targeting Protocols, Human Press, Totowa, N.J., 2000. All patents, patent applications, and references cited herein are incorporated by reference in their entirety.

[0262] 6. Embodiments The present invention provides the following non-limiting embodiments.

[0263] In one set of embodiments (Embodiment Set A), the following is provided: A1. A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population comprises iPSCs comprising a disrupted beta-2-microglobulin (B2M) gene. A2. The population of iPSCs according to Embodiment A1, wherein the disrupted B2M gene contains a deletion of at least a portion of the nucleotide sequence of SEQ ID NO: 1, or a deletion of about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleotide sequence of SEQ ID NO: 1. A3. The population of iPSCs according to Embodiment A1, wherein the nucleotide sequence of the B2M gene encodes an amino acid sequence that is about, at least about, or at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 12 - 16. A4. The population of iPSCs according to any one of Embodiments A1 - A3, wherein about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the iPSCs do not express detectable levels of B2M. A5. The population of iPSCs according to any one of Embodiments A1 - A4, wherein the disruption includes a deletion of at least one nucleotide base pair. A6. The population of iPSCs according to any one of Embodiments A1 - A4, wherein the disruption includes an insertion of at least one nucleotide base pair. A7. The population of iPSCs according to any one of Embodiments A1 - A6, wherein the disrupted B2M gene exhibits reduced B2M expression as compared to the non - disrupted B2M gene. A8. The population of iPSCs according to Embodiment A7, wherein the reduced B2M expression is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the expression of B2M in reference iPSCs. A population of iPSCs according to any one of embodiments A1 - A8, wherein the iPSCs containing a disrupted B2M gene exhibit reduced expression of HLA - A, HLA - B, and / or HLA - C as compared to the expression of HLA - A, HLA - B, and / or HLA - C in the reference iPSCs. A10. The population of iPSCs according to embodiment A9, wherein the reduced expression of HLA - A, HLA - B, and / or HLA - C is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the expression of HLA - A, HLA - B, and / or HLA - C in the reference iPSCs. A11. The population of iPSCs according to any one of embodiments A8 - A10, wherein the reference iPSCs are a population of iPSCs in which the B2M gene is not disrupted. A12. The population of iPSCs according to any one of embodiments A1 - A11, wherein the disrupted B2M gene is generated by contacting the population of iPSCs with an RNA - guided endonuclease or a nucleic acid encoding an RNA - guided endonuclease and a guide RNA (gRNA), and the gRNA binds to a target motif of the B2M gene. A13. The population of iPSCs according to embodiment A12, wherein the RNA - guided endonuclease is selected from the group consisting of MAD7, MAD2, C2c1, C2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, and Cas13c. The population of iPSCs according to embodiment A13, wherein the RNA-guided endonuclease is Cas12a (Cpf1). The population of iPSCs according to embodiment A13, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a. The population of iPSCs according to embodiment A13 or A14, wherein the RNA-guided endonuclease is MAD7. The population of iPSCs according to any one of embodiments A12 - A16, wherein the gRNA binds to at least a portion of the complementary sequence of SEQ ID NO: 1. The population of iPSCs according to any one of embodiments A12 - A16, wherein the gRNA binds to the complementary sequence of any one of SEQ ID NOs: 2 - 6 or 17. The population of iPSCs according to any one of embodiments A12 - A18, wherein the gRNA comprises the sequence of any one of SEQ ID NOs: 7 - 11 or 18. The population of iPSCs according to any one of embodiments A12 - A19, wherein the gRNA comprises the sequence of SEQ ID NO: 18. A21. The population of iPSCs according to any one of embodiments A12 - A20, wherein the gRNA consists of the sequence of any one of SEQ ID NOs: 7 - 11 or 18. The population of iPSCs according to any one of embodiments A4 - A21, wherein the iPSCs do not have a detectable RNA transcript encoding B2M. The population of iPSCs according to any one of embodiments A4 - A22, wherein the iPSCs do not have a detectable level of B2M protein. The population of iPSCs according to any one of embodiments A4 - A23, wherein the iPSCs do not express a detectable level of B2M protein when analyzed by flow cytometry. A25. The population of iPSCs according to any one of embodiments A12 - A24, wherein the gRNA consists of SEQ ID NO: 18. A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population comprises iPSCs containing a disrupted beta-2-microglobulin (B2M) gene, (i) at least about 50-100% of the iPSCs do not express a detectable level of B2M protein when measured by flow cytometry, (ii) the iPSCs containing the disrupted B2M gene exhibit reduced expression of HLA-A, HLA-B, and / or HLA-C as compared to the expression of HLA-A, HLA-B, and / or HLA-C in reference iPSCs in which the B2M gene is not disrupted. (iii) the disrupted B2M gene is generated by contacting the population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA), wherein the gRNA binds to a target motif of the B2M gene, a. the RNA-guided endonuclease is Cas12a (Cpf1), a population of induced pluripotent stem cells (iPSCs), wherein the gRNA binds to the complementary sequence of SEQ ID NO: 2. A27. The population according to embodiment A26, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a. A28. The population according to embodiment A26 or A27, wherein at least about 70% of the iPSCs do not express a detectable level of B2M protein when measured by flow cytometry. A29. The population according to any one of embodiments A26 - A28, wherein at least about 100% of the iPSCs do not express a detectable level of B2M protein when measured by flow cytometry. A30. A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population comprises iPSCs containing a disrupted beta-2-microglobulin (B2M) gene, (i) When at least about 50 - 100% of the iPSCs are measured by flow cytometry, they do not express detectable levels of the B2M protein. (ii) The iPSCs containing the disrupted B2M gene show reduced expression of HLA - A, HLA - B, and / or HLA - C compared to the expression of HLA - A, HLA - B, and / or HLA - C in reference iPSCs in which the B2M gene is not disrupted. (iii) The disrupted B2M gene is generated by contacting a population of iPSCs with an RNA - guided endonuclease or a nucleic acid encoding an RNA - guided endonuclease and a guide RNA (gRNA), and the gRNA binds to a target motif of the B2M gene. a. The RNA - guided endonuclease is MAD7. b. A population of induced pluripotent stem cells (iPSCs), wherein the gRNA binds to a sequence complementary to SEQ ID NO: 2. A31. The population according to embodiment A30, wherein the RNA - guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a. A32. The population according to embodiment A30 or A31, wherein at least about 70% of the iPSCs do not express detectable levels of the B2M protein when measured by flow cytometry. A33. The population according to any one of embodiments A30 - A32, wherein at least about 100% of the iPSCs do not express detectable levels of the B2M protein when measured by flow cytometry. A34. A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population contains iPSCs containing a disrupted beta - 2 - microglobulin (B2M) gene. (i) When at least about 50 - 100% of the iPSCs are measured by flow cytometry, they do not express detectable levels of the B2M protein. (ii) The iPSCs containing the disrupted B2M gene show reduced expression of HLA-A, HLA-B, and / or HLA-C as compared to the expression of HLA-A, HLA-B, and / or HLA-C in reference iPSCs in which the B2M gene is not disrupted. (iii) The disrupted B2M gene is generated by contacting a population of iPSCs with a RNA-guided endonuclease or a nucleic acid encoding a RNA-guided endonuclease, and a guide RNA (gRNA), wherein the gRNA binds to a target motif of the B2M gene. a. The RNA-guided endonuclease is Cas12a (Cpf1). b. A population of induced pluripotent stem cells (iPSCs), wherein the gRNA binds to a sequence complementary to SEQ ID NO: 3. A35. The population according to embodiment A34, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a. A36. The population according to embodiment A34 or A35, wherein at least about 70% of the iPSCs do not express detectable levels of B2M protein as measured by flow cytometry. A37. The population according to any one of embodiments A34 to A36, wherein at least about 100% of the iPSCs do not express detectable levels of B2M protein as measured by flow cytometry. A38. A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population comprises iPSCs containing a disrupted beta-2-microglobulin (B2M) gene. (i) At least about 50-100% of the iPSCs do not express detectable levels of B2M protein as measured by flow cytometry. (ii) The iPSCs containing the disrupted B2M gene show reduced expression of HLA-A, HLA-B, and / or HLA-C as compared to the expression of HLA-A, HLA-B, and / or HLA-C in reference iPSCs in which the B2M gene is not disrupted. (iii) The disrupted B2M gene is generated by contacting a population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA), wherein the gRNA binds to the target motif of the B2M gene, a. The RNA-guided endonuclease is MAD7, b. A population of induced pluripotent stem cells (iPSCs), wherein the gRNA binds to a sequence complementary to SEQ ID NO: 3. A39. The population according to embodiment A38, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a. A40. The population according to embodiment A38 or A39, wherein at least about 70% of the iPSCs do not express a detectable level of B2M protein as measured by flow cytometry. A41. The population according to any one of embodiments A38 to A40, wherein at least about 100% of the iPSCs do not express a detectable level of B2M protein as measured by flow cytometry. A42. A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population comprises iPSCs containing a disrupted beta-2-microglobulin (B2M) gene, (i) At least about 50-100% of the iPSCs do not express a detectable level of B2M protein as measured by flow cytometry, (ii) The iPSCs containing the disrupted B2M gene exhibit reduced expression of HLA-A, HLA-B, and / or HLA-C as compared to the expression of HLA-A, HLA-B, and / or HLA-C in reference iPSCs in which the B2M gene is not disrupted. (iii) The disrupted B2M gene is generated by contacting a population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA), wherein the gRNA binds to the target motif of the B2M gene, a. The RNA-guided endonuclease is Cas12a (Cpf1), b. A population of induced pluripotent stem cells (iPSCs) in which the gRNA binds to a sequence complementary to SEQ ID NO: 4. A43. The population according to embodiment A38, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a. A44. The population according to embodiment A42 or A43, wherein at least about 70% of the iPSCs do not express detectable levels of the B2M protein as measured by flow cytometry. A45. The population according to any one of embodiments A42 to A44, wherein at least about 100% of the iPSCs do not express detectable levels of the B2M protein as measured by flow cytometry. A46. A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population comprises iPSCs containing a disrupted beta-2-microglobulin (B2M) gene, (i) at least about 50-100% of the iPSCs do not express detectable levels of the B2M protein as measured by flow cytometry, (ii) the iPSCs containing the disrupted B2M gene exhibit reduced expression of HLA-A, HLA-B, and / or HLA-C as compared to the expression of HLA-A, HLA-B, and / or HLA-C in reference iPSCs in which the B2M gene is not disrupted. (iii) the disrupted B2M gene is generated by contacting the population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA), and the gRNA binds to a target motif of the B2M gene, a. The RNA-guided endonuclease is MAD7, b. A population of induced pluripotent stem cells (iPSCs) in which the gRNA binds to a sequence complementary to SEQ ID NO: 4. The population according to embodiment A46, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a. The population according to embodiment A46 or A47, wherein at least about 70% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry. The population according to any one of embodiments A46 - A48, wherein at least about 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry. A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population comprises iPSCs comprising a disrupted beta-2-microglobulin (B2M) gene, (iv) at least about 50 - 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry, (v) the iPSCs comprising the disrupted B2M gene exhibit reduced expression of HLA-A, HLA-B, and / or HLA-C as compared to the expression of HLA-A, HLA-B, and / or HLA-C in reference iPSCs in which the B2M gene is not disrupted. (vi) the disrupted B2M gene is generated by contacting the population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA), wherein the gRNA binds to a target motif of the B2M gene, a. the RNA-guided endonuclease is Cas12a (Cpf1), b. the gRNA binds to a sequence complementary to SEQ ID NO: 5, a population of induced pluripotent stem cells (iPSCs). The population of induced pluripotent stem cells (iPSCs) according to embodiment A46, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1). A population according to embodiment A50 or A51, wherein at least about 70% of the A52.iPSCs do not express detectable levels of B2M protein when measured by flow cytometry. A population according to any one of embodiments A50 - A52, wherein at least about 100% of the A53.iPSCs do not express detectable levels of B2M protein when measured by flow cytometry. A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population comprises iPSCs containing a disrupted beta-2-microglobulin (B2M) gene, (i) at least about 50 - 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry, (ii) the iPSCs containing the disrupted B2M gene exhibit reduced expression of HLA-A, HLA-B, and / or HLA-C as compared to the expression of HLA-A, HLA-B, and / or HLA-C in reference iPSCs in which the B2M gene is not disrupted. (iii) the disrupted B2M gene is generated by contacting the population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA), wherein the gRNA binds to a target motif of the B2M gene, a. the RNA-guided endonuclease is MAD7, b. the gRNA binds to a sequence complementary to SEQ ID NO: 5, a population of induced pluripotent stem cells (iPSCs). A population of induced pluripotent stem cells (iPSCs) according to embodiment A50, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1). A population according to embodiment A54 or A55, wherein at least about 70% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry. A population according to any one of embodiments A54 - A56, wherein at least about 100% of the A57.iPSCs do not express detectable levels of B2M protein when measured by flow cytometry. A58. A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population comprises iPSCs containing a disrupted beta-2-microglobulin (B2M) gene, (i) at least about 50 - 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry, (ii) the iPSCs containing the disrupted B2M gene exhibit reduced expression of HLA-A, HLA-B, and / or HLA-C compared to the expression of HLA-A, HLA-B, and / or HLA-C in reference iPSCs in which the B2M gene is not disrupted. (iii) the disrupted B2M gene is generated by contacting the population of iPSCs with a RNA-guided endonuclease or a nucleic acid encoding a RNA-guided endonuclease, and a guide RNA (gRNA), wherein the gRNA binds to a target motif of the B2M gene, a. the RNA-guided endonuclease is Cas12a (Cpf1), b. the gRNA binds to a sequence complementary to SEQ ID NO: 6, a population of induced pluripotent stem cells (iPSCs). A59. A population of induced pluripotent stem cells (iPSCs) according to embodiment A54, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1). A60. A population according to embodiment A58 or A59, wherein at least about 70% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry. A61. A population according to any one of embodiments A58 - A60, wherein at least about 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry. A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population comprises iPSCs containing a disrupted beta-2-microglobulin (B2M) gene, (i) at least about 50-100% of the iPSCs do not express a detectable level of B2M protein when measured by flow cytometry, (ii) the iPSCs containing the disrupted B2M gene exhibit reduced expression of HLA-A, HLA-B, and / or HLA-C compared to the expression of HLA-A, HLA-B, and / or HLA-C in reference iPSCs in which the B2M gene is not disrupted. (iii) the disrupted B2M gene is generated by contacting the population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA), and the gRNA binds to a target motif of the B2M gene, a. the RNA-guided endonuclease is MAD7, b. the gRNA binds to a sequence complementary to SEQ ID NO: 6, a population of induced pluripotent stem cells (iPSCs). A63. The population of induced pluripotent stem cells (iPSCs) according to embodiment A58, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1). A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population comprises iPSCs containing a disrupted beta-2-microglobulin (B2M) gene, (i) at least about 50-100% of the iPSCs do not express a detectable level of B2M protein when measured by flow cytometry, (ii) the iPSCs containing the disrupted B2M gene exhibit reduced expression of HLA-A, HLA-B, and / or HLA-C compared to the expression of HLA-A, HLA-B, and / or HLA-C in reference iPSCs in which the B2M gene is not disrupted. (iii) The disrupted B2M gene is generated by contacting a population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA), wherein the gRNA binds to a target motif of the B2M gene, a. The RNA-guided endonuclease is MAD7, A population of induced pluripotent stem cells (iPSCs) wherein the gRNA binds to a sequence complementary to SEQ ID NO: 17. A65. The population according to embodiment A62 or A63, wherein at least about 70% of the iPSCs do not express a detectable level of B2M protein as measured by flow cytometry. A66. The population according to any one of embodiments A62 - A65, wherein at least about 100% of the iPSCs do not express a detectable level of B2M protein as measured by flow cytometry. A67. The population according to any one of the preceding embodiments, wherein the disruption comprises an insertion of at least one nucleotide base pair, the insertion is an expression cassette, and the expression cassette encodes a therapeutic cargo, a therapeutic protein, a TCR, or a chimeric antigen receptor. A68. The population according to any one of the preceding embodiments, wherein the complementary sequence comprises any one of SEQ IDs 19 - 23.

[0264] In another set of embodiments (Embodiment Set B), the following are provided: B1. A method for producing induced pluripotent stem cells (iPSCs), comprising: (a) contacting a population of isolated cells with an activation culture comprising IL-15 and zoledronic acid; (b) culturing the population of isolated cells in the activation culture to enrich and / or activate γδ T cells in the population of isolated cells; (c) transducing the γδ T cells with a viral vector encoding one or more reprogramming factors; (d) Culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state, thereby producing a population of iPSCs, and (e) contacting the population of iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA), wherein the gRNA binds to a target motif of the beta-2-microglobulin (B2M) polynucleotide sequence in the population of iPSCs, and the contacting results in cleavage of the B2M polynucleotide sequence, a method. B2. The method according to embodiment B1, wherein the RNA-guided endonuclease is selected from the group consisting of MAD7, MAD2, C2c1, C2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, and Cas13c. B3. The method according to embodiment B2, wherein the RNA-guided endonuclease is Cas12a (Cpf1). B4. The method according to embodiment B2, wherein the RNA-guided endonuclease is MAD7. B5. The method according to any one of embodiments B1 to B4, wherein the target motif comprises a portion of SEQ ID NO: 1. B6. The method according to any one of embodiments B1 to B4, wherein the target motif comprises any one of the sequences of SEQ ID NOs: 2 to 6 or 17. B7. The method according to any one of embodiments B1 to B4, wherein the target motif consists of any one of the sequences of SEQ ID NOs: 2 to 6 or 17. The method according to any one of Embodiments B1 - B7, wherein the gRNA comprises any one of the sequences of SEQ ID NOs: 7 - 11 or 18. The method according to any one of Embodiments B1 - B7, wherein the gRNA comprises SEQ ID NO: 18. The method according to any one of Embodiments B1 - B7, wherein the gRNA consists of any one of the sequences of SEQ ID NOs: 7 - 11 or 18. The method according to any one of Embodiments B1 - B7, wherein the gRNA consists of SEQ ID NO: 18. The method according to any one of Embodiments B1 - B12, wherein cleavage of the B2M polynucleotide sequence results in reduced B2M expression in iPSCs as compared to the expression of B2M in the reference. The method according to Embodiment B13, wherein the reduced B2M expression is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the expression of B2M in the reference. The method according to Embodiment B13 or B14, wherein the reduced B2M expression is reduced by about or at least about 70% as compared to the expression of B2M in the reference. The method according to any one of Embodiments B1 - B15, wherein cleavage of the B2M polynucleotide sequence results in reduced HLA - A, HLA - B, and / or HLA - C expression as compared to the expression of HLA - A, HLA - B, and / or HLA - C in the reference. The method according to Embodiment B16, wherein the reduced HLA - A, HLA - B, and / or HLA - C expression is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the expression of HLA - A, HLA - B, and / or HLA - C in the reference. The method according to any one of embodiments B13 to B17, wherein the reference is an iPSC or a population of iPSCs without cleavage of the B2M polynucleotide sequence. The method according to any one of embodiments B1 to B18, wherein the activation culture further comprises IL-2. The method according to any one of embodiments B1 to B19, wherein the viral vector is a Sendai virus (SeV) vector. The method according to any one of embodiments B1 to B20, further comprising obtaining a population of cells isolated from a subject. The method according to any one of embodiments B1 to B21, wherein the cells in the isolated population of cells are peripheral blood mononuclear cells (PBMCs). The method according to any one of embodiments B1 to B22, wherein the isolated population of cells is a terminally differentiated cell. The method according to any one of embodiments B1 to B23, wherein the cells in the isolated population of cells are mammalian cells. The method according to any one of embodiments B1 to B24, wherein the cells in the isolated population of cells are human cells. The method according to any one of embodiments B22 to B25, wherein the isolated population of cells is cultured in the activation culture for a maximum of 13 days, a maximum of 10 days, a maximum of 9 days, a maximum of 8 days, a maximum of 7 days, a maximum of 6 days, a maximum of 5 days, a maximum of 4 days, a maximum of 3 days, a maximum of 2 days, or a maximum of 1 day. The method according to embodiment B26, wherein the isolated population of cells is cultured in the activation culture for a maximum of 3 days. The method according to embodiment B26, wherein the isolated population of cells is cultured in the activation culture for 3 days. The method according to any one of embodiments B1 to B28, wherein after culturing in the activation culture, the isolated population of cells contains less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, or less than 30% γδ T cells. The method according to embodiment B29, wherein after culturing in an activated culture, the isolated cell population contains less than 35% γδ T cells. The method according to any one of embodiments B1 - B30, further comprising concentrating γδ T cells in the isolated cell population after step (b). The method according to embodiment B31, wherein the γδ T cells are concentrated by cell - cell aggregate concentration. B33. At least a part of the γδ T cells is activated to γδ T cells by Vγ9 + The method according to any one of embodiments B1 - B32. B34. At least a part of the γδ T cells is activated to γδ T cells by Vγ9δ2 + The method according to any one of embodiments B1 - B32. The method according to any one of embodiments B1 - B34, wherein one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c - Myc. The method according to any one of embodiments B1 - B35, wherein in step (d), the transduced γδ T cells are cultured in the presence of one or more supporting cell layers. The method according to embodiment B36, wherein in step (d), the transduced γδ T cells are cultured in the presence of a monolayer of supporting cells. The method according to embodiment B36 or B37, wherein the supporting cell layer comprises mouse embryonic fibroblasts (MEF). The method according to any one of embodiments B1 - B38, further comprising isolating and / or purifying the produced iPSCs. The method according to any one of embodiments B1 - B39, further comprising differentiating the iPSCs ex vivo into cells of a desired cell type, thereby producing differentiated iPSCs. The method according to any one of embodiments B1 - B40, wherein the produced iPSCs are negative for Sendai virus (SeV) vectors. The generated iPSCs are obtained by the method according to any one of Embodiments B1 to B41, which are derived from γδ T cells. B43. The generated iPSCs have rearranged genes at the TRG locus and the TRD locus, and optionally, the generated iPSCs have a Vγ9 gene configuration and a Vδ2 gene configuration, according to the method of any one of Embodiments B1 to B41. B44. The generated iPSCs are not derived from αβ T cells, according to the method of any one of Embodiments B1 to B41. B45. The generated iPSCs do not produce or express TCRA and / or TCRB, or fragments thereof, such that there is no detectable or other surface expression of TCRA and TCRB, according to the method of any one of Embodiments B1 to B44. B46. The generated iPSCs are genomically stable without chromosomal loss, according to the method of any one of Embodiments B1 to B45. B47. The genomic stability of the generated iPSCs is determined by karyotype analysis, according to the method of Embodiment B46. B48. The generated iPSCs can proliferate in a medium without feeder cells after domestication, according to the method of any one of Embodiments B1 to B47. B49. The RNA-guided endonuclease is Cas12a (Cpf1), according to the method of any one of Embodiments B1 to B48. B50. A method for producing induced pluripotent stem cells (iPSCs), (a) contacting a population of isolated human PBMCs with an activation culture comprising IL-15, IL-2, and zoledronic acid; (b) culturing the population of isolated cells in the activation culture to enrich and / or activate at least a portion of the γδ T cells in the population of isolated cells that are γδ T cells activated by Vγ9δ2 + γδ T cells; (c) transducing the γδ T cells with a Sendai virus vector encoding one or more reprogramming factors; (d) Culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state, thereby producing a population of iPSCs, and (e) contacting the population of iPSCs with an RNA-guided endonuclease, which is Cas12a or MAD7, an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease, and a guide RNA (gRNA), wherein the gRNA binds to a target motif of the beta-2-microglobulin (B2M) polynucleotide sequence in the population of iPSCs, the contacting results in cleavage of the B2M polynucleotide sequence, and the cleavage of the B2M polynucleotide sequence results in reduced expression of B2M in the iPSCs as compared to the expression of B2M in a population of reference iPSCs that does not involve cleavage of the B2M polynucleotide sequence, the produced iPSCs are negative for Sendai virus (SeV) vectors, do not produce or express TCRA and / or TCRB, or fragments thereof, such that there is no detectable or other surface expression of TCRA and TCRB, a method. B51. The method according to any one of the preceding embodiments B1 to B48, further comprising selecting B2M-negative cells from the population of cells. B52. The method according to embodiment B51, wherein the selection is performed by fluorescence-activated cell sorting, dilution plating of single cells per well of a multi-well plate, magnetic bead enrichment, or a cartridge-based cell sorter. B53. The method according to any one of the preceding embodiments B1 to B52, further comprising introducing a vector encoding a knock-in cassette for inserting the B2M polynucleotide sequence. B54. The method according to embodiment B53, wherein the method further comprises introducing a vector encoding a transposase, recombinase, or integrase for insertion of the knock-in cassette. The method according to embodiment B54, wherein a transposase, recombinase, or integrase is conjugated to a targeting moiety, and the moiety comprises a zinc finger, a transcription activator-like effector, or a nuclease-deficient CRISPR / CAS molecule. The method according to any one of embodiments B53 to B55, wherein the knock-in cassette encodes a therapeutic cargo, a therapeutic protein, a TCR, or a chimeric antigen receptor. Induced pluripotent stem cells (iPSCs) produced according to the method according to any one of embodiments B1 to B56. A population of iPSCs according to embodiment B1, produced according to the method according to any one of embodiments B1 to B57. A composition comprising the iPSCs according to embodiment B57. Differentiated iPSCs produced according to the method according to embodiment B39. B2M-negative cells produced by any one of embodiments B1 to B56. B2M-negative induced pluripotent stem cells for use in the manufacture of a therapeutic composition for a subject in need thereof. B2M-negative induced pluripotent stem cells for use in the treatment of a subject in need thereof. B2M-negative induced pluripotent stem cells for use according to embodiment B58 or embodiment 59, wherein the cells further comprise a therapeutic cargo, a therapeutic protein, a TCR, or a chimeric antigen receptor. The method according to any one of embodiments B1 to B56, wherein the gRNA binds to a complementary sequence. The method according to B61, wherein the complementary sequence comprises any one of SEQ ID NOs: 19 to 24.

[0265] In another set of embodiments (Embodiment Set C), the following are provided: A method of producing induced pluripotent stem cells (iPSCs), comprising: (a) a step for performing a function of enriching and / or activating γδ T cells in a population of isolated cells; (b) A step of implementing a function to reprogram γδ T cells into a pluripotent state, thereby producing iPSCs, (c) A step of contacting iPSCs with an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease and a guide RNA (gRNA), wherein the gRNA binds to a target motif of the beta-2-microglobulin (B2M) polynucleotide sequence in the iPSCs, and the step of contacting results in cleavage of the B2M polynucleotide sequence. C2. Induced pluripotent stem cells (iPSCs) produced according to the method described in Embodiment C1. C3. An isolated population of induced pluripotent stem cells (iPSCs) containing pluripotent cells, wherein the pluripotent cells include means for expressing one or more reprogramming factors and / or the pluripotent cells include means for encoding rearrangement of the TRG gene and the TRD gene, and the pluripotent cells include means for cleaving the B2M gene. C4. An isolated induced pluripotent stem cell (iPSC), wherein the pluripotent cell includes means for expressing one or more reprogramming factors and / or the cell includes means for encoding rearrangement of the TRG and TRD genes, and the cell includes means for cleaving the B2M gene.

Examples

[0266] The following is a description of various methods and materials used in the research. These are described to provide a complete disclosure and explanation of how to make and use the invention to those skilled in the art, and are not intended to limit the scope that the inventors regard as their invention. Also, the following experiments were conducted and are not intended to represent all possible experiments. The exemplary descriptions written in the present tense are not necessarily those that have been carried out. Rather, it should be understood that the descriptions are those that can be carried out to generate data related to the teachings of the present invention. Although efforts have been made to ensure the accuracy of the numerical values used (e.g., amounts, percentages, etc.), some experimental errors and deviations should be taken into account.

[0267] 7.1. Example 1: Generation and Characterization of β2m Knockout in γδ iPSC Cells This example describes the generation of β2m knockout in γδ iPSC cell lines using nucleases. The γδ iPSC cells were generated as disclosed in International Publication No. WO 2021 / 257679 (International Application PCT / US2021 / 037594), which is hereby incorporated by reference in its entirety. In particular, the γδ iPSC cells utilized herein correspond to Clone B described in International Publication No. WO 2021 / 257679.

[0268] Coating of 6-well plates with iMatrix-511 6-well plates were pre-coated with iMatrix-511 laminin. The iMatrix-511 laminin was diluted by adding 5 μL of 0.5 mg / mL iMatrix-511 laminin to 1 mL of DPBS. Then, approximately 1 mL to approximately 1.5 mL of the diluted iMatrix-511 laminin was added to each well of the 6-well plate and cultured at 37 °C and 5% CO2 for 1 hour.

[0269] 7.1.1. Preparation of Reagents The reagents used in this example are provided in Table 2.

[0270] [Table 2]

[0271] Preparation of crRNA First, the vial containing Alt-R crRNA was centrifuged. Subsequently, each Alt-R crRNA was dissolved to 100 μM in nuclease-free IDTE buffer (Table 3).

[0272] [Table 3]

[0273] The vial was vortexed to obtain a homogeneous solution and incubated at room temperature for 15 minutes. The vial was vortexed again and spun down to collect the liquid at the bottom of the vial. Each crRNA was aliquoted in an amount of 5 μL and stored at -20 °C. Each crRNA was not used in more than 3 freeze-thaw cycles.

[0274] Preparation of enhancer To prepare the stock solution provided in Table 4, Alt-R nuclease electroporation enhancer was resuspended to 100 μM in IDTE buffer.

[0275] [Table 4]

[0276] 7.1.2. Transfection Assembly of RNP For each crRNA, the crRNA and nuclease were combined as provided in Table 5. The transfection mixture was incubated at room temperature for 20 - 30 minutes.

[0277] [Table 5]

[0278] Cell recovery The culture medium composition was prepared as shown in Table 6.

[0279]

Table 6

[0280] The cells were harvested when they reached about 70 - 80% confluence. The used culture medium was aspirated from the wells and discarded. Then, the cells were rinsed with 1 mL of DPBS per well, and 1 mL of ReLeSR was added to each well of a 6 - well tissue culture - treated plate. The plate was incubated at 37 °C for 45 - 60 seconds in a 5% CO2 incubator. After incubation, the ReLeSR was aspirated and discarded. The 6 - well tissue culture - treated plate containing the cells was further incubated at 5% CO2, 37 °C for 5 minutes.

[0281] The cells were washed away with approximately 1 mL of fresh StemFit Basic 04 complete growth medium, and the detached cells were collected into a 15 - mL centrifuge tube containing 3 mL of fresh complete growth medium (StemFit Basic 04). The cells were suspended and centrifuged at 800 RPM for 2 minutes at room temperature. After 2 minutes, the supernatant was discarded, and the pellet was resuspended in DPBS.

[0282] Then, the cells were counted using trypan blue and a hemocytometer. The formula for counting cells was: Total number of cells per mL=(Total number of cells counted for 4 squares / 4)×Dilution factor×10 4 cells / mL. Generally, 50,000 cells per tube were collected, pooled together, and centrifuged at 800 RPM for 2 minutes at room temperature. Then, without disturbing the pellet, the supernatant was removed. Then, for one electroporation, the cells were resuspended by adding 10 μL of resuspension buffer T per tube (containing 50,000 cells).

[0283] Approximately 8 μL of the cell suspension was mixed with 2 μL of the electroporation enhancer and 2 μL of the RNP complex. For the non-transfected control, only 2 μL of the enhancer was added to the cell suspension.

[0284] Neon transfection For Neon transfection, the electroporation settings were input into the system as follows: 1400 V, 20 milliseconds, and 1 pulse. The neon pipette station was set by filling the neon tube with 3 mL of the electrolysis buffer (E2) and inserting it into the station. A 10 μL neon tip was inserted into the Neon pipette, and 10 μL of the mixture was pipetted into the Neon tip while avoiding air bubbles. Then, the 10 μL neon tip containing the mixture was inserted into the station, and electroporation was initiated. After electroporation, the cells were transferred to wells containing 2 mL of StemFit Basic 04 complete growth medium containing a ROCK inhibitor (final concentration of 10 μM). The 6-well plates were treated as shown in Table 7.

[0285]

Table 7

[0286] Then, the cells were cultured overnight at 37 °C and 5% CO2. Daily, the cell morphology was observed under a microscope, the used medium was discarded, and the wells were replenished with fresh complete medium. When the cells reached confluence, the cells were harvested using ReLeSR for further downstream applications.

[0287] 7.1.3. Results γδ iPSCs were transfected with a) β2m_nuclease_gRNA1, b) β2m_nuclease_gRNA2, c) β2m_nuclease_gRNA3, d) β2m_nuclease_gRNA4, or e) β2m_nuclease_gRNA5 (Table 8). Table 8 relates to the gRNA sequences and target sequences. The gRNA sequences can be modified to increase the stability of short RNAs (e.g., gRNAs). In some embodiments, the modification is AltR1 and / or AltR2. The AltR1 and / or AltR2 modification is a terminal block, a modification at both ends of the gRNA sequence, or a modification at one end of the gRNA sequence.

[0288] [Table 8]

[0289] On the fifth day after electroporation, the phase-contrast images of the cells were recorded as shown in Figure 1. γδ iPSCs were electroporated with β2m knockout crRNA together with nuclease and imaged at 5-fold and 10-fold magnifications 5 days after electroporation (Figure 1). On the seventh day after electroporation, the cells were confirmed for β2m expression by flow cytometry (Figure 2). The cells were gated based on FSC-H and SSC-H (gate E1). Gate E1 cells were further selected for the live population based on Pacific Blue (live / dead) staining. Then, the live cells were gated for singlets based on FSC-H and FSC-A. The singlets were used to gate the β2m-specific population based on each FMO. Cells transfected with β2m_nuclease_gRNA4 and β2m_nuclease_gRNA5 did not show a significant reduction in β2m expression (Figure 2). However, cells transfected with β2m_nuclease_gRNA1, β2m_nuclease_gRNA2, and β2m_nuclease_gRNA3 showed a significant reduction in β2m expression 7 days after electroporation (Figure 2). Figure 3 shows 5-fold, 10-fold, and 20-fold γδ iPSC images 7 days after FACS sorting. Table 9 shows an overview of the knockout (KO) efficiency observed 7 days after each crRNA.

[0290]

Table 9

[0291] Furthermore, cells were expanded and cell sorting of the double-negative population (β2m, as well as both HLA-A, HLA-B, and HLA-C) from the total electroporation pool was performed on day 14 after electroporation (Figure 4). After FACS sorting, cells were expanded and reconfirmed for both β2m and HLA-A, HLA-B, HLA-C expression by flow cytometry on day 22 after electroporation. Cells were gated based on FSC-H and SSC-H (gate E1). Gate E1 cells were further selected for the viable population based on Pacific Blue (live / dead) staining. Then, live cells were gated for singlets based on FSC-H and FSC-A. Singlets were used to gate β2m and HLA A, HLA-B, HLA-C specific populations based on their respective FMOs. Flow cytometry analysis showed that cells electroporated with crRNA 1, 2, and 3 showed reductions of 93.2%, 96.6%, and 91.1% respectively, in both β2m and HLA-A, HLA-B, HLA-C protein levels (Figure 5).

[0292] This example demonstrates the successful generation of β2m gene knockout clones derived from γδ iPSC cell lines using CRISPR nuclease technology. The data presented herein show that robust knockout efficiency (up to 82.1%) was observed as early as 7 days after electroporation with β2m_nuclease_gRNA2. Three β2m KO clones were generated in γδ iPSCs using CRISPR nuclease technology with KO efficiency exceeding 90% within 22 days after electroporation. The time required for the generation of β2m knockout in γδ iPSCs using CRISPR nuclease technology was approximately 4 weeks.

[0293]

Table 10-1

[0294]

Table 10-2

[0295]

Table 10-3

[0296]

Table 10-4

[0297]

Table 10-5

[0298]

Table 10-6

[0299]

Table 10-7

[0300]

Table 10-8

[0301]

Table 10-9

[0302]

Table 10-10

[0303]

Table 10-11

[0304]

Table 10-12

[0305]

Table 10-13

[0306]

Table 10-14

[0307]

Table 10-15

[0308] 7.2 Example 2 Transgene Insertion in Human γδT Cell-Derived iPSCs A. Preparation of RNP Complex β2m_nuclease_gRNA6Alt-R crRNA (SEQ ID NO: 18) was synthesized (IDT) and dissolved at 200 μM in nuclease-free duplex buffer (IDT #11-05-01-12). The RNP complex was freshly prepared on the day of nucleofection by combining 1 μg of Alt-R® A.s. Cas12a (Cpf1) V3 (IDT #1081068), 200 pmol (1 μl of 200 μ) of crRNA in P3 buffer (Lonza #V4SP-3096), and 110 μg of PGA (Sigma #P4761) were incubated at room temperature for 30 minutes. After incubation, 3 μM of electroporation enhancer (IDT #1076301) was added to bring the total volume to 10 μL.

[0309] B. Generation of Edited Human iPSCs In this specification, human iPS cells derived from γδ T cells, referred to as "iPSC", were pretreated with 10 μM of the Y-27632 ROCK inhibitor (STEMCELL Technologies #72302) in Stemfit Basic 04 complete medium (Ajinomoto #Basic04CT). The iPS cells were collected (0.5e6 cells per reaction) and resuspended in 10 μL of P3 buffer containing 3 μM of an electroporation enhancer. 2 μg of the HDR template (PCR amplicon with GFP) was added to the cells and incubated at room temperature for 1 minute. The cells were combined with 10 μL of the RNP complex and nucleofected in a 96-well cuvette (Lonza #V4SP-3096) using the CA-137 program on the Lonza 4D system. Subsequently, the iPS cells were transferred to one well of a 24-well plate coated with 0.5 μg / cm2 of iMatrix-511 (Takara #T304) containing 0.5 mL of Stemfit Basic 04 medium with 10 μM of the Y-27632 ROCK inhibitor. The iPS cells were grown in a 6-well plate on the second day after nucleofection, and the medium was changed daily until the fifth day when the pluripotency markers, surface expression of HLA class I, and expression of GFP were measured by flow cytometry. As shown in Figure 6, 12.7% of the human iPS cells derived from γδ T cells were GFP-positive and B2M protein-negative after electroporation with the B2M-targeted RNP combined with the DNA plasmid repair template encoding GFP.

[0310] Therefore, targeted insertion of the transgene into B2M in human iPS cells derived from γδ T cells was achieved.

[0311] 7.3 Example 3 Production of iPS Cells Derived from γδ T Cells A. Preparation of the RNP Complex β2m_nuclease_gRNA6 Alt-R crRNA (SEQ ID NO: 18) was synthesized (IDT) and dissolved at 200 μM in nuclease-free duplex buffer (IDT #11-05-01-12). The RNP complex was freshly prepared on the day of nucleofection by combining 1 μg of Alt-R® A.s, Cas12a (Cpf1) V3 (IDT #1081068) or MAD7 (Aldevron Eureca-V MAD7), 200 pmol (1 μl of 200 μM) of crRNA in P3 buffer (Lonza #V4SP-3096), and 110 μg of PGA (Sigma #P4761), and incubated at room temperature for 30 minutes. After incubation, 3 μM of electroporation enhancer (IDT #1076301) was added to bring the total volume to 10 μL.

[0312] B. Generation of Edited Human iPSCs Human iPS cells, referred to herein as “iPSCs,” derived from human γδ T cells were pretreated with 10 μM of Y-27632 ROCK inhibitor (STEMCELL Technologies #72302) in Stemfit Basic 04 complete medium (Ajinomoto #Basic04CT). The iPSCs were collected (0.5e6 cells per reaction) and resuspended in 10 μL of P3 buffer containing 3 μM of electroporation enhancer. The cells were combined with 10 μL of the RNP complex and nucleofected in a 96-well cuvette (Lonza #V4SP-3096) using the CA-137 program on the Lonza 4D system. The iPSCs were then seeded onto a 0.5 μg / cm 2It was transferred to one well of a 24-well plate coated with iMatrix-511 (Takara #T304). The iPSCs were grown in a 6-well plate 2 days after nucleofection, and the medium was changed daily until day 7 when the surface expression of pluripotency markers and HLA class I was measured by flow cytometry. As shown in Figure 7, 83.2% and 87.9% of the human γδT cell-derived iPSC cells were negative for the B2M protein after electroporation with the B2M-targeted RNP generated using Cas12a or MAD7, respectively.

[0313] Therefore, electroporation with the B2M-targeted RNP achieved human γδT cell-derived iPSC cells negative for the B2M protein.

[0314] 7.4 Example 4. Human γδT Cell-Derived iPSCs Having CAR A. Preparation of RNP Complex β2m_nuclease_gRNA6 Alt-R crRNA (SEQ ID NO: 18) (Table 10) was synthesized (IDT) and dissolved at 200 μM in nuclease-free duplex buffer (IDT #11-05-01-12). The RNP complex was freshly prepared on the day of nucleofection by combining 1 μg of Alt-R® A.s. Cas12a (Cpf1) V3 (IDT #1081068) was incubated at room temperature for 30 minutes with the crRNA in 200 pmol (1 μl of 200 μM) of P3 buffer (Lonza #V4SP-3096) and 110 μg of PGA (Sigma #P4761). After incubation, 3 μM of electroporation enhancer (IDT #1076301) was added to make the total volume 10 μL.

[0315] B. Generation of Edited Human iPSCs In this specification, human iPS cells derived from γδT cells, referred to as "iPSC", were pretreated with 10 μM of the Y-27632 ROCK inhibitor (STEMCELL Technologies #72302) in Stemfit Basic 04 complete medium (Ajinomoto #Basic04CT). The iPSCs were collected (0.5e6 cells per reaction) and resuspended in 10 μL of P3 buffer containing 3 μM of an electroporation enhancer. 3.5 μg of an HDR template (DNA plasmid with GFP) was added to the cells and incubated at room temperature for 1 minute. The cells were combined with 10 μL of the Cas12a:crRNA RNP complex and nucleofected in a 96-well cuvette (Lonza #V4SP-3096) using the CA-137 program on the Lonza 4D system. Subsequently, the iPSCs were transferred to one well of a 24-well plate coated with 0.5 μg / cm 2 of iMatrix-511 (Takara #T304) containing 0.5 mL of Stemfit Basic 04 medium with 10 μM of the Y-27632 ROCK inhibitor. The iPSCs were grown in a 6-well plate on the second day after nucleofection, and the medium was changed daily until the fifth day when the expression of pluripotency markers, surface expression of HLA class I, and expression of BCMA CAR were measured by flow cytometry. As shown in Figure 8, 2.6% of the human iPS cells derived from γδT cells were positive for the CAR transgene and negative for the B2M protein after electroporation with the B2M-targeting RNP combined with the DNA plasmid repair template encoding the CAR.

[0316] Therefore, by treating with the B2M-targeting RNP combined with the DNA plasmid repair template encoding the CAR, it was achieved that human iPS cells derived from γδT cells were positive for the CAR transgene and negative for the B2M protein.

Claims

1. A population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are generated by reprogramming γδ T cells, and the population includes iPSCs containing a disrupted beta-2-microglobulin (B2M) gene.

2. (i) The disrupted B2M gene includes a deletion of at least a portion of the nucleotide sequence of SEQ ID NO: 1, or a deletion of about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleotide sequence of SEQ ID NO: 1, or The nucleotide sequence of the B2M gene encodes an amino acid sequence that is approximately, at least approximately, or at most approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of sequence numbers 12 to 16; (ii) About or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the iPSCs do not express B2M at a detectable level; (iii) The disruption comprises the deletion of at least one nucleotide base pair; (iv) The disruption includes the insertion of at least one nucleotide base pair; (v) The disrupted B2M gene exhibits reduced B2M expression compared to an undisrupted B2M gene, and optionally, the reduced B2M expression is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to B2M expression in a reference iPSC, and optionally, the reference iPSC is a population of iPSCs in which the B2M gene is not disrupted; (vi) The iPSC containing the disrupted B2M gene exhibits reduced expression of HLA-A, HLA-B, and / or HLA-C compared to the expression of HLA-A, HLA-B, and / or HLA-C in the reference iPSC, and optionally, the reduced expression of HLA-A, HLA-B, and / or HLA-C is compared to the expression of HLA-A, HLA-B, and / or HLA-C in the reference iPSC. Compared to the expression of, it is reduced by approximately or at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, and optionally, the reference iPSC is a population of iPSCs in which the B2M gene is not disrupted; and / or (vii) The disrupted B2M gene is generated by contacting the iPSC population with an RNA-induced endonuclease or a nucleic acid encoding the RNA-induced endonuclease and a guide RNA (gRNA), wherein the gRNA binds to the target motif of the B2M gene, and optionally the RNA-induced endonuclease binds to MAD7, MAD2, C2c1, C2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, A selection is made from the group consisting of Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, and Cas13c, and further selections are made as needed. (I) The RNA-induced endonuclease is Cas12a(Cpf1); and / or Acidaminococcus BV3L6 Cas12a(Cpf1); or (II) The RNA-induced endonuclease is MAD7, and further optionally, The RNA-inducing endonuclease is MAD7: Furthermore, optionally, the gRNA is selected (III) To bind with at least a portion of the complementary sequence of SEQ ID NO: 1, or to bind with any one of the complementary sequences of SEQ ID NOs: 2-6 or 17; and / or (IV) Contains any one sequence of sequence numbers 7-11 or 18; consists of any one sequence of sequence numbers 7-11 or 18; contains sequence number 18, or consists of sequence number 18 A group of iPSCs as described in claim 1.

3. A method for producing induced pluripotent stem cells (iPSCs), (a) Contacting a population of isolated cells with an activated culture containing IL-15 and zoledronic acid, (b) Culturing the isolated cell population in the activated culture to enrich and / or activate the γδ T cells in the isolated cell population, (c) Transducing the γδ T cells using a viral vector encoding one or more reprogramming factors, (d) Culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells into a pluripotent state, thereby producing a population of iPSCs, (e) Contacting the group of iPSCs with an RNA-induced endonuclease or a nucleic acid encoding the RNA-induced endonuclease, and a guide RNA (gRNA), The gRNA binds to a complementary sequence in the target motif of the beta-2-microglobulin (B2M) polynucleotide sequence in the iPSC population, A method wherein the contact results in the cleavage of the B2M polynucleotide sequence.

4. (i) The RNA-induced endonuclease is MAD7, MAD2, C2c1, C2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr 4. Selected from the group consisting of Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, and Cas13c, and arbitrarily selected, (I) The RNA-inducing endonuclease is Cas12a(Cpf1), and optionally the RNA-inducing endonuclease is Acidaminococcus BV3L6 Cas12a(Cpf1); or (II) The RNA-induced endonuclease is MAD7; (ii)(I) The target motif includes a portion of Sequence ID No. 1; (II) The target motif contains one of the sequences 2-6 or 17; (III) The target motif consists of one of the sequences 2-6 or 17; (IV) gRNA contains one of the sequences 7-11 or 18; (V) gRNA contains SEQ ID NO: 18; (VI)gRNA consists of one of the sequences 7-11 or 18; or (VII) The gRNA consists of sequence number 18; (iii) The cleavage of the B2M polynucleotide sequence results in reduced B2M expression in the iPSC compared to B2M expression in the reference, and optionally, the reduced B2M expression is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to B2M expression in the reference; (iv) The cleavage of the B2M polynucleotide sequence results in reduced HLA-A, HLA-B, and / or HLA-C expression compared to the expression of HLA-A, HLA-B, and / or HLA-C in the reference, and optionally, the reduced HLA-A, HLA-B, and / or HLA-C expression is reduced by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to the expression of HLA-A, HLA-B, and / or HLA-C in the reference; (v) The reference is an iPSC or a group of iPSCs that does not involve cleavage of the B2M polynucleotide sequence; (vi) The activated culture further comprises IL-2; (vii) The viral vector is a Sendai virus (SeV) vector; (viiii) The method further comprises obtaining the population of isolated cells from the subject; (ix) The cells in the aforementioned population of isolated cells are peripheral blood mononuclear cells (PBMCs); (x) The cells in the isolated population of cells are terminally differentiated cells; (xi) The cells in the population of isolated cells are mammalian cells; (xi) The cells in the aforementioned population of isolated cells are human cells; (xiii) The isolated population of cells is cultured in the activated culture for a maximum of 13 days, a maximum of 10 days, a maximum of 9 days, a maximum of 8 days, a maximum of 7 days, a maximum of 6 days, a maximum of 5 days, a maximum of 4 days, a maximum of 3 days, a maximum of 2 days, or a maximum of 1 day; optionally, the isolated population of cells is cultured in the activated culture for a maximum of 3 days; optionally, the isolated population of cells is cultured in the activated culture for a further 3 days; (xiv) After being cultured in the activated culture, the isolated cell population contains less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, or less than 30% γδ T cells, and optionally, after being cultured in the activated culture, the isolated cell population contains less than 35% γδ T cells; (xv) The process further comprises enriching the γδ T cells in the isolated cell population after step (b), wherein the γδ T cells are enriched by intercellular aggregate enrichment; (xvi) At least a portion of the γδ T cells are activated into Vγ9+ γδ T cells in step (b); (xvii) At least a portion of the γδ T cells are activated into Vγ9δ2+ γδ T cells in step (b); (xviiii) The one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28, and c-Myc; (xix) In step (d), the transduced γδ T cells are cultured in the presence of one or more supporting cell layers, and optionally, in step (d), the transduced γδ T cells are cultured in the presence of a single layer of supporting cells; and / or (II) The supporting cell layer comprises mouse embryonic fibroblasts (MEF); and / or (xx) Further comprising isolating and / or purifying the iPSC produced; The method according to claim 3

5. The method according to claim 3 or 4, further comprising differentiating the iPSCs ex vivo into cells of a desired cell type, thereby producing differentiated iPSCs.

6. (i) The produced iPSC is negative for the Sendai virus (SeV) vector; (ii) The iPSC produced is derived from a γδ T cell; (iii) The produced iPSC has rearrangement genes at the TRG and TRD loci, and optionally the produced iPSC has Vγ9 and Vδ2 gene configurations; (iv) The iPSCs produced are not derived from αβ T cells; (v) The produced iPSC does not produce or express TCRA and / or TCRB, or fragments thereof, and as a result there is no detectable or other surface expression of TCRA and TCRB; (vi) The produced iPSC is genomically stable without chromosome loss, and optionally, the genomic stability of the produced iPSC is determined by karyotype analysis; and / or (vii) The iPSCs produced can grow in a culture medium that does not contain supporting cells after acclimatization; The method according to claim 3 or 4.

7. Induced pluripotent stem cells (iPSCs) produced according to the method described in claim 3 or 4.

8. A population of iPSCs according to claim 1, wherein the iPSCs are produced according to the method described in claim 3 or 4.

9. A composition comprising the iPSC described in claim 7.

10. Differentiated IPSCs produced according to the method described in claim 5.

11. A method for producing induced pluripotent stem cells (iPSCs), (a) A step for carrying out the function of enriching and / or activating γδ T cells in a population of isolated cells, (b) A step of performing a function to reprogram the γδ T cells into a pluripotent state, thereby producing iPSCs, (c) The process includes bringing the iPSC into contact with an RNA-induced endonuclease or a nucleic acid encoding the RNA-induced endonuclease, and a guide RNA (gRNA), The gRNA binds to the target motif of the beta-2-microglobulin (B2M) polynucleotide sequence in the iPSC, A method wherein the contact step results in the cleavage of the B2M polynucleotide sequence.

12. Induced pluripotent stem cells (iPSCs) produced according to the method of claim 11.

13. A population of isolated induced pluripotent stem cells (iPSCs) comprising pluripotent cells, wherein the pluripotent cells include means for expressing one or more reprogramming factors, and / or the pluripotent cells include means for encoding the rearrangement of the TRG gene and the TRD gene, and the pluripotent cells include means for cleaving the B2M gene.

14. The population of iPSCs according to claim 1 or 2, wherein the disruption further comprises a knock-in of a polynucleotide encoding a transgene, the polynucleotide being inserted into the B2M gene, and optionally, (i) the transgene encoding a chimeric antigen receptor, a TCR, a therapeutic payload, or a therapeutic protein; or (ii) the polynucleotide being inserted into SEQ ID NO:

1.

15. The method according to claim 3 or 4, further comprising introducing a vector containing a gene encoding a transgene, wherein the gene is incorporated into a target gene, and optionally the target gene contains Sequence ID No. 1.

Images