Substances and methods for bioengineering iPSC populations

Abstract

Provided herein are methods of producing bioengineered pluripotent stem cells (iPSCs) and isolated populations thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 357,922, filed on July 1, 2022, which is incorporated herein by reference in its entirety. 1. Technical Field

[0003] In particular, provided herein are materials and methods for generating bioengineered induced pluripotent stem cells (iPSCs) and uses thereof. 2. Background technology

[0004] Pluripotent stem cells such as embryonic stem cells (ES) and induced pluripotent stem cells (iPSCs) have the proliferation and developmental capacity to differentiate and generate a variety of cell types in vivo. Therefore, the scientific potential of these cells is uncertain, but also extraordinary, especially after it is said that studies have revealed that the gene expression profile in somatic cells can be altered, thereby reprogramming them into pluripotent stem cells in an epigenetic manner (see, e.g., Takahashi, K. and Yamanaka, S, Nat. Rev. Mol. Cell Biol., 2016, 17 (3): 183-93).

[0005] Embryonic stem cells can be derived from the inner cell mass of a mammalian blastocyst, see, e.g., Human Genes and Genomes: Science, Health, Society (Rosenberg, LE and Rosenberg, DD, 1st ed., 2012). In addition, somatic cell nuclear transfer (SCNT)-mediated reprogramming has also been used to generate pluripotent ES cells and, in some cases, cloned animals (Wilmut, I. et al., Nature, 1997, 385: 810-813; Wakayama, T. et al., Nature, 1998, 394: 369-374). However, SCNT is plagued by various technical (e.g., epigenetic) obstacles, because the destruction of the embryo and the introduction of mammalian genetic information into unfertilized eggs are controversial (Matoba, S. and Zhang, Y., supra; Kastenberg, ZJ and Odorico, JS, Transplant Rev., 2008, 22 (3): 215-22).

[0006] Alternative technologies for reprogramming somatic cells into pluripotent stem cells remain of interest. When Yamanaka et al. reported that transcription factors Oct3 / 4, Sox2, Klf4, and c-Myc can confer pluripotency to adult somatic cells and be used to generate induced pluripotent stem cells (iPSCs), iPSC technology emerged as one of such alternatives (Takahashi, K. and 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). 3. Summary of the invention

[0007] In one aspect, provided herein is a population of induced pluripotent stem cells (iPSCs), wherein the iPSCs are optionally generated by reprogramming γδT cells, and the population comprises iPSCs containing a disrupted β-2-microglobulin (B2M) gene. In certain embodiments, the iPSCs are caused by two copies of the B2M gene (B2M - / - ) and was destroyed.

[0008] In some aspects, the nucleotide sequence of the B2M gene encodes the 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 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. In some aspects, the amino acid sequence 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 about or at least about 50 consecutive amino acids of any one of SEQ ID NOs: 12-16. In some aspects, the nucleotide sequence encodes the amino acid sequence of B2M, a portion thereof, or an isoform thereof.

[0009] In some aspects, the disrupted B2M gene comprises a deletion of at least a portion of the nucleotide sequence of SEQ ID NO: 1. In some aspects, 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 aspects, 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.

[0010] In some aspects, the destruction includes the deletion of at least one nucleotide base pair. In some aspects, the destruction includes the insertion of at least one nucleotide base pair. In some aspects, the destroyed B2M gene shows the B2M expression of reduction relative to the undestroyed B2M gene. In some aspects, compared with the B2M expression in reference iPSC, the B2M expression of the reduction 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%.

[0011] In some aspects, compared with HLA-A, HLA-B and / or HLA-C expression in a reference iPSC (e.g., a reference iPSC not comprising a disrupted B2M gene), the iPSC comprising a disrupted B2M gene exhibits reduced HLA-A, HLA-B and / or HLA-C expression. In some aspects, compared with HLA-A, HLA-B and / or HLA-C expression in a reference iPSC, 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%. In some aspects, the reference iPSC is an iPSC population that does not comprise a disrupted B2M gene. In some embodiments, the reference iPSC is an iPSC population in which the B2M gene is not disrupted.

[0012] In some aspects, the disrupted B2M gene is generated by contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), and wherein the gRNA binds to a complementary sequence of a target motif of the B2M gene. In some aspects, the RNA-guided endonuclease is selected from 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, The group consisting of 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 aspects, the endonuclease guided by the RNA is Cas12a (Cpf1). In some aspects, the endonuclease guided by the RNA is Acidaminococcus sp. BV3L6Cas12a (Cpf1). In some aspects, the endonuclease guided by the RNA is MAD7.

[0013] In some aspects, the gRNA binds to at least a portion of the complementary sequence of SEQ ID NO: 1. In some aspects, the gRNA binds to the complementary sequence of any one of SEQ ID NO: 2-6 or 17. In some aspects, the complementary sequence comprises any one of SEQ ID NO: 19-24. In some aspects, the gRNA comprises the sequence of any one of SEQ ID NO: 7-11 or 18. In some aspects, the gRNA comprises SEQ ID NO: 18. In some aspects, the gRNA consists of the sequence of any one of SEQ ID NO: 7-11 or 18. In some aspects, the gRNA consists of SEQ ID NO: 18. In some aspects, the complementary sequence comprises any one of SEQ ID NO: 19-24.

[0014] In one aspect, an induced pluripotent stem cell (iPSC) comprising a disrupted B2M gene (e.g., B2M - / -iPSCs) are iPSCs produced by a method comprising the following steps: (a) contacting an isolated population of cells with an activation culture; wherein the activation culture comprises IL-15 and zoledronic acid; (b) culturing the isolated population of cells in the activation culture to enrich and / or activate γδT cells in the isolated population of cells; (c) transducing γδT cells with a viral vector encoding one or more reprogramming factors; and (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state, thereby producing an iPSC population. In certain embodiments, iPSCs comprising a disrupted B2M gene are produced by the following steps: contacting the iPSC population thus produced 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 β-2-microglobulin (B2M) polynucleotide sequence in the iPSC population; wherein the contact results in cleavage of the B2M polynucleotide sequence, and wherein the B2M gene is disrupted.

[0015] In one aspect, provided herein is a method for producing induced pluripotent stem cells (iPSCs), wherein the method comprises: (a) contacting an isolated population of cells with an activation culture; wherein the activation culture comprises IL-15 and zoledronic acid; (b) culturing the isolated population of cells in the activation culture to enrich and / or activate γδT cells in the isolated population of cells; (c) transducing γδT cells with a viral vector encoding one or more reprogramming factors; (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state, thereby producing an iPSC population; and (e) contacting the iPSC population with an RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease and a guide RNA (gRNA); wherein the gRNA binds to a target motif of a β-2-microglobulin (B2M) polynucleotide sequence in the iPSC population; wherein the contact results in cleavage of the B2M polynucleotide sequence, and wherein the B2M gene is disrupted.

[0016] In some aspects, the RNA-guided endonuclease is selected from 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, The group consisting of 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 aspects, the RNA-guided endonuclease is Acidococcus species BV3L6 Cas12a (Cpf1). In some aspects, the RNA-guided endonuclease is Cas12a (Cpf1). In some aspects, the RNA-guided endonuclease is MAD7.

[0017] In some aspects, the target motif comprises a portion of SEQ ID NO: 1. In some aspects, the target motif comprises the sequence of any one of SEQ ID NO: 2-6 or 17. In some aspects, the target motif consists of the sequence of any one of SEQ ID NO: 2-6 or 17. In some aspects, the gRNA comprises the sequence of any one of SEQ ID NO: 7-11 or 18. In some aspects, the gRNA consists of the sequence of any one of SEQ ID NO: 7-11 or 18. In some aspects, the gRNA comprises SEQ ID NO: 18. In some aspects, the gRNA consists of SEQ ID NO: 18. In some aspects, the gRNA binds to the complementary sequence of any one of SEQ ID NO: 2-6 or 17. In some aspects, the complementary sequence comprises any one of SEQ ID NO: 19-24.

[0018] In some aspects, compared with the B2M expression in a reference, the cutting of the B2M polynucleotide sequence causes the B2M expression in iPSC to decrease. In some aspects, compared with the B2M expression in a reference, the B2M expression of the reduction decreases 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%. In some aspects, compared with the HLA-A, HLA-B and / or HLA-C expression in a reference, the cutting of the B2M polynucleotide sequence causes the expression of HLA-A, HLA-B and / or HLA-C to decrease. In some aspects, 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 HLA-A, HLA-B and / or HLA-C expression in the reference. In some aspects, the reference is an iPSC or an iPSC population that has not cut the B2M polynucleotide sequence.

[0019] In some aspects, the activated culture also includes IL-2. In some aspects, the viral vector is a Sendai virus (SeV) vector. In some aspects, the method also includes obtaining the separation population of the cell from a subject. In some aspects, the cell in the separation population of the cell is a peripheral blood mononuclear cell (PBMC). In some aspects, the cell in the separation population of the cell is a terminally differentiated cell. In some aspects, the cell in the separation population of the cell is a mammalian cell. In some aspects, the cell in the separation population of the cell is a human cell. In some aspects, the separation population of the cell is cultivated 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 aspects, the separation population of the cell is cultivated in the activated culture for up to 3 days. In some aspects, the separation population of the cell is cultivated in the activated culture for 3 days. In some aspects, after culturing in an activated culture, the isolated population of 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% of γδT cells. In some aspects, after culturing in an activated culture, the isolated population of cells comprises less than 35% of γδT cells. In some aspects, the method further comprises enriching the γδT cells in the isolated population of cells after step (b). In some aspects, the γδT cells are enriched by cell-cell clump enrichment. In some aspects, at least a portion of the γδT cells are activated to Vγ9 in step (b). + In some aspects, at least a portion of the γδT cells are activated in step (b) to become Vγ9δ2 + γδ T cells.

[0020] In some aspects, the one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28 and c-Myc. In some aspects, in step (d), the transduced γδT cells are cultured in the presence of one or more layers of feeder layers. In some aspects, in step (d), the transduced γδT cells are cultured in the presence of a monolayer feeder layer. In some aspects, the feeder layer includes mouse embryonic fibroblasts (MEF). In some aspects, the method also includes separating and / or purifying the iPSC produced. In some aspects, the method also includes differentiating iPSC in vitro into cells of desired cell types.

[0021] In some aspects, the iPSC produced is negative for Sendai virus (SeV) vectors. In some aspects, the iPSC produced is derived from γδT cells. In some aspects, the iPSC produced has rearranged genes of TRG and TRD loci; and wherein the iPSC produced optionally has Vγ9 and Vδ2 gene arrangements. In some aspects, the iPSC produced is not derived from αβT cells. In some aspects, the iPSC produced does not produce or express TCRA and / or TCRB or fragments thereof, so that there is no surface expression of TCRA and TCRB that can be detected or otherwise detected.

[0022] In some aspects, the iPSC produced is genome stable, without chromosome loss. In some aspects, the genomic stability of the iPSC produced is determined by karyotyping. In some aspects, the iPSC produced can grow in feeder-free culture medium after adoptive transfer. In some aspects, induced pluripotent stem cells (iPSC) are produced according to these methods. In some aspects, iPSC is produced according to these methods. In some aspects, a composition, the composition includes iPSC. In some aspects, the IPSC of differentiation is produced according to the method.

[0023] In one aspect, the present invention provides a method for producing induced pluripotent stem cells (iPSCs), comprising: (a) performing the function of enriching and / or activating γδT cells in an isolated population of cells; (b) performing the function of reprogramming the γδT cells to a pluripotent state to produce iPSCs; and (c) 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 β-2-microglobulin (B2M) polynucleotide sequence in the iPSCs; and wherein the contacting step results in the cleavage of the B2M polynucleotide sequence. In some aspects, induced pluripotent stem cells (iPSCs) are produced according to the method.

[0024] In one aspect, provided herein is an isolated population of induced pluripotent stem cells (iPSCs), the isolated population of iPSCs comprising pluripotent cells, wherein the pluripotent cells comprise means for expressing one or more reprogramming factors, and / or wherein the pluripotent cells comprise means for rearrangement of genes encoding TRG and TRD, and wherein the pluripotent cells comprise means for cleaving the B2M gene. 4. Description of the drawings

[0025] Figure 1 Representative phase contrast images of γδ iPSC cells 5 days after electroporation are depicted. γδ iPSCs were electroporated with crRNA knocking down β2m as well as nuclease and imaged at 5x and 10x magnification 5 days after electroporation.

[0026] Figure 2 Depicted are flow cytometry graphs showing β2m expression on electroporated γδ iPSCs transfected with different gRNAs compared to untransfected cells. 7 days after electroporation, flow cytometry was used to examine the reduction of β2m expression in bulk transfected cells. Figure 2 Representative FACS plots showing β2m expression in γδ iPSC B2M KO cells are shown. The corresponding percentages for each population can be seen on these plots.

[0027] Figure 3 Depicted are images of γδ iPSCs at 5X, 10X, and 20X magnification 7 days after FACS sorting.

[0028] Figure 4 Depicted is cell sorting of the γδ iPSC double negative population (β2m and HLA-A, HLA-B and HLA-C) from the total electroporated pool 14 days after electroporation.

[0029] Figure 5 Depicted are flow cytometry showing β2m and HLA-A, HLA-B, HLA-C expression on γδ iPSC cells electroporated with gRNAs 1, 2, and 3. 22 days after electroporation, flow cytometry was used to examine the reduction of β2m and HLA-A, HLA-B, and HLA-C expression in bulk transfected cells. Figure 5 Representative FACS plots showing β2m and HLA A, HLA-B, and HLA-C expression in γδ iPSC B2M KO cells compared to wild-type (WT) γδ iPSCs are shown.

[0030] Figure 6 Targeted insertion of a GFP transgene into B2M in human γδ T cell-derived iPSCs is exemplified (see especially Example 2).

[0031] Figure 7 B2M knockout in human γδ T cell-derived iPSCs is exemplified (see especially Example 3).

[0032] Figure 8 Targeted insertion of a CAR transgene into B2M in human γδ T cell-derived iPSCs is exemplified (see especially Example 4). 5. Specific implementation methods

[0033] The present disclosure provides, in part, iPSCs with knockout β2m and methods for producing iPSCs with knockout β2m, which are in turn derived from T cells, particularly from γδT 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 β-2-microglobulin (B2M) gene, and wherein the iPSCs are produced by reprogramming γδT cells.

[0034] γδT cells are a subset of T lymphocytes that express TCRs different from those expressed by αβT cells (the main subset of T lymphocytes in human peripheral blood) (Kalyan, S. and Kabelitz, D., Cell Mol. Immunol., 2013, 10(1): 21-29). Vγ9Vδ2T cells are the main subset of γδT cells and exhibit significant effector functions against tumor cells (Tyler, CJ et al., Cellular Immunology, 2015, 296(1): 10-21; Silva-Santos. B., Nat Rev Immunol., 2015, 15: 683-91).

[0035] 5.1. Definitions

[0036] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, size, size, amount, weight or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference quantity, level, value, number, frequency, percentage, size, size, amount, weight or length. The range of quantity, level, value, number, frequency, percentage, size, size, amount, weight or length can be ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% of a reference quantity, level, value, number, frequency, percentage, size, size, amount, weight or length. The term "about" associated with a reference value can include the value itself and a range of values, such as plus or minus 10% of the value. In some embodiments, the amount "about 10" includes 10 and any amount from 9 to 11. In some cases, a value disclosed throughout may be "about" the value, even though the term "about" is not expressly stated.

[0037] Unless otherwise indicated, the terms "at least," "at most," or "about" preceding a series of elements are to be understood to refer to every element in the series.

[0038] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0039] As used herein, and unless expressly indicated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0040] As used herein, the connection term "and / or" between multiple listed elements is understood to include both single options and combined options. For example, where two elements are connected by "and / or", the first option refers to the application of the first element without the second element. The second option refers to the application of the second element without the first element. The third option refers to the application of the first element and the second element together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" as used herein. The parallel applicability of more than one option is also understood to fall within the meaning and therefore meets the requirements of the term "and / or".

[0041] As used herein, the term "β-2 microglobulin", "B2M" or "β2m" refers to the β chain component of the MHC class I molecule. Human β-2 microglobulin is encoded by the B2M gene (e.g., NCBI Gene ID 567). The expression of β-2 microglobulin is essential for the assembly and function of MHC class I molecules on the cell surface.

[0042] As used herein, the term "MHC class I molecule" refers to the major histocompatibility complex (MHC) found on the cell surface, which displays peptide fragments of non-self proteins. MHC class I molecules are composed of two polypeptide chains. The alpha chain is composed of 3 polypeptides called alpha-1, alpha-2 and alpha-3 domains. The alpha chain is non-covalently linked to the beta chain composed of beta-2 microglobulin (B2M) through the alpha-3 domain. The alpha chain is polymorphic and encoded by 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.

[0043] As used herein, the term "deletion" or "knockout" refers to a genetic modification in which a site or region of genomic DNA is removed by any molecular biological method (such as the method described herein), such as by delivering an endonuclease and at least one gRNA to the site of genomic DNA. The term "deletion" or "knockout" includes the deletion of all or part of a target polynucleotide sequence in a manner that interferes with the function of the target polynucleotide sequence. In some embodiments, "deletion" or "knockout" may result in the complete or partial loss of target gene expression. Any number of nucleotides may be deleted. In some embodiments, deletion involves removal of at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, or more than at least 25 nucleotides. In some embodiments, deletion involves removal of 10 to 50, 25 to 75, 50 to 100, 50 to 200, or more than 100 nucleotides. In some embodiments, deletion involves removal of an entire target gene, such as a B2M gene. In some embodiments, deletion involves removing a portion of the target gene, such as all or part of the promoter and / or coding sequence of the B2M gene. In some embodiments, deletion involves removing the transcriptional regulatory factor of the target gene, such as the promoter region. In some embodiments, deletion involves removing all or part of the coding region, so that the product normally expressed by the coding region is no longer expressed, expressed as a truncated type or expressed at a low level. In some embodiments, relative to unmodified cells, deletion causes a reduction in gene expression. In some embodiments, knockout can be achieved by inducing insertion and deletion in the target polynucleotide sequence in the functional domain (e.g., DNA binding domain) of the target polynucleotide sequence to change the target polynucleotide sequence. The term "destruction" or "destroyed" refers to a change that causes a gene product to not show wild-type function and / or activity level. In some aspects, destruction refers to a change in a gene, which results in the production of such a non-wild-type gene product due to a destroyed gene. In certain embodiments, the destruction causes a gene to be truncated, such as a beta-2 microglobulin (B2M) gene. In certain embodiments, the destruction causes a gene to be deleted, such as a beta-2 microglobulin (B2M) gene. In certain embodiments, the destruction causes the gene to produce an inactive protein. In certain embodiments, the destruction causes the destruction of the reading frame of B2M by multiple out-of-frame deletions. In certain embodiments, the destruction causes the destruction of the reading frame of B2M by a single out-of-frame deletion. In certain embodiments, the destruction causes about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 nucleotides or nucleotide base pairs of insertion (e.g., change the insertion of the reading frame of a gene (e.g., B2M)). In certain embodiments, the destruction causes the destruction of the reading frame of B2M. In a specific embodiment, the gene is a B2M gene, and the destruction causes the B2M gene to produce an inactive B2M protein.In certain embodiments, the destruction causes the gene to express a reduced amount of gene product, such as a reduced amount of B2M polypeptide. In specific embodiments, the gene is a B2M gene, and the destruction causes the B2M gene to express a reduced amount of B2M polypeptide. In certain embodiments, the destruction causes the gene to express an undetectable amount of gene product, such as an undetectable amount of B2M polypeptide. In certain embodiments, the gene is a B2M gene and the destruction causes the B2M gene to express an undetectable amount of B2M polypeptide. A destroyed gene, such as a destroyed B2M gene, may refer to a gene comprising an insertion, deletion or substitution relative to a corresponding wild-type gene, so that relative to the expression of the wild-type gene, the destroyed gene expresses a reduced amount of (e.g., an undetectable amount of) functional protein. A gene may be destroyed, for example, by inserting, deleting or replacing at least one nucleotide / nucleic acid in an endogenous gene, thereby reducing or inhibiting the expression of a functional protein by the endogenous gene. In some embodiments, the terms "destruction," "destroyed," "knockout," or "deletion" are used interchangeably in the present disclosure.

[0044] As used herein, the term "endonuclease" generally refers to an enzyme that cuts the phosphodiester bond in a polynucleotide. In some embodiments, an endonuclease specifically cuts the phosphodiester bond in a DNA polynucleotide. In some embodiments, the endonuclease is a zinc finger nuclease (ZFN), a transcription activator such as an effector nuclease (TALEN), a homing endonuclease (HE), a meganuclease, a MegaTAL or a CRISPR (clustered regularly spaced short palindromic repeats) related endonuclease. The CRISPR cluster contains a spacer, a sequence complementary to a previous mobile element, and a nucleic acid targeted for invasion. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In some embodiments, the endonuclease is an RNA-guided endonuclease. In some aspects, the RNA-guided endonuclease is a CRISPR nuclease, such as a type II CRISPRCas9 endonuclease or a type V CRISPR Cpf1 (or Cas12a) endonuclease. In some aspects, the RNA-guided endonuclease is Acidinococcus species BV3L6 Cas12a (Cpf1). CRISPR-Cas systems can be characterized as Class 1 or Class 2 systems. Class 1 systems are characterized by multi-subunit effectors; that is, comprising multiple Cas proteins. Class 1 systems can be further characterized as Type I, Type III, and Type IV. Class 2 systems are characterized by a single effector protein with multiple domains. Class 1 systems can be further characterized as Type II, Type V, and Type VI. For example, Class 2 Type II systems comprise Cas9, while Class 2 Type V systems comprise Cpf1 (Cas12a). Other examples of Cas proteins include, but are not limited to, Cas9 proteins, Cas9-like proteins encoded by Cas9 orthologs, Cas9-like synthetic proteins, Cpf1 proteins, proteins encoded by Cpf1 orthologs, Cpf1-like synthetic proteins, C2c1 proteins, C2c2 proteins, C2c3 proteins, 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 endonucleases, 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, Cs In some aspects, the RNA-guided endonuclease is Acidococcus species BV3L6 Cas12a (Cpf1). In some embodiments, the endonuclease may introduce one or more single-strand breaks (SSBs) and / or one or more double-strand breaks (DSBs).

[0045] As used herein, the term "Cas12" or "Cas12 protein" refers to any Cas12 protein, including but not limited to Cas12 proteins such as 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%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) identical to the amino acid sequence of a functional Cas12 protein. In some aspects, the RNA-guided endonuclease is Acidococcus species BV3L6 Cas12a (Cpf1). In some embodiments, the Cas12 protein can be a Cas12 polypeptide substantially identical to a protein found in nature, or a Cas12 polypeptide having 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) and having substantially the same biological activity as the Cas12 protein found in nature. 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.

[0046] In some embodiments, the term "Cpf endonuclease" means a DNA endonuclease guided by RNA associated with CRISPR, which cuts a target DNA sequence when coupled to a guide RNA. The Cpf endonuclease is guided by a guide RNA to recognize and cut a specific target site in double-stranded DNA in the cell genome. In some embodiments, the CRISPR-Cpf system uses an aminoacidococcus species Cpf1 endonuclease, a Lachnospiraceae sp. Cpf1 endonuclease, or a Francisella novicide Cpf1 endonuclease, or a variant thereof. The Cpf1-crRNA complex cuts the target DNA by recognizing the protospacer adjacent motif (PAM) 5'-TTTN for aminoacidococcus species Cpf1 endonuclease and Lachnospiraceae species Cpf1 endonuclease and the PAM sequence 5'-TTN for Francisella novicide Cpf1. After recognizing a PAM, Cpf1 introduces a sticky-ended DNA double-strand break with a 4 to 5 nucleotide overhang distal to the 3' end of the targeted PAM, which is then repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR). It should be understood that the term "Cpf1 endonuclease" encompasses variants thereof.

[0047] As known to those of ordinary skill in the art, the term "Mad endonuclease" means a DNA endonuclease guided by RNA associated with CRISPR, which cuts a target DNA sequence when coupled to a guide RNA. Mad endonucleases are guided by guide RNA to recognize and cut specific target sites in double-stranded DNA in the cell genome. The CRISPR-Mad system is closely related to the V-type (Cpf1-like) of the CAS enzyme class 2 family. In some embodiments, the CRISPR-Mad system uses Eubacterium rectale MAD7 endonuclease or a variant thereof. In some embodiments, MAD7 is a class 2 VA CRISPR family identified in Eubacterium rectale. The MAD7-crRNA complex cuts the target DNA by recognizing the protospacer adjacent motif (PAM) 5'-YTTN. After recognizing PAM, MAD7 introduces a sticky-end DNA double-strand break with 4 to 5 nucleotide overhangs to the 3' end of the targeted PAM, which is then repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR). It should be understood that the term "Mad endonuclease" encompasses variants thereof. In some embodiments, the B2M target motif recognized or used by the CRISPR-Cpf1 (Cas12a) system is the same B2M target motif as when MAD7 is used. In some embodiments, the same guide nucleic acid or guide RNA can be used with Cpf1 (or Cas12a) and MAD7 nucleases. In some aspects, the RNA-guided endonuclease is Acidaminococcus species BV3L6 Cas12a (Cpf1).

[0048] As used herein, the term "guide RNA" or "gRNA" generally refers to a short ribonucleic acid that can interact with an endonuclease (e.g., bind to an endonuclease) and bind or hybridize to a target genomic site or region. In some embodiments, the gRNA is a single-molecule guide RNA (sgRNA). In some embodiments, the gRNA may include a spacer extension region. In some embodiments, the gRNA may include a tracrRNA extension region. In some embodiments, the gRNA is single-stranded. In some embodiments, the gRNA includes naturally occurring nucleotides. In some embodiments, the gRNA is a chemically modified gRNA. In some embodiments, the chemically modified gRNA is a gRNA comprising at least one nucleotide with a chemical modification (e.g., a 2'-O-methyl sugar modification). In some embodiments, the chemically modified gRNA includes a modified nucleic acid backbone. In some embodiments, the chemically modified gRNA includes a 2'-O-methyl-phosphorothioate residue. In some embodiments, the gRNA may be pre-complexed with a DNA endonuclease. In some embodiments, the gRNA sequence includes modifications. In some embodiments, these modifications increase the stability of the gRNA. In some embodiments, the gRNA sequence comprises AltR1 and / or AltR2. In some embodiments, AltR1 and AltR2 are proprietary (IDT) modifications for increasing the stability of short RNA (eg, gRNA).

[0049] As used herein, the term "genetic modification" generally refers to genetic editing or manipulation of genomic DNA sites using any molecular biology method (e.g., methods described herein), such as by delivering endonucleases and at least one gRNA to the sites of genomic DNA. Examples of genetic modifications include insertions, deletions, duplications, inversions, and translocations, and combinations thereof. In some embodiments, genetic modification is deletion. In some embodiments, genetic modification is insertion. In other embodiments, genetic modification is insertion-deletion mutation (or indel), which shifts the reading frame of the target gene, thereby causing the gene product to change or have no gene product.

[0050] In some embodiments, "change", "modification" or "at least partial deletion" of a gene (e.g., a B2M gene) results in reduced expression of a target polynucleotide sequence (e.g., compared to a cell or cell population that is not modified, altered, or at least partially deleted by B2M). The terms "reduced", "reduced" and "lower" are all used interchangeably herein, meaning a statistically significant reduction in amount (e.g., two standard deviations (2SD) below normal). In some embodiments, "reduced", "reduced" or "lower" means a reduction of at least about 5% compared to a reference level, for example, a reduction of 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, "reduced", "reduced" or "lower" is any reduction between 10% and 100% compared to a reference level. In some embodiments, "reduced", "reduced" or "lower" means a reduction of at least about 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times compared to a reference level. In some embodiments, the reduced or reduced expression causes the target gene or target polynucleotide sequence in a cell or cell population to be at an undetectable level, as determined by a method used by those skilled in the art or a method disclosed in the present disclosure (e.g., FACS). In some embodiments, the reduced B2M expression is reduced relative to a reference. In some embodiments, the reference is an iPSC or an iPSC population without a B2M polynucleotide sequence cut.

[0051] In some embodiments, the terms "increase", "enhance" and "elevate" are all used interchangeably herein, meaning an increase of at least about 5% compared to a reference level, for example, an increase of 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", "enhance" or "elevate" is any increase between 10% and 100% compared to a reference level. In some embodiments, "increase", "enhance" or "elevate" means an increase of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times compared to a reference level.

[0052] As used herein, the term "polynucleotide", which can be used interchangeably with the term "nucleic acid", generally refers to a biomolecule comprising two or more nucleotides. Generally, the polynucleotides of the present disclosure are composed of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine) naturally present in DNA or RNA joined by phosphodiester bonds. In some embodiments, polynucleotides are hybrid DNA / RNA molecules. In some embodiments, the term encompasses molecules comprising nucleosides or nucleoside analogs, which contain 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 application relates to polynucleotides, it should be understood that both DNA and RNA are provided and that single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided in each case. As used herein, "polynucleotide sequence" can refer to polynucleotide material itself and / or sequence information (i.e., letter sequences used as base abbreviations) that characterize specific nucleic acids in a biochemical manner. Unless otherwise indicated, the polynucleotide sequences provided herein are provided in a 5' to 3' direction. 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 in the cell genome. In some embodiments, the polynucleotide is an exogenous polynucleotide that is not integrated into the genomic DNA. In some embodiments, the polynucleotide is an exogenous polynucleotide that is integrated into the 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.

[0053] As used herein, "cell culture medium" (also referred to herein as "culture medium" or "culture") is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation. Cell culture medium may contain any of the following in any appropriate combination: salts, buffers, amino acids, glucose or other sugars, antibiotics, serum or serum replacements, and other components such as peptide growth factors, etc. Cell culture media commonly used for specific cell types are known to those skilled in the art. Some non-limiting examples are provided herein.

[0054] As used herein, "cell line" refers to a generally or substantially identical cell population that is typically derived from a single progenitor cell or derived from a defined and / or substantially identical progenitor cell population. A cell line may have been or may be able to be maintained in culture for an extended period (e.g., months, years, an indefinite period of time). It may have undergone a spontaneous or induced transformation process, thereby giving the cells an unlimited culture lifespan. Cell lines include all cell lines recognized in the art. It should be understood that cells acquire mutations and possible epigenetic changes over time, such that at least some of the properties of the individual cells of a cell line may differ from one another.

[0055] As used herein, the terms "differentiation" and "differentiation" refer to the process by which unspecialized (or undirected) or less specialized cells acquire the characteristics of specialized cells, such as blood cells or muscle cells. Differentiated or differentiation-induced cells are cells that occupy a more specialized (or directed) position in a cell lineage. When a cell reaches a certain point in the differentiation pathway, it is directed: under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and under normal circumstances cannot differentiate into a different cell type or revert to a poorly differentiated cell type.

[0056] As used herein, the term "encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA) for serving as a template for synthesizing other polymers and macromolecules in a biological process, and these polymers and macromolecules have a limited nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a limited amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene encodes the protein. The coding strands that are identical to the mRNA sequence and are usually provided in the sequence table and the non-coding strands used as templates for transcription of a gene or cDNA can all be referred to as encoding the protein, or other products of the gene or cDNA.

[0057] As used herein, the term "exogenous" is intended to mean that the mentioned molecule or the mentioned activity is introduced into the host cell. For example, the molecule can be introduced by introducing the encoding nucleic acid into the host genetic material, such as by integrating into the host chromosome or as a non-chromosomal genetic material such as a plasmid. Therefore, when used for the expression of encoding nucleic acids, the term refers to the introduction of encoding nucleic acids into cells in an expressible form. The term "endogenous" refers to the mentioned molecule or activity present in the host cell. Similarly, when used to refer to the expression of encoding nucleic acids, the term refers to the expression of encoding nucleic acids contained in the cell rather than exogenous.

[0058] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a stem cell generated from a differentiated adult cell that has been induced or altered (i.e., reprogrammed) to become a cell capable of differentiating into tissues having all three germ layers or dermis: mesoderm, endoderm, and ectoderm.

[0059] As used herein, the term "isolated" and the like, when used to refer to a cell, is intended to mean a cell that is substantially free of at least one component when the cell in question is found in nature. The term includes cells that have been removed from some or all of the components found in its natural environment. The term also includes cells that have been removed from at least one, some or all of the components when the cell is found in a non-naturally occurring environment. Thus, an isolated cell is partially or completely separated from other substances found in nature or grown, stored or present in a non-naturally occurring environment. Specific examples of isolated cells include partially pure cells, substantially pure cells, and cells cultured in a non-naturally occurring culture medium.

[0060] As used herein, the term "purify" and the like refers to increasing purity. For example, the purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (eg, compared to a reference).

[0061] As used herein, the term "pluripotency" refers to the ability of a cell to form all lineages of a body or soma (i.e., the embryo itself). For example, embryonic stem cells are a class of pluripotent stem cells that can form cells from each of the three germ layers, i.e., ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential, ranging from incomplete or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot produce a complete organism to more primitive, more capable cells (e.g., embryonic stem cells) that can produce a complete organism.

[0062] As used herein, the term "population" refers to a group of cells including two or more T lymphocytes when used to refer to T lymphocytes. The separation population of T lymphocytes may have only one type of T lymphocytes, or two or more types of T lymphocytes. The separation population of T lymphocytes may be a homogeneous population of one type of T lymphocytes or a heterogeneous population of two or more types of T lymphocytes. The separation population of T lymphocytes may also be a heterogeneous population of cells (such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc.) with T lymphocytes and at least one other than T lymphocytes. The heterogeneous population may have 0.01% to about 100% of T lymphocytes. Therefore, the separation population of T lymphocytes may have at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of T lymphocytes. The separation population of T lymphocytes may include only one type of T lymphocytes, or a mixture of more than one type of T lymphocytes. The separation population of T lymphocytes may include one or more or all different types of T lymphocytes, including but not limited to those disclosed herein. The separation population of T lymphocytes may include all known types of T lymphocytes. In the separation population of T lymphocytes including more than one type of T lymphocytes, the proportion of each type of T lymphocyte may be in the range of 0.01% to 99.99%. The separation population may also be a clonal population of T lymphocytes, wherein all T lymphocytes of the population are clones of a single T lymphocyte.

[0063] A "recombinant" polynucleotide is one that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context different from that in which it occurs in nature, e.g., separated from nucleotide sequences to which it is normally adjacent in nature, or adjacent (or contiguous) to nucleotide sequences to which it is not normally adjacent. For example, the sequence in question can be cloned into a vector, or recombined with one or more additional nucleic acids.

[0064] As used herein, "reprogramming" refers to the process of changing or reversing the differentiation state of somatic cells. Before reprogramming, cells can be partially differentiated or terminally differentiated. Reprogramming includes that the differentiation state of somatic cells (e.g., T cells) is completely reversed to a pluripotent state. Reprogramming also includes that the differentiation state of somatic cells is partially reversed to make the cells more easily fully reprogrammed to a pluripotent state when performing additional operations such as those described herein. Such contact can cause cells to express specific genes, which contribute to reprogramming. In certain embodiments of the present disclosure, the reprogramming of somatic cells makes somatic cells in a pluripotent and ES-like state. The resulting cells are referred to herein as reprogrammed pluripotent somatic cells or induced pluripotent stem cells (iPSC). In some embodiments, reprogramming also includes that the differentiation state of somatic cells is partially reversed to a pluripotent state.

[0065] Reprogramming is different from simply maintaining an existing undifferentiated state of a cell that is already pluripotent or maintaining an existing incompletely differentiated state of a cell that is already a pluripotent cell (e.g., hematopoietic stem cell). Reprogramming is also different from promoting the 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 can be implemented on fully differentiated cells and / or cells of a particular type (e.g., γδT cells), rather than on cells that are already pluripotent or multipotent.

[0066] As used herein, "reprogramming factor" refers to, for example, a gene, RNA or protein that promotes or contributes to cell reprogramming in vitro. Examples of reprogramming factors of interest for reprogramming somatic cells to pluripotency in vitro are Oct3 / 4, Klf4, c-Myc, Nanog, Sox2 and Lin28, as well as any gene / protein that can replace one or more of these reprogramming factors in, for example, a method for reprogramming somatic cells in vitro.

[0067] As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably, and refer to the main types of leukocytes that complete thymic maturation and have multiple roles in the immune system, including the identification of specific exogenous antigens in vivo and the activation and inactivation of other immune cells. T lymphocytes can be any T lymphocytes, such as cultured T lymphocytes, such as primary T lymphocytes, or T lymphocytes from cultured T cell lines, such as Jurkat, SupT1, etc., or T lymphocytes obtained from mammals. T lymphocytes can be CD3+ cells. T lymphocytes can be any type of T lymphocytes and can be in any developmental stage, 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, initial T cells, regulatory T cells, γδT cells (γδT cells), etc. T lymphocytes can be T regulatory cells, which include nTreg (natural Treg), iTreg (induced Treg), CD8 + Treg, Tr1 regulatory cells and Th3 cells. Other types of helper T cells include cells such as Th3 (Treg), Th17, Th9 or Tfh cells. Other 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 EMRACells). T lymphocytes may 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). T lymphocytes may also be differentiated from stem cells, permanent hemogenic endothelium, CD34+ cells, HSC (hematopoietic stem cells and progenitor cells), hematopoietic multipotent progenitor cells, or T cell progenitor cells.

[0068] As used herein, the term "γδ T cell" refers to a T cell having a T cell receptor comprising a γ chain and a δ chain on its surface.

[0069] As used herein, the term "selective marker" refers to a gene, RNA or protein that confers a selective phenotype to a cell when expressed, such as resistance to cytotoxic agents or cell growth inhibitors (e.g., antibiotic resistance), nutritional prototrophy, or expression of a specific protein that can be used as a basis for distinguishing cells expressing proteins from cells that do not express proteins. Proteins whose expression can be easily detected, such as fluorescent or luminescent proteins or enzymes that act on substrates to produce colored, fluorescent or luminescent substances ("detectable markers"), constitute a subset of selective markers. The presence of a selective marker connected to a natural expression control element of a gene that is typically selectively or specifically expressed in pluripotent cells makes it possible to identify and select somatic cells that have been reprogrammed to a pluripotent state. Can use a variety of selective marker genes, such as neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyl transferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyl transferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyl transferase (HPRT) and hisD gene. Detectable markers include green fluorescent protein (GFP), sapphire, yellow, red, orange and cyan fluorescent protein and any variant of these. Luminescent proteins such as luciferase (for example, firefly or Renilla luciferase) are also useful. As apparent to those skilled in the art, as used herein, the term "selective marker" can refer to the expression product of a gene or this gene, for example, a protein encoded.

[0070] In some embodiments, the selective marker gives the cell expressing it relative to the proliferation and / or survival advantage of the cell not expressing it or expressing it at a significantly lower level.When the cell is maintained under certain conditions, i.e., under "selective conditions", such proliferation and / or survival advantage usually occurs.In order to ensure effective selection, the cell group can be maintained under certain conditions and maintained enough time periods so that the cells not expressing the mark do not proliferate and / or do not survive and are eliminated from the cell group, or their number is reduced to only a very small part of the cell group.The method for selecting the cell expressing the mark by maintaining the cell group under selective conditions to substantially or completely eliminate the cell not expressing the mark that gives proliferation and / or survival advantage is referred to as "positive selection" in this article, and the mark is referred to as "can be used for positive selection".Negative selection and the mark for negative selection are also of interest in some methods described herein.Relative to the cell not expressing the mark or expressing the mark at a significantly lower level, the expression of these marks gives the cell proliferation and / or survival defect of expressing the mark (or, considering in another way, the cell not expressing the mark has proliferation and / or survival advantage relative to the cell expressing the mark). Thus, when maintained under selective conditions for a sufficient period of time, cells expressing the marker can be substantially or completely eliminated from the cell population.

[0071] As used herein, "feeder cells" or "feeder layers" are terms describing a type of cell co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, expand or differentiate, because the feeder cells provide stimulation, growth factors and nutrients for supporting the second cell type. Feeder cells are optionally from species different from the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood derived cells or transformed leukemia cells support the expansion and maturation of natural killer cells. When co-cultured with other cells, feeder cells can usually be inactivated by irradiation or treatment with anti-mitotic agents such as mitomycin to prevent them from growing beyond the cells they support. Feeder cells may include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts) and leukemia cells. Without limiting the foregoing, a specific feeder cell type may be a human feeder layer, such as human skin fibroblasts. Another feeder cell type may be mouse embryonic fibroblasts (MEF). Typically, various feeder cells can be used in part to direct differentiation toward a certain lineage, enhance proliferation capacity, and promote maturation into specialized cell types, such as effector cells.

[0072] As used herein, a "feeder-free" (FF) environment refers to an environment such as culture conditions, cell culture, or culture medium that is substantially free of feeder or stromal cells and / or is not preconditioned by culturing feeder cells. In some embodiments, a feeder-free culture medium comprising a recombinant protein surface is provided. One skilled in the art will appreciate the use of culture media such as iMatrix / laminin, for example, in a feeder-free environment.

[0073] "Preconditioning" culture medium refers to a culture medium harvested after culturing feeder cells for a period of time (such as at least one day) in a culture medium. Preconditioning culture medium contains many medium substances, including growth factors and cytokines secreted by the feeder cells cultivated in the culture medium. In some embodiments, the feeder-free environment does not contain both feeder layers and stromal cells, and is not preconditioned by culturing feeder cells. In some embodiments, additional steps are performed to filter the medium, thereby removing non-human elements.

[0074] The term "pluripotency-related gene" refers to a gene whose expression under normal conditions (e.g., in the absence of genetic engineering or other manipulation designed to change gene expression) occurs in pluripotent stem cells and is generally limited to pluripotent stem cells and is itself essential for its functional identity. It should be understood that the polypeptide encoded by the gene associated with the pluripotency function can be present in the oocyte as a maternal factor. The gene can be expressed by at least some cells of the embryo, such as at least a portion of the entire preimplantation period and / or in the germ cell precursors of the adult.

[0075] The term "pluripotency factor" is used to refer to the expression product of pluripotency-related genes, such as polypeptides encoded by the gene. In some embodiments, pluripotency factors are factors that are generally not expressed in the somatic cell types that constitute the body of adult animals (except germ cells or their precursors). For example, pluripotency factors can be factors whose average level in ES cells is at least 50 times or 100 times higher than the average level in those terminally differentiated cell types present in adult mammals. In some embodiments, pluripotency factors are factors necessary for maintaining the viability or pluripotency of ES cells in vivo and / or ES cells derived using conventional methods. Therefore, if the gene encoding the factor is knocked out or suppressed (that is, its expression is eliminated or significantly reduced), ES cells will not form, die, or in some embodiments will not differentiate. In some embodiments, suppressing the expression of genes whose functions are related to pluripotency in ES cells (causing, for example, the average steady-state level of RNA transcripts and / or proteins encoded by the gene to be reduced by at least 50%, 60%, 70%, 80%, 90%, 95% or more) produces living but no longer pluripotent cells. In some embodiments, the gene is characterized in that its expression in ES cells is reduced (resulting in, for example, a reduction in the average steady-state level of RNA transcripts and / or protein encoded by the gene by at least 50%, 60%, 70%, 80%, 90%, 95% or more) as the cells differentiate into terminally differentiated cells.

[0076] As used herein, "pluripotency inducing gene" refers to a gene whose expression contributes to reprogramming somatic cells to a pluripotent state. "Pluripotency inducing factor" refers to the expression product of a pluripotency inducing gene. A pluripotency inducing factor may be, but need not be, a pluripotency factor. The expression of an exogenously introduced pluripotency inducing factor may be transient, i.e., it may be required during at least part of the reprogramming period in order to induce pluripotency and / or establish a stable pluripotent state, but pluripotency need not be maintained thereafter. For example, the factor may induce the expression of endogenous genes whose functions are associated with pluripotency. These genes may then maintain the reprogrammed cells in a pluripotent state.

[0077] "Polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. Peptides are relatively short polypeptides, typically between about 2 and 60 amino acids in length. The polypeptides used herein typically contain the most common amino acids in proteins, such as 20 L-amino acids. However, other amino acids and / or amino acid analogs known in the art may be used. One or more amino acids in a polypeptide may be modified, for example, by adding chemical entities such as carbohydrate groups, phosphate groups, fatty acid groups, connectors for conjugation, functionalization, etc. A polypeptide having a non-polypeptide portion covalently or non-covalently bound thereto is still considered a "polypeptide". Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, and synthesized by chemical methods such as conventional solid phase peptide synthesis. As used herein, the term "polypeptide sequence" or "amino acid sequence" may refer to the polypeptide material itself and / or the sequence information (i.e., a sequence of letters or three-letter codes used as abbreviations of the amino acid names) that characterizes the polypeptide biochemically. Unless otherwise indicated, the polypeptide sequences provided herein are provided in the direction of N-terminus to C-terminus.

[0078] 5.2. Abbreviations

[0079] A list of abbreviations used in this disclosure is provided in Table 1 below.

[0080] Table 1. Abbreviations

[0081]

[0082]

[0083] Target genes In one aspect, provided herein is a method for changing a target polynucleotide sequence (e.g., B2M) in a cell (e.g., an iPSC derived from a γδT cell (Sections 5.6 and 5.7)), comprising contacting the target polynucleotide sequence with a genome editing method (Section 5.4) of the present disclosure. In some embodiments, iPSCs used in the present disclosure (e.g., knocking out B2M using a gene editing system) and methods for preparing the same are provided in Sections 5.6 and 5.7 of the present disclosure.

[0084] In one aspect, provided herein is a method for changing a target polynucleotide sequence in a cell (e.g., an IPSC derived from a γδT cell) using a CRISPR / Cas system (see Section 5.4). Any CRISPR / Cas system capable of changing a target polynucleotide sequence in a cell can be used in the present disclosure (e.g., changing B2M in an IPSC derived from a γδT cell). In one aspect, provided herein is a method for changing a target polynucleotide sequence in a cell (e.g., an IPSC derived from a γδT cell) using a TALEN system (see Section 5.4). In one aspect, provided herein is a method for causing a zinc finger system (see Section 5.4) to change a target polynucleotide sequence in a cell (e.g., an IPSC derived from a γδT cell). It should be understood that although examples of methods utilizing CRISPR / Cas (e.g., cpf1) are described in detail herein, the present disclosure is not limited to the use of these methods / systems. Other methods of targeting, such as B2M, known to those skilled in the art can be used herein to reduce or eliminate expression in target cells.

[0085] In one aspect, provided herein is a method for altering a target B2M polynucleotide sequence in a cell, comprising contacting at least a portion of a B2M polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein or endonuclease of the present disclosure and at least one ribonucleic acid (e.g., one or two ribonucleic acids), wherein the at least one ribonucleic acid guides the Cas protein or endonuclease to a target motif of the target B2M polynucleotide sequence and hybridizes thereto. In some embodiments, the target B2M polynucleotide sequence is cleaved.

[0086] In some embodiments, changing the target polynucleotide sequence causes the knockout or expression reduction of the target polynucleotide sequence (e.g., B2M) or a portion thereof (e.g., compared to a reference). In some embodiments, the reference is an iPSC or an iPSC colony without cutting the B2M polynucleotide sequence. In some embodiments, changing or knocking out the target polynucleotide sequence is performed in vitro. In some embodiments, changing or knocking out the target polynucleotide sequence is performed in vitro.

[0087] In some embodiments, the target polynucleotide sequence is beta-2-microglobulin (also referred to as B2M or β2m) or a portion thereof. The terms "β-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 a direct homolog 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., mammals, humans, mice, rats, pigs, or any other species).

[0088] In some embodiments, the disruption of B2M or B2M knockout is due to the 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% of the target polynucleotide sequence.

[0089] In some embodiments, the disruption of B2M or B2M knockout is due to 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, the disruption of B2M or B2M knockout is due to 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, wherein such insertion changes the reading frame of B2M. In some embodiments, the disruption of B2M or B2M knockout is due to 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.

[0090] In some embodiments, the present disclosure provides a method for destroying a gene or polynucleotide sequence encoding an amino acid sequence of B2M. In some embodiments, the amino acid sequence of B2M is any one of SEQ ID NO: 12-16. In some embodiments, the amino acid sequence of B2M is any one of UnitProtKB P61769, UnitProtKB F5H6I0, UnitProtKBH0YLF3, UnitProtKB J3KNU0 and / or UnitProtKB Q16446. In some embodiments, the amino acid sequence 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 NO: 12-16. In some embodiments, the amino acid sequence 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 about or at least about 50 consecutive amino acids of any one of SEQ ID NOs: 12-16. 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 at most 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 amino acids (e.g., any one of SEQ ID NOs: 12-16). In some embodiments, the nucleotide sequence encodes an amino acid sequence of B2M, a portion thereof, or an isoform thereof.

[0091] In some embodiments, changing the target polynucleotide sequence includes contacting at least a portion of the polynucleotide sequence (e.g., B2M) with a nucleic acid endonuclease of the present disclosure and at least one ribonucleic acid (e.g., gRNA), wherein the at least one ribonucleic acid guides the nucleic acid endonuclease to the target motif of the target B2M polynucleotide sequence and hybridizes with it. In some embodiments, the target motif is a part of a target polypeptide. In some embodiments, the target motif is a part of the NCBI reference sequence: NG_012920.2. In some embodiments, the target motif is a part of a target polypeptide. In some embodiments, the target motif is a part of B2M. In some embodiments, the target motif is a part of SEQ ID NO:1. In some embodiments, the target motif is about, at least about, or at most 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, a sequence of about 15 NT to about 25 NT, a sequence of about 15 NT to about 30 NT, a sequence of about 15 NT to about 35 NT, a sequence of about 15 NT to about 40 NT, a sequence of about 15 NT to about 45 NT, a sequence of about 15 NT to about 50 NT, a sequence of about 20 NT to about 25 NT, a sequence of about 20 NT to about 30 NT, a sequence of about 20 NT to about 35 NT, a sequence of about 20 NT to about 40 NT, a sequence of about 20 NT to about 45 NT, or a sequence of 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 at most about 20 NT. In some embodiments, the target motif is about 21 NT. In some embodiments, the target motif is about 22 NT. In some embodiments, the target motif is about 23 NT. In some embodiments, the target motif is about 24 NT. In some embodiments, the target motif is about 25 NT. In some embodiments, the target motif is about 26 NT. In some embodiments, the target motif is about 27 NT. In some embodiments, the target motif is about 28 NT. In some embodiments, the target motif is about 29 NT. In some embodiments, the target motif is about 30 NT.

[0092] In some embodiments, the target motif comprises at least one mutation, substitution, addition and / or deletion relative to a portion of a target polypeptide. In some embodiments, the target motif is about, at least about or at most about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a portion of a target polypeptide. In some embodiments, the target motif comprises one or more than one nucleotide modification (e.g., nucleotide substitution, addition, deletion and / or insertion) relative to a portion of a target polypeptide or is composed of it. In some embodiments, the target motif comprises about 1, 2, 3, 4, 5 or more than 5 nucleotide modifications relative to a portion of a target polypeptide or is composed of it.

[0093] In some embodiments, the target motif is selected to minimize off-target effects of CRISPR / Cas systems or any gene or genome editing disclosed herein. It will be appreciated by those skilled in the art that a variety of techniques can be used to select suitable target motifs to minimize off-target effects (e.g., bioinformatics analysis). In some embodiments, the target motif is selected so that when compared to the genomic nucleotide sequence in the cell, it contains at least one mismatch (e.g., at least two, three, four or more mismatches).

[0094] In some embodiments, the target motif comprises 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 comprises 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 comprises 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 at most 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 comprises a sequence comprising at least one, two, three, four, five, or more than five nucleotide mismatches compared to any of SEQ ID NOs: 2-6 or 17. In some embodiments, the complementary sequence comprises any of SEQ ID NOs: 19-24.

[0095] 5.4. Gene or genome editing

[0096] In some embodiments, genome editing methods as described herein, such as CRISPR-nuclease systems, can be used to genetically modify cells as described herein, such as to produce cells that knock out B2M (iPSC cells that knock out B2M derived from γδT cells). In some embodiments, genome editing methods as described herein, such as CRISPR-nuclease systems, are used to genetically modify cells as described herein, such as to introduce at least one genetic modification in or near at least one gene (e.g., B2M).

[0097] In some embodiments, it is known to the technician that any known genome editing method can be used to produce the cell of knocking out B2M disclosed herein.In some embodiments, the genome editing method described herein includes using a site-directed nuclease to cut deoxyribonucleic acid (DNA) at a precise target position in the genome, so as to produce a method for single-stranded or double-stranded DNA breaks at a specific position in the genome.Such breaks can and regularly be repaired by natural, endogenous cell processes, such as homology-directed repair (HDR) and non-homologous end joining (NHEJ).NHEJ directly joins the DNA ends produced by double-strand breaks, sometimes with the loss or addition of nucleotide sequences, which may destroy or enhance gene expression.HDR uses homologous sequences or donor sequences as templates, for inserting a defined DNA sequence at a breakpoint.Homologous sequences can be in endogenous genomes, such as sister chromatids. Alternatively, the donor sequence can be an exogenous polynucleotide, such as a plasmid, a single-stranded oligonucleotide, a double-stranded oligonucleotide, a duplex oligonucleotide, or a virus, which has regions highly homologous to the locus cleaved by the nuclease (e.g., left and right homology arms), but it can also contain additional sequences or sequence changes, including deletions that can be incorporated into the cleaved target locus. A third repair mechanism can be microhomology-mediated end joining (MMEJ), also known as "alternative NHEJ," in which 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 flanking the DNA break site to drive a more favorable DNA end joining repair outcome (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).

[0098] Each of these genome editing mechanisms can be used to produce the desired genetic modification. A step in the genome editing process can be to produce one or two DNA breaks in the target locus near the site of the expected mutation or change, the latter being a double-strand break or two single-strand breaks. This can be achieved by using an endonuclease as described herein.

[0099] In some embodiments, the target gene or target polypeptide (such as B2M) of the present disclosure is destroyed or at least partially missing by the CRISPR-Cas system. In some embodiments, the CRISPR / Cas system for changing the target polynucleotide sequence in the cell includes RNA binding protein, endonuclease or exonuclease, helicase and / or polymerase. In some embodiments, the CRISPR- endonuclease system includes an endonuclease and at least one guide nucleic acid, and the at least one guide nucleic acid guides the DNA cutting of the endonuclease by hybridizing with the recognition site (or target motif of target polynucleotide) in genomic DNA. In some embodiments, the gRNA of the embodiments herein is combined with the complementary strand of the target gene. In some aspects, the complementary sequence includes any one of SEQ ID NO: 19-24. In some embodiments, the CRISPR- endonuclease system includes an endonuclease and at least one ribonucleic acid (for example, guide RNA (gRNA)), and the at least one ribonucleic acid guides the DNA cutting of the endonuclease by hybridizing with the recognition site (or target motif of target polynucleotide) in genomic DNA. It will be appreciated by those skilled in the art that gRNA is guided to the target motif and binds to its complementary sequence in the target motif. In some aspects, the complementary sequence includes any one of SEQ ID NO: 19-24. In some embodiments herein, by contacting the iPSC colony with the RNA-guided endonuclease or the nucleic acid encoding the RNA-guided endonuclease and the guide RNA (gRNA), a destroyed B2M gene is produced, and wherein the gRNA is combined with the target motif of the B2M gene. The target motif includes a double-stranded nucleic acid region, wherein the gRNA is bound to one of the chains, i.e., the complementary chain of 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 a CRISPR-MAD7 system. In some embodiments, the CRISPR system includes a nuclease (e.g., Cas9, Cpf1, or MAD7), and one or two non-coding RNAs - crispr RNA (crRNA) and trans-activating RNA (tracrRNA) to target DNA cleavage.

[0100] 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 guide an endonuclease (Cas protein) to a target motif of a target polynucleotide sequence and hybridize therewith. In some embodiments, at least one ribonucleic acid includes tracrRNA. In some embodiments, at least one ribonucleic acid includes CRISPR RNA (crRNA). In some embodiments, CRISPR RNA (crRNA) is or includes about 17 to 20 nucleotide sequences complementary to a target DNA (target motif of a target polynucleotide). In some embodiments, tracrRNA is used as a binding scaffold for an endonuclease (e.g., Cas9, Cpf1, MAD7, or any other endonuclease disclosed herein). In some embodiments, a single ribonucleic acid includes a guide RNA that guides an endonuclease or Cas protein to a target motif of a target polynucleotide sequence in a cell and hybridizes therewith. In some embodiments, at least one ribonucleic acid in the ribonucleic acid includes a guide RNA that guides an endonuclease or Cas protein to a target motif of a target polynucleotide sequence in a cell and hybridizes therewith. In some embodiments, the one to two ribonucleic acids all include guide RNA, which guides the endonuclease or Cas protein to the target motif of the target polynucleotide sequence in the cell and hybridizes with it. In some embodiments, at least one ribonucleic acid of the present disclosure can be selected to hybridize with a variety of different target motifs (e.g., different target motifs in the target polynucleotide). In some embodiments, at least one ribonucleic acid of the present disclosure can be selected to hybridize with a variety of different target motifs (depending on the specific CRISPR / Cas system adopted) 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 to two ribonucleic acids) can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, when compared with all other genomic nucleotide sequences in the cell, the at least one ribonucleic acid (e.g., one to two ribonucleic acids) hybridizes with a target motif containing at least two mismatches. In some embodiments, when compared with all other genomic nucleotide sequences in the cell, the at least one ribonucleic acid (e.g., one to two ribonucleic acids) hybridizes with a target motif containing at least one mismatch. In some embodiments, the at least one ribonucleic acid (e.g., one to two ribonucleic acids) is designed to hybridize with a target motif next to a deoxyribonucleic acid motif, and the deoxyribonucleic acid motif is recognized by an endonuclease or Cas protein. In some embodiments, the at least one ribonucleic acid (e.g., one to two ribonucleic acids) is designed to hybridize with a target motif next to a deoxyribonucleic acid motif, and the deoxyribonucleic acid motif is recognized by an endonuclease or Cas protein flanking a mutant allele between the target motifs.

[0101] In some embodiments, the 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, the 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, the at least one ribonucleic acid (e.g., guide RNA) is not complementary to and / or hybridizes with a sequence on the opposite strand of a target polynucleotide sequence. In some embodiments, the 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 may bind to a complementary sequence in a target motif. The target motif comprises a double-stranded nucleic acid, wherein the gRNA will bind to a strand in a strand complementary thereto. In some embodiments, the at least one ribonucleic acid (e.g., guide RNA) is complementary to and / or hybridizes with a offset target motif of a target polynucleotide sequence. In some aspects, the complementary sequence comprises any one of SEQ ID NO: 19-24.

[0102] In some embodiments, the CRISPR endonuclease is Cas9 and / or Cpf1 (e.g., Lachnospiraceae bacteria (L.bacterium) ND2006 Cpf1 and / or Acidiococcus species BV3L6 Cpf1 (Example 1, Section 7)), and / or MAD7, and CRISPR / MAD7 is used in various embodiments (especially in Example 1 of the present disclosure, for example, MAD7 in which the same B2M target motif and gRNA sequence (Section 7 of the present disclosure) is used with other V-type CRISPR enzymes). In some embodiments, since MAD7 is a Cas12a-like endonuclease, the B2M target motif and / or guide nucleic acid (e.g., gRNA) used or recognized by Cpf1 or Cas-12a is the same as the B2M target motif and / or guide nucleic acid (e.g., gRNA) used by MAD7. In some embodiments, the B2M target motif recognized or used by the CRISPR-Cpf1 system is the same as the B2M target motif used by the CRISPR-MAD7 system. In some embodiments, the guide nucleic acid (e.g., gRNA) recognized or used by the CRISPR-Cpf1 system is the same as the guide nucleic acid (e.g., gRNA) used by the CRISPR-MAD7 system. In some embodiments, the B2M target motif and guide nucleic acid (e.g., gRNA) recognized or used by the CRISPR-Cpf1 system are the same as the B2M target motif and guide nucleic acid (e.g., gRNA) used by the CRISPR-MAD7 system. In some embodiments, the CRISPR nuclease is MAD7. In some embodiments, the nuclease used in the methods of the present disclosure is MAD7 of Inscripta.TM Nuclease. In some embodiments, the nuclease used in the methods of the present disclosure is a nuclease of Inscripta. In some embodiments, the CRISPR endonuclease is Cas9 (CRISPR-associated protein 9). In some embodiments, the Cas9 endonuclease is from Streptococcus pyogenes. In some embodiments, other Cas9 homologs are used, such as Staphylococcus aureus (S. aureus) Cas9, N. meningitidis (N. meningitidis) Cas9, S. thermophilus (S. thermophilus) CRISPR 1Cas9, S. thermophilus CRISPR 3Cas9 or T. denticola (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, a modified version of an endonuclease (e.g., a homolog thereof, a recombinant of a naturally occurring molecule thereof, a codon-optimized version thereof, or a modified version thereof) may be used. In some embodiments, the endonuclease is any one or more endonucleases disclosed herein. In some embodiments, the endonuclease is any one or more endonucleases known to the technician. In some embodiments, an exogenous Cas protein can be introduced into a cell in the form of a polypeptide. In certain embodiments, the Cas protein can be conjugated or fused to a cell-penetrating polypeptide or a cell-penetrating peptide. As used herein, "cell-penetrating polypeptide" and "cell-penetrating peptide" refer to polypeptides or peptides that promote the uptake of molecules into cells, respectively. In some embodiments, the cell-penetrating polypeptide may contain a detectable label.

[0103] In some embodiments, the endonuclease or Cas protein can be conjugated or fused to a charged protein (e.g., a protein carrying a positive charge, a negative charge, or an overall neutral charge). Such a connection can be covalent. In some embodiments, the endonuclease or Cas protein can be fused to a superpositively charged GFP to significantly improve the ability of the Cas protein to penetrate 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 penetrating peptides. In some embodiments, the endonuclease or Cas protein includes a Cas polypeptide fused to a cell penetrating peptide.

[0104] In some embodiments, the endonuclease is connected to at least one nuclear localization signal (NLS). The at least one NLS may be located within or within 50 amino acids of the amino terminus of the endonuclease, and / or the at least one NLS may be located within or within 50 amino acids of the carboxyl terminus of the endonuclease.

[0105] In some embodiments, the CRISPR-endonuclease system comprises 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 to a wild-type endonuclease (e.g., Cpf1, MAD7, Cas9, and / or any other endonuclease disclosed herein). In some embodiments, the endonuclease is about or at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to 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 consecutive amino acids. In some embodiments, the endonuclease is at most about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to 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 consecutive amino acids. In some embodiments, the endonuclease has 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 consecutive amino acids in the HNH nuclease domain of the endonuclease. In some embodiments, the endonuclease has at most 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 consecutive amino acids in the HNH nuclease domain of the endonuclease. In some embodiments, the endonuclease is at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identical to a wild-type endonuclease (e.g., Cpf1, MAD7, Cas9 and / or any other endonuclease of the disclosure) over about or at least about 10 consecutive amino acids in the RuvC nuclease domain of the endonuclease.In some embodiments, the endonuclease has at most 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 disclosed herein) on about or at least about 10 consecutive amino acids in the RuvC nuclease domain of the endonuclease. The present disclosure provides a guide RNA (gRNA) that can guide the activity of the relevant endonuclease to a specific target site in a polynucleotide. In some embodiments, the guide RNA includes a spacer sequence hybridized with a target nucleic acid sequence of interest, and a CRISPR repeat sequence. In some embodiments, for example, in a CRISPR II type system, the gRNA also includes a second RNA called a tracrRNA sequence. In some embodiments, in a CRISPR II type guide RNA (gRNA), the CRISPR repeat sequence and the tracrRNA sequence hybridize to each other to form a duplex. In some embodiments, in a CRISPR V type system, the gRNA includes a crRNA forming a duplex. In some embodiments, the gRNA can bind to the endonuclease so that the gRNA and the endonuclease form a complex. The gRNA can provide target specificity to the complex through its association with the endonuclease.

[0106] In some embodiments, the tracrRNA sequence comprises nucleotides that hybridize with the CRISPR repeat sequence in the cell. The tracrRNA sequence and the CRISPR repeat sequence can form a duplex, i.e., a double-stranded structure of base pairing. At the same time, the tracrRNA sequence and the CRISPR repeat sequence can bind to the RNA-guided endonuclease. In some embodiments, at least a portion of the tracrRNA sequence can hybridize with 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 about 7 nucleotides to about 100 nucleotides. For example, the tracrRNA sequence can be about 7 nucleotides (NT) to about 50 NT, about 7 NT to about 40 NT, about 7 NT to about 30 NT, about 7 NT to about 25 NT, about 7 NT to about 20 NT, about 7 NT to about 15 NT, about 8 NT to about 40 NT, about 8 NT to about 30 NT, about 8 NT to about 25 NT, about 8 NT to about 20 NT, about 8 NT to about 15 NT, about 15 NT to about 100 NT, about 15 NT to about 80 NT, about 15 NT to about 50 NT, about 15 NT to about 40 NT, about 15 NT to about 30 NT, or about 15 NT to about 25 NT. In some embodiments, the length of the tracrRNA sequence can be about 9 nucleotides. In some embodiments, the tracrRNA sequence can be about 12 nucleotides.

[0107] In some embodiments, the tracrRNA sequence can be at least about 60% identical to a reference tracrRNA (e.g., wild-type, tracrRNA from Streptococcus pyogenes) sequence over a segment of at least 6, 7, or 8 consecutive nucleotides. For example, the tracrRNA sequence can be at least about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 98% identical, about 99% identical, or 100% identical to a reference tracrRNA sequence over a segment of at least 6, 7, or 8 consecutive nucleotides.

[0108] 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 a Cas protein or endonuclease (e.g., Cas9, Cpf1, MAD7, or any endonuclease or Cas protein disclosed herein). In some embodiments, the method includes a technique for introducing nucleic acid into γδiPSC cells. The process of introducing nucleic acid into cells can be achieved 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, neon transfection is used for transfection. In some embodiments, Lonza Nucleofection is used for transfection. In some embodiments, a non-viral system (e.g., neon transfection) is used to introduce nucleic acid into cells. In some embodiments, a viral system (e.g., adeno-associated virus) is used to introduce nucleic acid into cells. In some embodiments, the method includes electroporating γδiPSC cells to introduce genetic material, including, for example, DNA, RNA, and / or mRNA. In some embodiments, the technique for introducing a protein or nucleic acid may include introducing the protein or nucleic acid by: electroporation; microinjection; viral delivery; exosomes; liposomes; gene gun; jet injection; hydrodynamic injection; ultrasound; magnetic field-mediated gene transfer; electric pulse-mediated gene transfer; use of nanoparticles, including, for example, lipid-based nanoparticles; incubation with an endosomal lytic agent; use of cell-penetrating peptides; or any other suitable technique. In some embodiments, the method includes electroporating γδ iPSC cells, including, for example, using a neon transfection system (Thermo Fisher Scientific Inc.).

[0109] 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.

[0110] In some embodiments, the Cas protein or endonuclease is complexed with at least one ribonucleic acid (e.g., one to two 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.

[0111] In some embodiments, the endonuclease and gRNA can be applied to the cell separately. In some embodiments, the endonuclease can be pre-compounded with one or more guide RNAs or one or more crRNAs and tracrRNA. The pre-compounded material can then be applied to the cell. Such pre-compounded materials are referred to as ribonucleoprotein particles (RNPs). The endonuclease in RNP can be, for example, Cpf1 endonuclease, MAD7 endonuclease, Cas9 endonuclease, or any endonuclease disclosed herein. In some aspects, the endonuclease guided by the RNA is Acidococcus species BV3L6 Cas12a (Cpf1). In some embodiments, the endonuclease can be flanked by one or more nuclear localization signals (NLS) at the N-terminus, C-terminus, or both ends of the N-terminus and the C-terminus. In some embodiments, the weight ratio of the genome targeting nucleic acid to the endonuclease in the RNP can be 1: 1, 2: 1, 1: 2 or any suitable ratio.

[0112] In some embodiments, the gRNA can be a double-molecule guide RNA. In some embodiments, the gRNA can be a single-molecule guide RNA (sgRNA).

[0113] In some embodiments, the gRNA comprises a sequence that hybridizes with a sequence in a target polynucleotide. In some embodiments, the nucleotide sequence of the gRNA may vary according to the sequence of the target nucleic acid of interest. In some embodiments, the gRNA comprises a variable length sequence with 17 to 30 nucleotides, wherein at least a portion of the sequence hybridizes with a sequence in a target polynucleotide. In some embodiments, the gRNA sequence may be designed to hybridize with a target polynucleotide at 5' of the PAM of the endonuclease used in the system.

[0114] In some embodiments, the gRNA comprises another portion (e.g., a stability control sequence, an endoribonuclease binding sequence, or a ribozyme). This portion can reduce or increase the stability of the nucleic acid targeting the nucleic acid. In some embodiments, the portion can be a transcription terminator fragment (i.e., a transcription termination sequence. In some embodiments, the portion can function in eukaryotic cells. The portion can function in prokaryotic cells. In some embodiments, the portion can function in both eukaryotic cells and prokaryotic cells. Non-limiting examples of suitable portions include: a 5' cap (e.g., a 7-methylguanylate cap (m7G)), a riboswitch sequence (e.g., allowing for regulation of stability and / or regulation of accessibility of proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.), a modification or sequence that provides tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a portion that facilitates fluorescence detection, a sequence that allows fluorescence detection, etc.), and / or a modification or sequence that provides a protein binding site (e.g., proteins that act on DNA, including transcription activators, transcription repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and the like).

[0115] In some embodiments, the part that gRNA hybridizes with the sequence or target motif in the target polynucleotide is referred to as an intermolecular. In some embodiments, the part (intermolecular) that gRNA hybridizes with the sequence or target motif in the target polynucleotide includes about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more than about 25 nucleotides. In some embodiments, the part that gRNA hybridizes with the sequence or target motif in the target polynucleotide includes less than about 25 nucleotides. In some embodiments, the part that gRNA hybridizes with the sequence or target motif in the target polynucleotide, or gRNA includes more than about 20 nucleotides. In some embodiments, the part that gRNA hybridizes with the sequence or target motif in the target polynucleotide, or gRNA includes 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, the portion of the gRNA that hybridizes to a sequence or target motif in a target polynucleotide, or the gRNA comprises at most 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, the sequence or target motif in the target polynucleotide sequence comprises about, at least about, or at most about 20 bases 5' of the first nucleotide immediately adjacent to the PAM.

[0116] In some embodiments, the portion of the gRNA that hybridizes to a sequence or target motif in a target polynucleotide has a length of at least about 6 nucleotides (NT). In some embodiments, the portion of the gRNA that hybridizes to a sequence or target motif in a target polynucleotide, or the gRNA is about or at least about 6 NT, about or at least about 10 NT, about or at least about 15 NT, about or at least about 18 NT, about or at least about 19 NT, about or at least about 20 NT, about or at least about 21 NT, about or at least about 22 NT, about or at least about 23 NT, about or at least about 24 NT, about or at least about 25 NT, about or at least about 30 NT, about or at least about 35 NT, about or at least about 40 NT, about or at least about 45 NT, about or at least about 50 NT, or more than about 50 NT. In some embodiments, the portion of the gRNA that hybridizes to a sequence or target motif in a target polynucleotide, or the gRNA is about 6 NT to about 40 NT, about 6 NT to about 35 NT, about 6 NT to about 30 NT, about 6 NT to about 29 NT, about 6 NT to about 28 NT, about 6 NT to about 27 NT, about 6 NT to about 26 NT, about 6 NT to about 25 NT, about 6 NT to about 24 NT, about 6 NT to about 23 NT, about 6 NT to about 22 NT, about 6 NT to about 21 NT, about 6 NT to about 20 NT, about 10 NT to about 50 NT, about 10 NT to about 40 NT, about 10 NT to about 35 NT, about 10 NT to about 30 NT, about 10 NT to about 30 NT, about 10 NT to about 29 NT, about 10 NT to about 28 NT, about 10 NT to about 27 NT, about 10 NT to about 26 NT, about 10 NT to about 25 NT, about 10 NT to about 24 NT, about 10 NT to about 23 NT, about 10 NT to about 22 NT, about 10 NT to about 21 NT, about 10 NT to about 20 NT, about 19 NT to about 23 NT, about 19 NT to about 24 NT, about 19 NT to about 25 NT, about 19 NT to about 30 NT, about 19 NT to about 35 NT, about 19 NT to about 40 NT, about 19 NT to about 45 NT, about 19 NT to about 50 NT, about 19 NT to about 60 NT, about 20 NT to about 25 NT, about 20 NT to about 30 NT, about 20 NT to about 35 NT, about 20 NT to about 40 NT, about 20 NT to about 45 NT, about 20 NT to about 50 NT, or about 20 NT to about 60 NT.

[0117] In some embodiments, the percent complementarity between the gRNA or a portion of a gRNA (e.g., a 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 percentage of 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 length of this portion of the gRNA and the target nucleic acid may differ by 1 to 6 nucleotides, which can be considered one or more bulges.

[0118] In some embodiments, the gRNA or a portion of a gRNA (e.g., a 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 a gRNA (e.g., a 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 a gRNA (e.g., a 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 a gRNA (e.g., a 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 a gRNA (e.g., a spacer or crRNA) is about, at least about, or at most 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 a gRNA (e.g., a spacer or crRNA) is about, at least about, or at most 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 a gRNA (e.g., a spacer or crRNA) comprises a sequence comprising at least one, two, three, four, five, or more than five nucleotide mismatches compared to any one of SEQ ID NOs: 7-11 or 18.

[0119] 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 with a chemical modification (e.g., 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 a 2'-O-methyl-thiophosphate residue. In some embodiments, chemical modifications enhance stability, reduce the likelihood or degree of an innate immune response, and / or enhance other attributes, as described in the art.

[0120] In some embodiments, the modified gRNA comprises a modified backbone, such as phosphorothioate, phosphotriester, morpholino, methylphosphonate, short chain alkyl or cycloalkyl intersugar linkages, or short chain heteroatom or heterocyclic intersugar linkages.

[0121] In some embodiments, the modified gRNA comprises one or more substituted sugar moieties, such as one of the following at position 2': OH, SH, SCH3, F, OCN, OCH3, OCH3 O(CH2)n CH3, O(CH2)n NH2, or O(CH2)nCH3, wherein n is 1 to about 10; C1 to 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;

[0122] SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkylaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; intercalator; 2'-O-(2-methoxyethyl); 2'-methoxy (2'-O-CH3); 2'-propoxy (2'-OCH2 CH2CH3); and 2'-fluoro (2'-F). Similar modifications can 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 and the internucleoside bond (i.e., the backbone) of the nucleotide unit can be replaced by different groups.

[0123] In some embodiments, gRNA includes additional or alternative nucleobase (or "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases that are only occasionally or transiently found in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidine, 5-methylcytosine (also known as 5-methyl-2'deoxycytosine or 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentiobiosyl HMC, as well as synthetic nucleobases such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine, and 2,6-diaminopurine.

[0124] In some embodiments, the modified nucleobases may include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil and cytosine. Pyrimidine, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo (specifically 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.

[0125] In some embodiments, the method of modifying genes or genome editing disclosed herein can be performed using zinc finger nucleases (ZFNs). Zinc finger nucleases (ZFNs) are modular proteins composed of engineered zinc finger DNA binding domains connected to the catalytic domain of type II endonuclease FokI. Because FokI functions as a dimer, a pair of ZFNs are engineered to bind to homologous target "half-site" sequences on opposite DNA strands, and there is a precise interval between them so that catalytically active FokI dimers can be formed. After dimerization of the FokI domain, a DNA double-strand break is generated between the ZFN half-sites, which serves as the starting step for genome editing.

[0126] In some embodiments, the DNA binding domain of each ZFN consists of 3 to 6 zinc fingers with a rich Cys2-His2 architecture, wherein each finger primarily recognizes a nucleotide triplet on one strand of the target DNA sequence, although cross-strand interactions with the fourth nucleotide may also occur. Changing the amino acid of the finger at the position of the key contact with the DNA will change the sequence specificity of a given finger. Therefore, a four-finger zinc finger protein will selectively recognize a 12 bp target sequence, wherein the target sequence is a combination of triplet preferences contributed by each finger, although the triplet preference may be affected to varying degrees by adjacent fingers. By simply modifying a single finger, ZFN can easily retarget almost any genomic address. In some embodiments, proteins with 4 to 6 fingers are used, each recognizing 12 to 18 bp. Therefore, a pair of ZFNs will generally recognize a combined target sequence of 24 to 36 bp, excluding the typical 5 to 7 bp spacers between half sites. These binding sites can be further separated by larger spacers (comprising 15 to 17 bp).

[0127] A variety of ZFN-based systems have been described in the art, modifications of which are frequently reported, and many references describe rules and parameters for guiding ZFN design; see, for example, Segal et al., Proc Natl Acad Sci, 199996(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.

[0128] In some embodiments, the method of modifying genes or genome editing disclosed herein can be performed using transcription activator-like effector nucleases (TALENs). TALEN represents another form of modular nuclease, in which, like ZFN, the engineered DNA binding domain is connected to the FokI nuclease domain, and a pair of TALENs are operated in series to achieve targeted DNA cutting. The main difference from ZFN is the nature of the DNA binding domain and the associated target DNA sequence recognition characteristics. The TALEN DNA binding domain is derived from TALE protein, which was originally described in plant bacterial pathogens Xanthomonas species (Xanthomonas sp.). TALE consists of a tandem array of 33 to 35 amino acid repeats, wherein each repeat recognizes a single base pair in a target DNA sequence, and the length of the target DNA sequence is generally up to 20 bp, thereby obtaining a total target sequence of up to 40 bp in length. The nucleotide specificity of each repeat is determined by a repeat variable di-residue (RVD), which includes only two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine and thymine are mainly recognized by four RVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly. A variety of TALEN-based systems have been described in the art, and modifications thereof are frequently reported; see, for example, Boch, Science, 2009 326(5959):1509-12; Mak et al., Science, 2012, 335(6069):716-9; and Moscou et al., Science, 2009, 326(5959):1501. The use of TALENs based on the "Golden Gate" platform or cloning scheme has been described by 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.

[0129] In some embodiments, the method of modifying gene or genome editing disclosed herein can be performed using homing endonucleases (HE). Homing endonucleases (HE) are sequence-specific endonucleases that have longer recognition sequences (14 to 44 base pairs) and typically cut DNA with high specificity at unique sites in the genome. According to the structural classification of HE families, there are at least six known HE families, including GIY-YIG, His-Cis box, HNH, PD-(D / E)xK, and Vsr-like, which are derived from a wide range of hosts, including eukaryotes, protists, bacteria, archaea, cyanobacteria, and bacteriophages. Like ZFN and TALEN, HE can be used to generate DSB at the target locus, which serves as the starting step for genome editing. In addition, some natural and engineered HEs only cut single strands of DNA, thereby acting in the form of site-specific nickases. A variety of HE-based systems have been described in the art, and modifications thereof are frequently reported; see, for example, the following reviews: 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.

[0130] In some embodiments, the methods of modifying genes or genome editing disclosed herein can be performed using the MegaTAL or Tev-mTALEN platforms. The MegaTAL and Tev-mTALEN platforms use a fusion of a TALE DNA binding domain with a catalytically active HE, utilizing both the tunable DNA binding and specificity of TALE and the cleavage sequence specificity of HE; see, e.g., Boissel et al., Nucleic Acids Res., 2014, 42: 2591-2601; Kleinstiver et al., G3, 2014, 4: 1155-65; and Boissel and Scharenberg, Methods Mol. Biol., 2015, 1239: 171-96.

[0131] In some embodiments, the MegaTev architecture is a fusion of a meganuclease (Mega) with a nuclease domain derived from the GIY-YIG homing endonuclease I-Teel (Tev). The two active sites are separated by about 30 bp on the DNA substrate and produce two DSBs with incompatible sticky ends; see, e.g., Wolfs et al., Nucleic Acids Res., 2014, 42, 8816-29. It is expected that other combinations of existing nuclease-based methods will continue to develop and can be used to achieve targeted genome modifications as described herein.

[0132] 5.5. iPSCs derived from γδT cells with B2M knockout

[0133] iPSC derived from γδT cells express HLA class I, which is one of the most polymorphic human antigens, and is therefore often recognized by allogeneic T cells, resulting in allogeneic rejection. Therefore, such iPSCs (and immunotherapeutics based on them) have limited applicability in clinical settings without additional intervention (such as host immune system suppression / exhaustion). B2M is necessary for the surface expression of HLA-I molecules. Therefore, destroying B2M can lead to reduction / elimination of the surface expression of HLA-I molecules. The resulting iPSCs are therefore improved because they are more suitable for use in allogeneic environments (e.g., developing so-called "off-the-shelf" therapies).

[0134] In some embodiments, the genetically modified cells of the present disclosure (e.g., knockout B2M derived from γδT cells and / or knockout HLA-A, HLA-B and / or HLA-C iPSC) include at least one genetic modification introduced within or near at least one gene (e.g., B2M), the at least one genetic modification causing B2M and / or one or more MHC-I human leukocyte antigens (e.g., HLA-A, HLA-B and / or HLA-C) expression to be reduced or altered relative to unmodified cells. In some embodiments, compared to unmodified cells or unmodified cell populations (e.g., compared to iPSCs derived from γδT cells and not comprising B2M knockout and / or HLA-A, HLA-B and / or HLA-C knockout), the genetically modified cells of the present disclosure or their populations have reduced immunogenicity or reduced immune responses. In some embodiments, compared with unmodified cell colonies, the genetically modified cell colonies disclosed herein have a reduction 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%, 100% or more than 100% (lower) immunogenicity or immune response. In some embodiments, the immunogenicity of cells is assessed using any suitable method known to technicians. In some embodiments, cells are analyzed to detect the presence of antibodies on the cell surface, for example, by staining with anti-IgM antibodies. In some embodiments, immunogenicity is assessed by PBMC cell lysis assays. In some embodiments, cell colonies are incubated with peripheral blood mononuclear cells (PBMCs), and then PBMCs are assessed for cell lysis. In some embodiments, immunogenicity is assessed by natural killer (NK) cell lysis assays. In some embodiments, cell colonies are incubated with NK cells, and then the cleavage of NK cells to cells is assessed. In some embodiments, immunogenicity is assessed by CD8+ T cell lysis assay. In some embodiments, cell colonies are incubated with CD8+ T cells, and then the cleavage of CD8+ T cells to cells is assessed. In some embodiments, compared with unmodified cells or unmodified cell colonies (for example, compared with iPSC derived from γδT cells and not comprising B2M knockouts and / or HLA-A, HLA-B and / or HLA-C knockouts), the genetically modified cells of the present disclosure or their colonies have increased viability or increased survival rate. In some embodiments, the genetically modified cell population of the present disclosure has an increase in viability or survival 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%, 100%, or more than 100% (higher) compared to an unmodified cell population.In some embodiments, the increased viability or increased survival of cells is assessed using any suitable method known to those skilled in the art. In some embodiments, cell viability or survival is determined using flow cytometry, high content imaging, tetrazolium reduction (MTT) assay, resazurin reduction assay, protease activity marker assay, and / or ATP detection assay.

[0135] In some embodiments, the genetically modified cells of the present disclosure include introducing at least one genetic modification in or near at least one gene, which reduces the expression of one or more MHC-I human leukocyte antigens relative 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 in or near at least one gene, which changes the expression of one or more MHC-I human leukocyte antigens relative to unmodified cells.

[0136] In some embodiments, the genome of the modified cell is to reduce the expression of beta-2-microglobulin (B2M). The HLA-I protein is closely related to the B2M in the endoplasmic reticulum, which is necessary for the formation of functional, cell surface expressed HLA-I molecules. In some embodiments, the gRNA targets sites within the B2M gene, as described in sections 5.3 and 5.4. In some embodiments, B2M expression is not detected in the genetically modified cell population disclosed herein (e.g., not detected by conventional methods (e.g., FACS)). In some embodiments, compared with an unmodified cell population, the genetically modified cell population disclosed herein has a completely or at least partially reduced B2M expression. In some embodiments, B2M expression in a genetically modified cell population 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 B2M expression in an unmodified cell population.

[0137] In some embodiments, the genetically modified cells of the present disclosure do not have detectable levels of B2M. In some embodiments, the genetically modified cells do not have detectable RNA transcripts encoding B2M. In some embodiments, the genetically modified cells do not have detectable levels of B2M protein. In some embodiments, when analyzed by flow cytometry, iPSCs do not express detectable levels of B2M protein.

[0138] In some embodiments, the genetically modified cells of the present disclosure include genomic modifications of one or more MHC-I genes. In some embodiments, the genetically modified cells of the present disclosure include genomic modifications of one or more polynucleotide sequences that regulate MHC-I expression. 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. 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 as cytolytic CD8+ T cells. MHC-I proteins are closely related to beta-2-microglobulin (B2M) in the endoplasmic reticulum, which is necessary for the formation of functional MHC-I molecules on the cell surface.

[0139] In some embodiments, the expression of one or more MHC-I human leukocyte antigens is reduced relative to unmodified cells by directly targeting (e.g., genetic deletion) at least one base pair in the MHC-I gene to achieve. In some embodiments, the expression of one or more MHC-I human leukocyte antigens is reduced relative to unmodified cells by targeting (e.g., genetic deletion) B2M genes to achieve. In some embodiments, the expression of one or more MHC-I human leukocyte antigens is reduced relative to unmodified cells by targeting (e.g., genetic deletion) at least one transcriptional regulatory factor of MHC-I to achieve. In some embodiments, the genome of the modified cell is to delete all or part of the promoter region of HLA-A, HLA-B and / or HLA-C genes. In some embodiments, the genome of the modified cell is to delete all or part of the gene encoding the MHC-I transcriptional regulatory factor. In some embodiments, the genome of the modified cell is to delete all or part of the promoter region of the gene encoding the MHC-I transcriptional regulatory factor.

[0140] In some embodiments, HLA-A, HLA-B and / or HLA-C expression is not detected in the genetically modified cell population of the present disclosure (e.g., not detected by conventional methods (e.g., FACS)). In some embodiments, the genetically modified cell population of the present disclosure has completely or at least partially reduced HLA-A, HLA-B and / or HLA-C expression compared to an unmodified cell population. In some embodiments, the HLA-A expression in the genetically modified cell population 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 HLA-A expression in the unmodified cell population. In some embodiments, HLA-B expression in the genetically modified cell population 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 HLA-B expression in the unmodified cell population. In some embodiments, HLA-C expression in the genetically modified cell population 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 HLA-C expression in the unmodified cell population. In some embodiments, the expression of B2M, HLA-A, HLA-B and / or HLA-C in the genetically modified cell population 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 / or HLA-C in the unmodified cell population. In some embodiments, the expression of B2M, HLA-A, HLA-B and HLA-C in the genetically modified cell population 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 the unmodified cell population. In some embodiments, the expression of B2M, HLA-A, HLA-B and HLA-C is detected by FACS.

[0141] 5.6. Methods for generating induced pluripotent stem cells (iPSCs)

[0142] In some embodiments, the iPSCs and methods for preparing the same are disclosed in WO2021 / 257679 (PCT / US2021 / 037594), which is incorporated herein by reference in its entirety. In one aspect, provided herein is a method for reprogramming somatic cells (e.g., T cells) to a poorly differentiated state. The resulting cells are referred to herein as reprogrammed somatic cells. Reprogrammed somatic cells can be reprogrammed somatic cells in different differentiation states. In some embodiments, the reprogrammed somatic cells are induced pluripotent stem cells (iPSCs). The present disclosure is based in part on the surprising discovery that a combination of multiple factors, such as a combination of zoledronic acid and interleukin-15 (IL-15), can activate γδT cells, and thus improve the efficiency of inducing pluripotency in non-pluripotent mammalian T cells transformed with transcription factors. Therefore, in one aspect, the present disclosure provides methods for reprogramming γδT cells (e.g., Vγ9) in non-pluripotent mammalian γδT cells. + A method for inducing pluripotency in γδ T cells) comprising contacting peripheral blood mononuclear cells (PBMCs) with an activation culture medium comprising IL-15 and zoledronic acid.

[0143] In some embodiments, the iPSC disclosed herein and its preparation method are disclosed in WO2021 / 176373 (PCT / IB2021 / 051779), which is incorporated herein by reference in its entirety. In some embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising: (a) contacting a separate population of cells with an activated culture; wherein the activated culture comprises IL-15 and zoledronic acid; (b) culturing a separate population of cells in the activated culture to enrich and / or activate γδT cells in the separate population of cells; (c) transducing γδT cells with a viral vector encoding one or more reprogramming factors; (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state, thereby producing an iPSC population; (e) contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA). In some embodiments, the gRNA binds to a target motif of a beta-2-microglobulin (B2M) polynucleotide sequence in the iPSC population. In some embodiments, the contact results in cleavage of the B2M polynucleotide sequence.

[0144] In some embodiments, provided herein is a method for reprogramming somatic cells, comprising: (a) contacting an isolated population of cells with an activation culture; wherein the activation culture comprises IL-15 and zoledronic acid; (b) culturing an isolated population of cells in the activation culture to enrich and / or activate γδT cells in an isolated population of cells; (c) transducing γδT cells with one or more viral vectors encoding one or more reprogramming factors; and (d) culturing transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a poorly differentiated state. In some embodiments, the poorly differentiated state is a pluripotent state. In some embodiments, the poorly differentiated state is a pluripotent state.

[0145] In some more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising: (a) contacting an isolated population of cells with an activation culture; wherein the activation culture comprises IL-15 and zoledronic acid; (b) culturing the isolated population of cells in the activation culture to enrich and / or activate γδT cells in the isolated population of cells; (c) transducing γδT cells with one or more viral vectors encoding one or more reprogramming factors; and (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0146] In certain embodiments, the activated culture further comprises one or more additional agents or compounds, for example to increase the efficiency of activation or induction. In one embodiment, the activated culture further comprises interleukin-2 (IL-2).

[0147] Thus, in some embodiments, provided herein is a method for reprogramming somatic cells, comprising: (a) contacting an isolated population of cells with an activation culture; wherein the activation culture comprises IL-15, zoledronic acid, and IL-2; (b) culturing the isolated population of cells in the activation culture to enrich for and / or activate γδT cells in the isolated population of cells; (c) transducing γδT cells with one or more viral vectors encoding one or more reprogramming factors; and (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a poorly differentiated state.

[0148] In some more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising: (a) contacting an isolated population of cells with an activation culture; wherein the activation culture comprises IL-15, zoledronic acid, and IL-2; (b) culturing the isolated population of cells in the activation culture to enrich and / or activate γδT cells in the isolated population of cells; (c) transducing γδT cells with one or more viral vectors encoding one or more reprogramming factors; and (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0149] In some embodiments, the method also includes delivering a donor template comprising a knock-in box, which includes a cargo sequence for inserting into the target gene. In some embodiments, an endonuclease is introduced to edit the knock-in gene and insert it into the B2M gene. In some embodiments, the endonuclease includes a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) or an endonuclease of a CRISPR / CAS system.

[0150] In some embodiments, a transposase, a recombinase or an integrase is introduced to insert the knock-in gene into the B2M gene. In some embodiments, a transposase, a recombinase or an integrase is conjugated to a targeting moiety (such as a zinc finger, a transcription activator-like effector (TALE) or a nuclease-deficient CRISPR / CAS molecule). In some embodiments, the introduction of the construct encoding the goods includes the introduction of a vector DNA and a transposase. In some embodiments, zinc finger nucleases are used as gene editors in any of the embodiments described herein. Specific embodiments can use transcription activators such as nucleases (TALEN) as gene editors. In some embodiments, targeted genetic engineering methods, such as CRISPR nuclease systems, can be used.

[0151] Those skilled in the art will appreciate that the transposon system can be used to insert a cassette encoding a cargo into the B2M gene. In some embodiments, the Sleeping Beauty transposon system is used to insert an expression cassette into the B2M, wherein the expression cassette encodes a transgene. In some embodiments, the MOS1 transposase system is used to insert an expression cassette into the B2M gene, wherein the expression cassette encodes a transgene. In some embodiments herein, the PiggyBac transposase is used to insert an expression cassette into the B2M gene, wherein the expression cassette encodes a transgene. In some embodiments, the ISY100 transposase is used to insert an expression cassette into the B2M gene, wherein the expression cassette encodes a transgene. In some embodiments, the expression cassette further comprises a transcriptional regulatory element. In some embodiments, the transposase described herein further comprises a fusion protein comprising a nuclease-inactivated Cas protein, a TALE, or a zinc finger protein. In some embodiments, the transgene encodes a therapeutic cargo, a protein, a TCR, or a chimeric antigen receptor.

[0152] In some embodiments, the method further includes generating DSB in the B2M gene, and inserting a nucleic acid construct in or near the target gene. In some embodiments, the double-strand break is performed by an endonuclease, and the polynucleotide is inserted by HDR. In some embodiments, the method further includes providing a nucleic acid for insertion into the target gene. In some embodiments, the nucleic acid is a carrier encoding goods. In some embodiments, the goods include therapeutic proteins. In some embodiments, the nucleic acid includes genes encoding therapeutic proteins. In some embodiments, the nucleic acid includes genes encoding TCR. In some embodiments, the nucleic acid encodes a CAR construct (CAR) for insertion into the target gene. In some embodiments, the target is in the B2M gene. In some embodiments, the target is in SEQ ID NO: 1. In some embodiments, CAR includes an extracellular domain containing an antigen recognition region, a transmembrane domain, and an intracellular domain containing at least one costimulatory domain. In some embodiments, the nucleic acid also includes a promoter, at least one gene regulatory element, or a combination thereof.

[0153] In certain embodiments, the method further comprises obtaining an isolated population of the cells from a subject. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0154] In certain embodiments, the isolated population of cells is peripheral blood cells, umbilical cord blood cells or bone marrow cells. In one embodiment, the isolated population of cells is peripheral blood mononuclear cells (PBMC).

[0155] Methods for identifying reprogrammed mammalian somatic cells with 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 expressing appropriate selective markers. In some embodiments, the pluripotency characteristics of reprogrammed somatic cells are further assessed. The presence of pluripotency characteristics indicates that the somatic cell has been reprogrammed to a pluripotent state.

[0156] The differentiation state of a cell is a continuous spectrum, at one end of the spectrum is a terminal differentiation state, at the other end is a dedifferentiation state (pluripotent state). As used herein, reprogramming refers to the process of changing or reversing the differentiation state of a somatic cell, and the somatic cell can be partially differentiated or terminally differentiated. Reprogramming includes complete reversal and partial reversal of the differentiation state of a somatic cell. In other words, as used herein, the term "reprogramming" includes any movement of the differentiation state of a cell along a spectrum to a low differentiation state. For example, reprogramming includes reversing a pluripotent cell back to a pluripotent cell, and reversing a terminally differentiated cell back to a pluripotent cell or a pluripotent cell. In one embodiment, the reprogramming of a somatic cell causes the somatic cell to be transformed back to a pluripotent state. In another embodiment, the reprogramming of a somatic cell causes the somatic cell to be transformed back to a pluripotent state. Therefore, as used herein, the term "low differentiation state" is a relative term, including a complete dedifferentiation state and a partial differentiation state.

[0157] The term "pluripotency characteristics" refers to a number of features associated with pluripotency, including, for example, the ability to differentiate into all types of cells and the unique expression patterns of pluripotent cells, including expression of pluripotency genes, expression of other ES cell markers, and a unique expression profile at the global level known as the "stem cell molecular signature" or "stemness."

[0158] The ES cell of the present invention can be used for the pluripotency of the present invention.Therefore, in order to assess the pluripotency characteristics of reprogrammed somatic cells, the different growth characteristics and ES cell-like morphology of these cells can be analyzed.In some embodiments, the cell can be injected subcutaneously into the SCID mouse of immune incompetence to induce teratoma (standard determination of ES cells).ES-like cells can be differentiated into embryoid bodies (another ES-specific characteristics).In addition, ES-like cells can be differentiated in vitro by adding some known growth factors that drive differentiation into specific cell types.The self-renewal ability marked by inducing telomerase activity is another pluripotency characteristic that can be monitored.

[0159] In some embodiments, functional assays of reprogrammed somatic cells can be performed by introducing them into blastocysts to determine whether the cells are able to generate all cell types. If the reprogrammed cells are able to form several cell types of the body, they are multipotent; if the reprogrammed cells are able to form all cell types of the body, including germ cells, they are pluripotent.

[0160] In other embodiments, the expression of individual pluripotency genes in reprogrammed somatic cells can be examined to assess their pluripotency characteristics.

[0161] In addition, the expression of other ES cell markers can be assessed. Stage-specific embryonic 1 5 antigen-1, -3 and -4 (SSEA-1, SSEA-3, SSEA-4) are glycoproteins specifically expressed in early embryonic development and are markers of ES cells (Solter and Knowles, 1978, Proc. Natl. Acad. Sci. USA 75:5565-5569; Kannagi et al., 1983, EMBO J 2:2355-2361).

[0162] Elevated expression of 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 / progenitor cell markers include the 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 Biol. Chem. 274:1566-1572). Dev. 2:1647-1654; Subramania'n et al., 1998, Differentiation 64:11-18).

[0163] In some embodiments, the expression profile of reprogrammed somatic cells can be used to assess their pluripotency characteristics. It is known that pluripotent cells (such as embryonic stem cells) and multipotent cells (such as adult stem cells) have unique global gene expression profile patterns. This unique pattern is called "stem cell molecule signature" or "stemness". See, for example, Ramalho-Santos et al., Science 298:597-600 (2002); Ivanova et al., Science 298:601-604.

[0164] Somatic cells can be reprogrammed to obtain a full set of pluripotency characteristics, and are therefore multipotent. Alternatively, somatic cells can be reprogrammed to obtain only a subset of pluripotency characteristics. In another alternative, somatic cells can be reprogrammed to be multipotent.

[0165] Activation culture

[0166] In certain embodiments, the isolated population of the cell is cultured in an activated culture for a first period of time. In certain embodiments, the first period of time is 1 day to 20 days. In certain embodiments, the first period of time is 1 day to 17 days. In certain embodiments, the first period of time is 1 day to 15 days. In certain embodiments, the first period of time is 1 day to 13 days. In certain embodiments, the first period of time is 1 day to 11 days. In certain embodiments, the first period of time is 1 day to 9 days. In certain embodiments, the first period of time is 1 day to 7 days. In certain embodiments, the first period of time is 1 day to 5 days. In certain embodiments, the first period of time is 1 day to 3 days. In certain embodiments, the first period of time is 12 hours to 72 hours. In certain embodiments, the first period of time is 12 hours to 60 hours. In certain embodiments, the first period of time is 12 hours to 48 hours. In certain embodiments, the first period of time is 12 hours to 36 hours. In certain embodiments, the first period of time is 12 hours to 24 hours. In certain embodiments, the first period of time is 8 hours to 16 hours. In certain embodiments, the first time period is 4 hours to 8 hours. In certain embodiments, the first time period is 2 hours to 4 hours. In certain embodiments, the first time period is 4 hours to 8 hours. In certain embodiments, the first time period is 50 hours to 80 hours. In certain embodiments, the first time period is 4 hours to 8 hours. In certain embodiments, the first time period is 55 hours to 75 hours. In certain embodiments, the first time period is 4 hours to 8 hours. In certain embodiments, the first time period is 60 hours to 75 hours. In certain embodiments, the first time period is 4 hours to 8 hours. In certain embodiments, the first time period is 70 hours to 75 hours. In certain embodiments, the first time period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.

[0167] In certain embodiments, the separation population of this cell is cultivated in activation culture and is not longer than a certain time period.For example, the separation population of this cell is cultivated in activation culture for at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days or at most 1 day.In a specific embodiment, the separation population of this cell is cultivated in activation culture for at most 5 days.In a specific preferred embodiment, the separation population of this cell is cultivated in activation culture for at most 3 days.In a specific preferred embodiment, the separation population of this cell is cultivated in activation culture for about 3 days.

[0168] In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 100% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 95% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 90% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 85% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 80% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 75% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 70% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5%-65% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5%-60% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 55% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 50% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 45% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 40% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 35% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 15% to 35% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 25% to 35% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 30% to 35% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 30% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 25% of γδT cells. In certain embodiments, after culturing the first time period in the activated culture, the isolated population of the cell comprises 5% to 20% of γδT cells.In certain embodiments, after culturing in the activated culture for the first period of time, the isolated population of cells comprises 5%-15% γδ T cells.

[0169] In certain embodiments, after culturing in activated culture for a first period of time, the isolated population of 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% of γδ T cells. In certain embodiments, after culturing in activated culture for a first period of time, the isolated population of 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% of γδ T cells.

[0170] In one embodiment, after culturing the first time period in the activated culture, the isolated population of the cell comprises less than about 60% of γδT cells. In another embodiment, after culturing the first time period in the activated culture, the isolated population of the cell comprises less than about 55% of γδT cells. In another embodiment, after culturing the first time period in the activated culture, the isolated population of the cell comprises less than about 50% of γδT cells. In another embodiment, after culturing the first time period in the activated culture, the isolated population of the cell comprises less than about 45% of γδT cells. In another embodiment, after culturing the first time period in the activated culture, the isolated population of the cell comprises less than about 40% of γδT cells. In another embodiment, after culturing the first time period in the activated culture, the isolated population of the cell comprises less than about 35% of γδT cells.

[0171] In certain embodiments, after culturing in an activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 100% of TCRVγ9+ T cells. In certain embodiments, after culturing in an activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 95% of TCRVγ9+ T cells. In certain embodiments, after culturing in an activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 90% of TCRVγ9+ T cells. In certain embodiments, after culturing in an activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 85% of TCRVγ9+ T cells. In certain embodiments, after culturing in an activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 80% of TCRVγ9+ T cells. In certain embodiments, after culturing in an activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 75% of TCRVγ9+ T cells. In certain embodiments, after culturing in an activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 70% of TCRVγ9+ T cells. In certain embodiments, after culturing in an activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5%-65% TCRVγ9+ T cells. In certain embodiments, after culturing in an activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5%-60% TCRVγ9+ T cells. In certain embodiments, after culturing in an activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 55% TCRVγ9+ T cells. In certain embodiments, after culturing in an activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 50% TCRVγ9+ T cells. In certain embodiments, after culturing in an activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 45% TCRVγ9+ T cells. In certain embodiments, after culturing in activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 40% of TCRVγ9+ T cells. In certain embodiments, after culturing in activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 35% of TCRVγ9+ T cells. In certain embodiments, after culturing in activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 30% of TCRVγ9+ T cells. In certain embodiments, after culturing in activated culture for a first period of time, the γδT cells in the isolated population of the cell comprise 5% to 25% of TCRVγ9+ T cells.In certain embodiments, after culturing in activated culture for a first period of time, the γδ T cells in the isolated population of cells comprise 5% to 20% TCRVγ9+ T cells. In certain embodiments, after culturing in activated culture for a first period of time, the γδ T cells in the isolated population of cells comprise 5%-15% TCRVγ9+ T cells.

[0172] In certain embodiments, after culturing in activated culture for a first period of time, the γδ T cells in the isolated population of cells comprise 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 + In certain embodiments, after culturing in activated culture for a first period of time, the γδ T cells in the isolated population of cells comprise 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.

[0173] In one embodiment, after culturing the first time period in the activated culture, the γδT cells in the isolated population of the cell comprise less than about 60% TCRVγ9+ T cells. In one embodiment, after culturing the first time period in the activated culture, the γδT cells in the isolated population of the cell comprise less than about 55% TCRVγ9+ T cells. In another embodiment, after culturing the first time period in the activated culture, the γδT cells in the isolated population of the cell comprise less than about 50% TCRVγ9+ T cells. In another embodiment, after culturing the first time period in the activated culture, the γδT cells in the isolated population of the cell comprise less than about 45% TCRVγ9+ T cells. In another embodiment, after culturing the first time period in the activated culture, the γδT cells in the isolated population of the cell comprise less than about 40% TCRVγ9+ T cells. In another embodiment, after culturing the first time period in the activated culture, the γδT cells in the isolated population of the cell comprise less than about 35% TCRVγ9+ T cells.

[0174] In certain embodiments, the method further comprises enriching the isolated population of cells for γδ T cells. In certain embodiments, the γδ T cells are enriched by cell-cell aggregate enrichment.

[0175] In certain embodiments, at least a portion of the γδ T cells activated in step (b) are Vγ9 + γδ T cells.

[0176] In certain embodiments, at least a portion of the γδ T cells activated in step (b) are Vγ9δ2 + γδ T cells.

[0177] cell

[0178] The present disclosure is based on the discovery that isolated populations of cells (e.g., isolated populations of γδ T cells) can be activated and reprogrammed to pluripotency by activation (e.g., in the presence of zoledronic acid and IL-15) and introduction of transcription factors (e.g., via Sendai virus vectors).

[0179] The isolated populations of cells of the present disclosure include any T cell of the body that is not a stem cell, germ cell or iPSC. Non-limiting examples of non-iPSCs are T cells derived from any tissue of the body, including internal organs, skin, bone, blood, neural tissue, and connective tissue.

[0180] In certain embodiments, the separation colony of this cell is hemocyte.In certain embodiments, hemocyte is suitably peripheral blood mononuclear cell (PMBC), and can be included in all types of hemocytes existing in the whole differentiation process from hematopoietic stem cell to final differentiation into peripheral blood.In one embodiment, hemocyte includes such as hematopoietic stem cell, lymphoid stem cell, lymphoid dendritic cell progenitor cell, lymphoid dendritic cell, T lymphocyte progenitor cell, T cell, B lymphocyte progenitor cell, B cell, plasma cell, NK progenitor cell, NK cell, monocyte and macrophage.

[0181] In some embodiments, the isolated population of cells is peripheral blood mononuclear cells (PBMC), peripheral blood leukocytes (PBL), tumor infiltrating lymphocytes (TIL), or a combination thereof. In some embodiments, the isolated population of cells is peripheral blood mononuclear (PBMC) cells.

[0182] In certain embodiments, the isolated population of cells is a T cell. 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 cell memory TSCM cells, central memory TCM cells, effector memory TEM cells, effector memory TEMRA cells, γδT cells and any combination thereof.

[0183] In some embodiments, the isolated population of cells is derived from a cell type that is readily available and requires minimal invasiveness, such as fibroblasts, skin cells, umbilical cord blood cells, peripheral blood cells, and kidney epithelial cells.

[0184] In certain embodiments, the cell separation population is a terminally differentiated cell. In certain embodiments, the cell separation population is a terminally differentiated T cell. In certain embodiments, the cell separation population is a terminally differentiated PBMC cell. In certain embodiments, the cell separation population is a terminally differentiated γδ T cell.

[0185] The isolated populations of cells disclosed herein may be derived from mammals, preferably humans, but including but not limited to non-human primates, murines (i.e., mice and rats), canines, felines, equines, bovines, ovines, porcines, caprines, and the like.

[0186] In certain embodiments, the isolated population of cells are mammalian cells.

[0187] In certain embodiments, the isolated population of cells are human cells.

[0188] In some embodiments, the isolated population of cells are human PBMC cells.

[0189] Pluripotency-related gene introduction

[0190] The present disclosure also relates to introducing an endogenous locus as a pluripotency-related gene into an activated cell population. 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 with at least one sgRNA targeting a desired locus. In some embodiments, such pluripotency-related genes can be introduced by expressing from a recombinant expression cassette introduced into a target cell. In some embodiments, such pluripotency-related genes can be introduced by incubating cells in the presence of an exogenous reprogramming transcription factor polypeptide.

[0191] In certain embodiments, the expression vector for introducing pluripotency-related genes includes modified viral polynucleotides such as from adenovirus, Sendai virus, herpes virus or retrovirus such as lentiviral vectors. 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, Sendai virus vectors are used.

[0192] In order to address the safety issues caused by the target cell genome carrying integrated exogenous sequences, many modified genetic protocols have been developed and can be used in the production methods described herein. These protocols produce iPS cells with potentially reduced risks and include non-integrating adenoviruses that deliver reprogramming genes (Stadtfeld, M. et al., Science, 2008, 322:945-949), transient transfection of reprogramming plasmids (Okita, K. et al., Science, 2008, 322:949-953), piggyBac transposition system (Woltjen, K. et al., Nature, 2009, 45 8:766-770; Yusa et al., Nat. Methods, 2009, 6:363-369; Kaji, K. et al. (2009)), Cre excisable virus (Soldner, F. et al., Cell, 2009, 136:964-977), and oriP / EBNA1-based episomal expression system (Yu, J. et al., Science, 2009, 324(5928):797-801).

[0193] Non-limiting examples of pluripotency-associated 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.

[0194] In certain embodiments, the isolated population of cells endogenously expresses at least one or more proteins selected 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, the isolated population of cells does not endogenously express any reprogramming transcription factor.

[0195] In certain embodiments, the reprogramming factors include Oct3 / 4, Sox2, Klf4, and c-Myc.

[0196] In certain embodiments, the reprogramming factors are Oct3 / 4, Sox2, KLF4, c-Myc, and Lin28.

[0197] In certain embodiments, the reprogramming factors are Oct3 / 4, Sox2, Klf4, c-Myc.

[0198] Exogenously introduced pluripotency genes can be carried out in several ways. In one embodiment, the exogenously introduced pluripotency genes can be expressed from a chromosomal locus different from the endogenous chromosomal locus of the pluripotency gene. Such chromosomal loci can be loci with open chromatin structure and contain somatic cell non-essential genes. In other words, the desired chromosomal locus contains genes whose destruction does not cause cell death. Exemplary chromosomal loci include, for example, mouse ROSA 26 locus and type II collagen (Col2a1) locus (see Zambrowicz et al., 1997).

[0199] Exogenously introduced pluripotency genes can be expressed from inducible promoters, so that their expression can be regulated as desired.

[0200] In another embodiment, the exogenously introduced pluripotency gene can be transiently transfected into the cell alone 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 bodies (embryonic) cells, morula-derived cells, teratomas (teratocarcinomas) cells, and embryonic stem cells differentiated from pluripotent parts of the late embryonic development process.

[0201] The cDNA library is prepared by conventional techniques. In short, mRNA is isolated from the organism of interest. RNA-guided DNA polymerase is used for first-strand synthesis using mRNA as a template. Second-strand synthesis is performed using DNA-guided DNA polymerase, which produces a cDNA product. After conventional treatment to promote cDNA cloning, the cDNA is inserted into an expression vector so that the cDNA is operably linked to at least one regulatory sequence. The selection of expression vectors used in conjunction with the cDNA library is not limited to a specific vector. Any expression vector suitable for mouse cells is suitable. In one embodiment, the promoter driving the expression of 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 their expression when operably linked to a DNA sequence can be used in these vectors to express cDNA. Such useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, the immediate early promoter of adenovirus or cytomegalovirus, the lac system, the trp system, the TAC or TRC system, the T7 promoter (whose expression is directed by T7 RNA polymerase), the major operator and promoter region of bacteriophage 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 gene expression of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. In addition, the copy number of the vector, the ability to control the copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

[0202] Exogenously introduced pluripotency genes can be expressed by inducible promoters. As used herein, the term "inducible promoter" refers to a promoter that does not direct the expression of an operably linked gene (including cDNA) or directs low-level expression in the absence of an inducer (such as a chemical and / or biological agent) and that responds to an inducer whose ability to direct expression is enhanced. 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. PNAS 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), promoters that respond to chemicals such as glucose, lactose, galactose or antibiotics.

[0203] Tetracycline inducible promoters are examples of inducible promoters that respond to antibiotics. See Gossen et al., 2003. Tetracycline inducible promoters include a minimal promoter that is operably linked to one or more tetracycline operators. The presence of one of tetracycline or its analogs causes a transcriptional activator to bind to the tetracycline operator sequence, which activates the minimal promoter and thus activates transcription of the associated cDNA. Tetracycline analogs include any compound that shows structural homology to tetracycline and is capable of activating a tetracycline inducible promoter. Exemplary tetracycline analogs include, for example, doxycycline, chlortetracycline, and anhydrotetracycline.

[0204] Thus, in one embodiment, the present disclosure provides somatic cells carrying at least one pluripotency gene expressed as a transgene under an inducible promoter. It is possible that somatic cells with such inducible pluripotency transgenes are more susceptible to reprogramming.

[0205] Any of the engineered somatic cells disclosed herein can be used in the method. In one embodiment, the somatic cells used in the method comprise only one endogenous pluripotency gene connected to a first selective marker, and a selection step is performed to select the expression of the first selective marker. In an alternative embodiment, the somatic cells used in the method comprise any number of endogenous pluripotency genes, each of which is connected to different selective markers, and a selection step is performed to select at least one subset of these selective markers. For example, a selection step can be performed to select all selective markers connected to various endogenous pluripotency genes.

[0206] In an alternative embodiment, the somatic cells used in the method comprise a selectable marker linked to an endogenous pluripotency gene and an additional pluripotency gene expressed as a transgene under an inducible promoter. For these cells, the method of reprogramming may include inducing expression of the pluripotency transgene and selecting for expression of the selectable marker.

[0207] In certain embodiments, in step (d) described in the above method, the transduced γδT cells are cultured in the presence of one or more feeder layers. In certain embodiments, in step (d), the transduced γδT cells are cultured in the presence of a monolayer of feeder cells. In certain embodiments, the feeder layer comprises mouse embryonic fibroblasts (MEF). In certain embodiments, in step (d), the transduced γδT cells are cultured in the presence of a monolayer of feeder layers. In certain embodiments, in step (d), the transduced γδT cells are cultured in the presence of mitotically inactivated mouse embryonic fibroblasts (MEF). In certain embodiments, in step (d), the transduced γδT cells are cultured under feeder-free conditions. In certain embodiments, in step (d), the transduced γδT cells are cultured in a plate coated with iMatrix-511. In certain embodiments, after step (d), the method further comprises a step (e) of separating and / or purifying the generated iPSC.

[0208] In certain more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising: contacting an isolated population of cells with an activation culture; wherein the activation culture comprises IL-15, zoledronic acid, and IL-2; culturing the isolated population of cells in the activation culture to enrich and / or activate γδT cells in the isolated population of cells; transducing γδT cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors; and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0209] In certain more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising: obtaining an isolated population of cells (e.g., terminally differentiated cells, such as peripheral blood mononuclear cells (PBMCs)) from a subject (e.g., a human); contacting the isolated population of cells with an activation culture; wherein the activation culture comprises IL-15, zoledronic acid, and IL-2; culturing the isolated population of cells in the activation culture to enrich and / or activate γδT cells in the isolated population of cells; transducing the γδT cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors; and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0210] In other more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising: obtaining an isolated population of cells (e.g., terminally differentiated cells, such as peripheral blood mononuclear cells (PBMCs)) from a subject (e.g., a human); contacting the isolated population of cells with an activation culture; wherein the activation culture comprises 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 γδT cells in the isolated population of cells; transducing the γδT cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors; and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0211] In other more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising: obtaining an isolated population of cells (e.g., terminally differentiated cells, such as peripheral blood mononuclear cells (PBMCs)) from a subject (e.g., a human); contacting the isolated population of cells with an activation culture; wherein the activation culture comprises 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% γδT cells; transducing the γδT cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors; and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0212] In other more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising: obtaining an isolated population of cells (e.g., terminally differentiated cells, such as peripheral blood mononuclear cells (PBMCs)) from a subject (e.g., a human); contacting the isolated population of cells with an activation culture; wherein the activation culture comprises 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% γδT cells; further enriching for γδT cells by cell-cell aggregate enrichment; transducing γδT cells with a Sendai virus (SeV) vector encoding one or more reprogramming factors; and culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0213] In other more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising: obtaining an isolated population of cells (e.g., terminally differentiated cells, such as peripheral blood mononuclear cells (PBMCs)) from a subject (e.g., a human); contacting the isolated population of cells with an activation culture; wherein the activation culture comprises 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% γδT cells; optionally further enriching for γδT cells by cell-cell aggregate enrichment; transducing γδT 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 γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0214] In other more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising: obtaining an isolated population of cells (e.g., terminally differentiated cells, such as peripheral blood mononuclear cells (PBMCs)) from a subject (e.g., a human); contacting the isolated population of cells with an activation culture; wherein the activation culture comprises 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% γδT cells; optionally further enriching for γδT cells by cell-cell aggregate enrichment; transducing γδT 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 γδT cells in the presence of a monolayer of feeder cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0215] In other more specific embodiments, provided herein is a method for producing induced pluripotent stem cells (iPSCs), comprising: obtaining an isolated population of cells (e.g., terminally differentiated cells, such as peripheral blood mononuclear cells (PBMCs)) from a subject (e.g., a human); contacting the isolated population of cells with an activation culture; wherein the activation culture comprises 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% γδT cells; optionally further enriching for γδT cells by cell-cell aggregate enrichment; transducing γδT 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 γδT cells in the presence of a monolayer of feeder cells comprising mouse embryonic fibroblasts (MEFs) under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state.

[0216] iPSCs derived from γδ T cells

[0217] In certain embodiments, the iPSCs produced are derived from γδT cells. In certain embodiments, the iPSCs produced have rearranged genes of TRG and TRD loci. In certain embodiments, the iPSCs produced do not produce or express TCRA and / or TCRB or fragments thereof, so that there is no detectable or otherwise detectable surface expression of TCRA and TCRB.

[0218] In certain embodiments, the iPSCs generated are not derived from αβ T cells.

[0219] In certain embodiments, the generated iPSCs are negative for Sendai virus (SeV) vectors.

[0220] In certain embodiments, the generated iPSCs are genomically stable without chromosome loss.In one embodiment, the genomic stability of the generated iPSCs is determined by karyotyping.

[0221] In certain embodiments, the generated iPSCs are capable of growth and maintenance in feeder-free medium following adoptive transfer.

[0222] In certain embodiments, the method also includes differentiating the produced iPSC into a desired cell type in vitro or in vitro. In certain embodiments, the method also includes differentiating the produced iPSC into a desired cell type in vitro. In certain embodiments, the method also includes differentiating the produced iPSC into a desired cell type in vitro.

[0223] 5.7. T cell-derived induced pluripotent stem cells (iPSCs)

[0224] Also provided herein are isolated populations of induced pluripotent stem cells (iPSCs) having novel characteristics. In some embodiments, the isolated population of iPSCs comprises pluripotent cells expressing one or more reprogramming factors and comprises nucleotide sequences encoding TRG and TRD gene rearrangements.

[0225] In certain embodiments, the isolated population of iPSCs is generated according to the methods described herein (eg, in Section 5.6).

[0226] In certain embodiments, the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc and Lin28.

[0227] In certain embodiments, the reprogramming factors include Oct3 / 4, Sox2, Klf4, and c-Myc.

[0228] In certain embodiments, the reprogramming factors are Oct3 / 4, Sox2, KLF4, c-Myc, and Lin28.

[0229] In certain embodiments, these reprogramming factors are Oct3 / 4, Sox2, Klf4, and c-Myc.

[0230] In certain embodiments, the isolated population of iPSC is derived from γδT cells. In certain embodiments, the isolated population of iPSC has rearranged genes of TRG and TRD loci. In certain embodiments, the isolated population of iPSC does not produce PCR products from TCRG and TCRD loci.

[0231] In certain embodiments, the isolated population of iPSC is not derived from αβT cells. In certain embodiments, the isolated population of iPSC does not have rearranged genes of TRA and TRB loci. In certain embodiments, the isolated population of iPSC does not produce PCR products from TCRA and TCRB loci.

[0232] In certain embodiments, the isolated population of iPSCs is negative for Sendai virus (SeV) vectors.

[0233] In certain embodiments, the segregating population of iPSCs is genomically stable without chromosome loss.In one embodiment, the genomic stability of the segregating population of iPSCs is determined by karyotyping.

[0234] In certain embodiments, the isolated population of iPSCs is capable of growing and maintaining in feeder-free medium following adoptive transfer.

[0235] In some embodiments, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein (i) the pluripotent cells comprise nucleotide sequences encoding rearranged TRG and TRD genes or rearranged genes having TRG and TRD loci, (ii) the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc and Lin28, (iii) the isolated population of iPSCs is negative for Sendai virus (SeV) vectors; (iv) the isolated population of iPSCs is derived from γδT cells and not from αβT cells; (v) the isolated population of iPSCs does not produce PCR products from TCRA and TCRB loci; (vi) the isolated population of iPSCs is genomically stable without chromosome loss, for example, as determined by karyotyping; and / or (vii) the isolated population of iPSCs is capable of growth and maintenance in a feeder-free culture medium after adoptive transfer.

[0236] In a specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding TRG and TRD gene rearrangements, and wherein the reprogramming factors are selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc and Lin28.

[0237] In another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding TRG and TRD gene rearrangements, and wherein the reprogramming factors are Oct3 / 4, Sox2, Klf4, c-Myc and Lin28.

[0238] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding TRG and TRD gene rearrangements, and wherein the reprogramming factors are Oct3 / 4, Sox2, Klf4 and c-Myc.

[0239] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding a rearrangement of the TRG and TRD genes, and the isolated population of iPSCs is negative for Sendai virus (SeV) vectors.

[0240] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding TRG and TRD gene rearrangements, the reprogramming factors being 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.

[0241] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding TRG and TRD gene rearrangements, and the isolated population of iPSCs is derived from γδT cells and not from αβT cells.

[0242] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding TRG and TRD gene rearrangements, and the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci.

[0243] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding a rearrangement of TRG and TRD genes or a rearranged gene having TRG and TRD loci, and the isolated population of iPSCs is genomically stable without chromosome loss, e.g., as determined by karyotyping.

[0244] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise a nucleotide sequence encoding a rearrangement of TRG and TRD genes or have rearranged genes of the TRG and TRD loci, and the isolated population of iPSCs is capable of growing and maintaining in a feeder-free culture medium after adoptive transfer.

[0245] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding rearranged TRG and TRD genes or rearranged genes having TRG and TRD loci, the reprogramming factors being selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc and Lin28, the isolated population of iPSCs being negative for Sendai virus (SeV) vectors; the isolated population of iPSCs being derived from γδT cells and not from αβT cells; the isolated population of iPSCs not producing PCR products from the TCRA and TCRB loci; the isolated population of iPSCs being genomically stable without chromosome loss, e.g., as determined by karyotype analysis; and the isolated population of iPSCs being able to grow and be maintained in a feeder-free culture medium after adoptive transfer.

[0246] In yet another specific embodiment, provided herein is an isolated population of induced pluripotent stem cells (iPSCs) comprising pluripotent cells expressing one or more reprogramming factors, wherein the pluripotent cells comprise nucleotide sequences encoding rearranged TRG and TRD genes or rearranged genes having TRG and TRD loci, the reprogramming factors being selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc and Lin28, the isolated population of iPSCs being negative for Sendai virus (SeV) vectors; the isolated population of iPSCs not producing PCR products from the TCRA and TCRB loci; the isolated population of iPSCs being genomically stable without chromosome loss, e.g., as determined by karyotype analysis; and the isolated population of iPSCs being able to grow and be maintained in a feeder-free culture medium after adoptive transfer.

[0247] 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 nucleotide sequences encoding rearranged TRG and TRD genes or rearranged genes having TRG and TRD loci, the isolated population of iPSCs is negative for Sendai virus (SeV) vectors; the isolated population of iPSCs is derived from γδT cells and not from αβT cells; the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci; the isolated population of iPSCs is genomically stable without chromosome loss, for example, as determined by karyotype analysis; and the isolated population of iPSCs is capable of growing and maintaining in a feeder-free culture medium after adoptive transfer.

[0248] 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 nucleotide sequences encoding rearranged TRG and TRD genes or rearranged genes having TRG and TRD loci, the isolated population of iPSCs is negative for Sendai virus (SeV) vectors; the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci; the isolated population of iPSCs is genomically stable without chromosome loss, for example, as determined by karyotyping; and the isolated population of iPSCs is capable of growing and maintaining in a feeder-free culture medium after adoptive transfer.

[0249] 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 nucleotide sequences encoding rearranged TRG and TRD genes or rearranged genes having TRG and TRD loci, the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci; the isolated population of iPSCs is genomically stable without chromosome loss, e.g., as determined by karyotyping; and the isolated population of iPSCs is capable of growth and maintenance in a feeder-free culture medium after adoptive transfer.

[0250] 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 a rearrangement of TRG and TRD genes or a rearranged gene having TRG and TRD loci, the isolated population of iPSCs does not produce PCR products from the TCRA and TCRB loci; and the isolated population of iPSCs is genomically stable without chromosome loss, for example, as determined by karyotyping.

[0251] Also provided herein are reprogrammed somatic cells produced by the methods of the present disclosure, including reprogrammed pluripotent somatic cells such as iPSCs.

[0252] In some embodiments, the reprogrammed somatic cells of the present invention are ES-like cells, and thus differentiation can be induced according to known methods for differentiating ES cells to obtain desired cell types. For example, such cells can be induced to differentiate into hematopoietic stem cells, myocytes, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, urethral cells, etc. by culturing iPSCs in a differentiation medium and under conditions that provide cell differentiation. The culture medium and methods that lead to embryonic stem cell differentiation and suitable culture conditions are known in the art.

[0253] In some specific embodiments, iPSCs are induced to differentiate into hematopoietic stem cells, e.g., as described in Palacios et al., Proc. Natl. Acad. Sci., USA, 92:7530-37 (1995), which teaches the generation of hematopoietic stem cells from embryonic cell lines by subjecting the stem cells to an induction procedure comprising initially culturing aggregates of such cells in a suspension medium lacking retinoic acid, followed by culturing in the same medium containing retinoic acid, and then transferring the cell aggregates to a substrate that provides cell attachment.

[0254] In other specific embodiments, iPSC differentiation is induced according to the method described in Pedersen, J. Reprod. Fertil. Dev., 6:543-52 (1994), which cites many articles disclosing methods for differentiating embryonic stem cells in vitro to produce various differentiated cell types (including hematopoietic cells, muscle, cardiac muscle, neural cells, etc.).

[0255] In other specific embodiments, iPSC differentiation is induced according to Bain et al., Dev. Biol., 168:342-357 (1995), which teaches the in vitro differentiation of embryonic stem cells to generate neural cells with neuronal properties.

[0256] These references are examples of reported methods of obtaining differentiated cells from embryonic or stem-like cells. These references and in particular the disclosures therein regarding methods of differentiating embryonic stem cells are incorporated herein by reference in their entirety.

[0257] Therefore, using known methods and culture media, those skilled in the art can culture embryonic or stem-like cells of a subject to obtain desired differentiated cell types, such as neural cells, muscle cells, hematopoietic cells, etc. In addition, the use of inducible Bcl-2 or Bcl-x1 may help to enhance the in vitro development of specific cell lineages.

[0258] The iPSCs provided herein can be used to obtain any desired differentiated cell type.

[0259] The iPSC produced according to the present disclosure can be used to produce genetically engineered or transgenic differentiated cells. In essence, this will be achieved by introducing one or more desired genes, or removing all or part of one or more endogenous genes of the iPSC produced according to the present invention, and differentiating these cells into the desired cell type. The preferred method for realizing such modification is by homologous recombination, because such technology can be used to insert, delete or modify one or more genes at one or more specific sites of the stem cell-like cell genome.

[0260] The iPSCs disclosed herein can be used, for example, as an in vitro differentiation model, particularly for studying genes involved in early developmental regulation. Differentiated cell tissues and organs using iPSCs can be used for drug research.

[0261] Also provided herein is a composition comprising iPSC of the present disclosure. In some embodiments, the composition comprises iPSCs knocking out B2M or iPSC populations knocking out B2M. In some embodiments, iPSCs are produced by reprogramming γδT cells. In some embodiments, a composition is provided herein, comprising derived immune cells with enhanced functions of a separated population or subpopulation differentiated from iPSCs produced according to the methods provided herein.

[0262] 5.8. Methods for Identifying Agents That Reprogram or Help Reprogram Somatic Cells

[0263] In another aspect, provided herein are methods for identifying an agent that reprograms a somatic cell (eg, a T cell) to a less differentiated state, alone or in combination with one or more other agents. The present disclosure also provides agents identified according to the methods provided herein.

[0264] In one embodiment, the method comprises contacting somatic cells with an activation culture comprising IL-15, zoledronic acid and / or IL-2; contacting the somatic cells 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) relative to the reprogramming that would occur if the cells were not contacted with the candidate agent.

[0265] In some embodiments, provided herein is a method for identifying an agent that reprograms a somatic cell (e.g., a T cell) to a low-differentiation state, either alone or in combination with one or more agents, comprising (a) contacting a separate population of cells with an activation culture; wherein the activation culture comprises IL-15 and zoledronic acid; (b) culturing a separate population of cells in the activation culture to enrich and / or activate γδT cells in a separate population of cells; (c) contacting a separate population of cells with a candidate agent; (d) transducing γδT cells with one or more viral vectors encoding one or more reprogramming factors; (e) culturing transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a low-differentiation state, and (f) determining whether at least some of the somatic cells in these somatic cells are reprogrammed to a low-differentiation state. In some embodiments, the low-differentiation state is a multipotent state. In some embodiments, the low-differentiation state is a pluripotent state. In certain embodiments, the activation culture further comprises one or more additional agents or compounds, for example, to increase the efficiency of activation or induction. In one embodiment, the activation culture further comprises interleukin-2 (IL-2).

[0266] In some embodiments, IL-15, zoledronic acid and / or IL-2 are present in the cell culture medium together with the candidate agent, while in other embodiments, IL-15, zoledronic acid and / or IL-2 are not present together with the candidate agent (e.g., the cells are sequentially exposed to the agent). In certain embodiments, the cells are maintained in culture for 1 day to 20 days. In certain embodiments, the cells are maintained in culture for 1 day to 17 days. In certain embodiments, the cells are maintained in culture for 1 day to 15 days. In certain embodiments, the cells are maintained in culture for 1 day to 13 days. In certain embodiments, the cells are maintained in culture for 1 day to 11 days. In certain embodiments, the cells are maintained in culture for 1 day to 9 days. In certain embodiments, the cells are maintained in culture for 1 day to 7 days. In certain embodiments, the cells are maintained in culture for 1 day to 5 days. In certain embodiments, the cells are maintained in culture for 1 day to 3 days. In certain embodiments, the cells are maintained in culture for 12 hours to 72 hours. In certain embodiments, the cell is maintained in culture for 12 hours to 60 hours. In certain embodiments, the cell is maintained in culture for 12 hours to 48 hours. In certain embodiments, the cell is maintained in culture for 12 hours to 36 hours. In certain embodiments, the cell is maintained in culture for 12 hours to 24 hours. In certain embodiments, the cell is maintained in culture for 8 hours to 16 hours. In certain embodiments, the cell is maintained in culture for 4 hours to 8 hours. In certain embodiments, the cell is maintained in culture for 2 hours to 4 hours. The cell can be maintained in culture for, for example, 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, etc., during which they are contacted with IL-15, zoledronic acid and / or IL-2 and candidate agent for all or part of the time. In some embodiments, an agent is identified as an agent that reprograms cells if the number of reprogrammed cells or colonies comprising predominantly reprogrammed cells after the period of time is at least 2, 5, or 10 times greater than when the cells were not contacted with the agent.

[0267] Candidate agent can be any molecule or supramolecular complex, such as peptide, small organic or inorganic molecule, polysaccharide, polynucleotide etc., which will be tested for reprogramming or promoting or enhancing the ability of reprogrammed cells. As will be appreciated by those skilled in the art, candidate agent can be obtained from a variety of sources, including libraries of synthetic or natural compounds. In some embodiments, candidate agent is a synthetic compound. Many technologies can be used for the random and directional synthesis of various organic compounds and biomolecules. In some embodiments, candidate modulator is provided as a mixture of natural compounds in the form of bacteria, fungi, plant and animal extracts, fermentation broth, conditioned medium etc., which is obtainable or easily produced.

[0268] In some embodiments, screening compound libraries. Libraries are generally collections of compounds that can be presented or displayed, so that compounds can be identified in screening assays. In some embodiments, the compounds in the library are contained in separate holes (e.g., holes of microtiter plates), containers, tubes, etc., to facilitate convenient transfer to separate holes or containers for contacting cells, performing cell-free assays, etc. The library can be composed of molecules with common structural features, which are different or can be completely random in the number or type of groups attached to the main structure. Libraries include, but are not limited to, for example, phage display libraries, peptide libraries, polyribosome libraries, aptamer libraries, synthetic small molecule libraries, natural compound libraries, and chemical libraries. Methods for preparing molecular libraries are well known in the art, and many libraries can be obtained from commercial or non-commercial sources. Interested libraries include synthetic organic combinatorial libraries. Libraries such as synthetic small molecule libraries and chemical libraries can include a structural diversity collection of chemical molecules. Small molecules include organic molecules that generally have multiple carbon-carbon bonds. The library may include a ring carbon or heterocyclic structure and / or an aromatic or polyaromatic structure substituted by one or more functional groups. In some embodiments, the small molecule has 5 to 50 carbon atoms, for example, between 7 and 30 carbons. In some embodiments, the compound is a macrocyclic compound. Interested libraries also include peptide libraries, random oligonucleotide libraries, etc. Libraries of peptoids and non-peptide synthetic parts can be synthesized. Such libraries can be further synthesized, containing non-peptide synthetic parts that are less degraded by enzymes than their naturally occurring counterparts. Small molecule combinatorial libraries can also be generated. The combinatorial library of small molecule organic compounds can include a collection of closely related analogs that are different from each other in one or more diversity aspects and are synthesized by organic techniques using a multi-step method. Combinatorial libraries can include a large 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 arranged so that each compound has a common molecular core and one or more variable structural diversity elements. The compounds in such compound arrays are produced in parallel in separate reaction vessels, wherein each compound is identified and tracked by its spatial address. In some embodiments, mixtures containing two or more compounds, extracts or other preparations obtained from natural sources (which may contain dozens or more compounds) and / or inorganic compounds, etc. are screened.

[0269] In one embodiment, the methods of the present disclosure are used to screen for "approved drugs". An "approved drug" is any compound (this term includes biomolecules such as proteins and nucleic acids) that has been approved by the FDA or a similar government agency in another country for use in humans for any purpose. This can be a particularly useful class of compounds to screen because it represents a group of compounds that are considered safe and, at least in the case of FDA-approved drugs, are therapeutic agents for at least one purpose. Therefore, it is likely that these drugs are safe at least for other purposes.

[0270] Representative examples of libraries that can be screened include the DIVERSet library available from ChemBridge Corporation, 16981 Via Tazon, San Diego, Calif. 92127 TM . DIVERSet contains between 10,000 and 50,000 drug-like, manually synthesized small molecules. Compounds are pre-selected to form a "universal" library that covers the greatest pharmacophore diversity with a minimum number of compounds and is suitable for high-throughput or lower-throughput screening. For descriptions of additional libraries, see, e.g., Tan et al., Am. Chem Soc. 120, 8565-8566, 1998; Floyd CD, Leblanc C, Whittaker M, Prog Med Chem 36:91-168, 1999. Many libraries are commercially available, for example, from AnalytiCon USA Inc., PO 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, santonine, didymo-D-glucitol, hydroxypicolinic acid, andrographolide, libraries based on piperazine-2-carboxylic acid, cytosine, etc. are commercially available.

[0271] In some embodiments, the candidate agent is a cDNA from a cDNA expression library prepared from a cell (e.g., a pluripotent cell). Such cells can be embryonic stem cells, oocytes, blastomeres, teratocarcinomas, embryonic germ cells, inner cell mass cells, etc.

[0272] It should be understood that the candidate reprogramming agent to be tested is generally a reprogramming agent not present in a standard culture medium, or if present, then exists in a lower amount than for the present invention.It should also be understood that useful reprogramming agents or other forms of reprogramming processes do not need to be able to reprogram all types of somatic cells, nor do they need to be able to reprogram all somatic cells of a given cell type.Without limitation, such a candidate agent is used, which results in enrichment of reprogrammed cells in a population by 2, 5, 10, 50, 100 or more times (i.e., the fraction of reprogrammed cells in a population is 2, 5, 10, 50 or 100 times in the starting population of cells that are processed in the same manner but not contacted with the candidate agent).

[0273] In some embodiments, the screening methods provided herein are used to identify agents or combinations of agents that replace Klf4 in reprogramming cells to a pluripotent state. In some embodiments, the method is used to identify agents that replace Sox2 in reprogramming cells to a pluripotent state. In some embodiments, the method is used to identify agents that replace Oct3 / 4 in reprogramming cells to a pluripotent state. In some embodiments, the method is used to identify agents that replace c-Myc in reprogramming cells to a pluripotent state. In some embodiments, the method is used to identify agents that replace Lin28 in reprogramming cells to a pluripotent state. In some embodiments, the method is implemented using human cells. In some embodiments, the method is implemented using mouse cells. In some embodiments, the method is implemented using non-human primate cells.

[0274] In another aspect, provided herein are methods for identifying genes expressed by endogenous pluripotency genes in activated somatic cells (eg, T cells).

[0275] In some embodiments, the method includes (a) contacting an isolated population of cells with an activation culture; wherein the activation culture comprises IL-15 and zoledronic acid; (b) culturing an isolated population of cells in the activation culture to enrich and / or activate γδT cells in the isolated population of cells; (c) transducing γδT cells with one or more viral vectors encoding one or more candidate reprogramming factors; (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a poorly differentiated state, and (e) determining whether at least some of the somatic cells are reprogrammed to a poorly differentiated state. In some embodiments, the poorly differentiated state is a pluripotent state. In some embodiments, the poorly differentiated state is a pluripotent state.

[0276] In some more specific embodiments, the method comprises (a) contacting an isolated population of cells with an activation culture; wherein the activation culture comprises IL-15 and zoledronic acid; (b) culturing the isolated population of cells in the activation culture to enrich and / or activate γδT cells in the isolated population of cells; (c) transducing γδT cells with one or more viral vectors encoding one or more candidate reprogramming factors; and (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state, and (e) determining whether at least some of the somatic cells are reprogrammed to a pluripotent state.

[0277] In certain embodiments, the activated culture further comprises one or more additional agents or compounds, for example to increase the efficiency of activation or induction. In one embodiment, the activated culture further comprises interleukin-2 (IL-2).

[0278] Thus, in some embodiments, the methods provided herein include (a) contacting an isolated population of cells with an activation culture; wherein the activation culture comprises IL-15, zoledronic acid, and IL-2; (b) culturing the isolated population of cells in the activation culture to enrich and / or activate γδT cells in the isolated population of cells; (c) transducing γδT cells with one or more viral vectors encoding one or more candidate reprogramming factors; (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a poorly differentiated state; and (e) determining whether at least some of the somatic cells are reprogrammed to a poorly differentiated state.

[0279] In some more specific embodiments, the methods provided herein include: (a) contacting an isolated population of cells with an activation culture; wherein the activation culture comprises IL-15, zoledronic acid and IL-2; (b) culturing the isolated population of cells in the activation culture to enrich and / or activate γδT cells in the isolated population of cells; (c) transducing γδT cells with one or more viral vectors encoding one or more candidate reprogramming factors; (d) culturing the transduced γδT cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state; and (e) determining whether at least some of the somatic cells are reprogrammed to a pluripotent state.

[0280] In other embodiments, the method includes, for example, culturing somatic cells as provided herein in the presence of IL-15, zoledronic acid and / or IL-2, then transfecting the somatic cells of the present disclosure with a cDNA library prepared from an ES cell or oocyte, selecting cells expressing a first selective marker, and evaluating the expression of a first endogenous pluripotency gene in transfected cells expressing the first selective marker. Expression of the first endogenous pluripotency gene indicates that the cDNA encodes a gene that activates the expression of the endogenous pluripotency gene in a somatic cell.

[0281] The method of the present invention is applicable to identifying genes that activate at least two endogenous pluripotency genes expressed in somatic cells. The somatic cells used in the method also contain a second endogenous pluripotency gene connected to a second selective marker. The method can be modified to select transfected cells expressing two selective markers, wherein the expression of the first endogenous pluripotency gene and the second endogenous pluripotency gene is evaluated. The expression of the first endogenous pluripotency gene and the second endogenous pluripotency gene indicates that the cDNA encodes genes that activate at least two pluripotency genes expressed in somatic cells.

[0282] The method of the present invention is also applicable to identifying genes that activate the expression of at least three endogenous pluripotency genes in somatic cells. The somatic cells used in the method also contain a third endogenous pluripotency gene connected to a third selective marker. The method is modified to select transfected cells expressing all three selective markers, wherein the expression of all three endogenous pluripotency genes is evaluated. The expression of all three endogenous pluripotency genes indicates that the cDNA encodes genes that activate the expression of at least three pluripotency genes in somatic cells.

[0283] Unless otherwise indicated, the implementation of the present invention will adopt the conventional techniques of mouse genetics, developmental biology, cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the technical scope of the art. These techniques are described in the literature. See, for example, Current Protocols in Cell Biology, edited by Bonifacino, Dasso, Lippincott-Schwartz, Harford and Yamada, John Wiley and Sons, Inc., New York, 1999; Manipulating the Mouse Embryos, A Laboratory Manual, 3rd Edition, Hogan et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2003; Gene Targeting: A Practical Approach, IRL Press at Oxford University Press, Oxford, 1993; and Gene Targeting Protocols, Human Press, Totowa, NJ, 2000. All patents, patent applications and references cited herein are incorporated by reference in their entirety.

[0284] 6. Implementation Plan

[0285] The present invention provides the following non-limiting embodiments.

[0286] In one set of embodiments (Group A embodiments), there is provided:

[0287] A1. An induced pluripotent stem cell (iPSC) population, wherein the iPSC is generated by reprogramming γδ T cells, and the population comprises iPSCs containing a disrupted β-2-microglobulin (B2M) gene

[0288] A2. A population of iPSCs according to embodiment A1, wherein the disrupted B2M gene comprises 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.

[0289] A3. A population of iPSCs according to embodiment A1, wherein 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 SEQ ID NOs: 12-16.

[0290] A4. A population of iPSCs according to any one of embodiments A1 to 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.

[0291] A5. The iPSC population according to any one of embodiments A1 to A4, wherein the disruption comprises a deletion of at least one nucleotide base pair.

[0292] A6. The iPSC population according to any one of embodiments A1 to A4, wherein the disruption comprises an insertion of at least one nucleotide base pair.

[0293] A7. The iPSC population according to any one of embodiments A1 to A6, wherein the disrupted B2M gene exhibits reduced B2M expression relative to an undisrupted B2M gene.

[0294] A8. A 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% compared to B2M expression in a reference iPSC.

[0295] A9. A population of iPSCs according to any one of embodiments A1 to A8, wherein the iPSCs comprising a disrupted B2M gene exhibit reduced HLA-A, HLA-B and / or HLA-C expression compared to HLA-A, HLA-B and / or HLA-C expression in a reference iPSC.

[0296] A10. The iPSC population of embodiment A9, 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% compared to the HLA-A, HLA-B and / or HLA-C expression in a reference iPSC.

[0297] A11. The iPSC population according to any one of embodiments A8 to A10, wherein the reference iPSCs are an iPSC population in which the B2M gene is not disrupted.

[0298] A12. An iPSC population according to any one of embodiments A1 to A11, wherein the disrupted B2M gene is produced by contacting the iPSC population with an RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene.

[0299] A13. The iPSC population according to embodiment A12, wherein the RNA-guided endonuclease is selected from 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, A group consisting of 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.

[0300] A14. A population of iPSCs according to embodiment A13, wherein the RNA-guided endonuclease is Cas12a (Cpf1).

[0301] A15. A population of iPSCs according to embodiment A13, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 (Cas12a).

[0302] A16. The iPSC population of embodiment A13 or A14, wherein the RNA-guided endonuclease is MAD7.

[0303] A17. A population of iPSCs according to any one of embodiments A12 to A16, wherein the gRNA binds to at least a portion of the complementary sequence of SEQ ID NO: 1.

[0304] A18. The iPSC population of any one of embodiments A12 to A16, wherein the gRNA binds to the complementary sequence of any one of SEQ ID NO: 2-6 or 17.

[0305] A19. An iPSC population according to any one of embodiments A12 to A18, wherein the gRNA comprises the sequence of any one of SEQ ID NO: 7-11 or 18.

[0306] A20. An iPSC population according to any one of embodiments A12 to A19, wherein the gRNA comprises the sequence shown in SEQ ID NO: 18.

[0307] A21. The iPSC population according to any one of embodiments A12 to A20, wherein the gRNA consists of the sequence of any one of SEQ ID NO: 7-11 or 18.

[0308] A22. The iPSC population of any one of embodiments A4 to A21, wherein the iPSCs have no detectable RNA transcripts encoding B2M.

[0309] A23. The iPSC population of any one of embodiments A4 to A22, wherein the iPSCs do not have detectable levels of B2M protein.

[0310] A24. The iPSC population of any one of embodiments A4 to A23, wherein the iPSCs do not express detectable levels of B2M protein when analyzed by flow cytometry.

[0311] A25. The iPSC population according to any one of embodiments A12 to A24, wherein the gRNA consists of SEQ ID NO: 18.

[0312] A26. 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, wherein:

[0313] (i) at least about 50% to 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry;

[0314] (ii) the iPSCs comprising a 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 a reference iPSC in which the B2M gene is not disrupted.

[0315] (iii) generating the disrupted B2M gene by contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene, wherein:

[0316] a. The RNA-guided endonuclease is Cas12a (Cpf1); and the gRNA binds to the complementary sequence of SEQ ID NO: 2.

[0317] A27. A population according to embodiment A26, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 (Cas12a).

[0318] A28. A population according to embodiment A26 or A27, wherein at least about 70% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0319] A29. A population according to any one of embodiments A26 to A28, wherein at least about 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0320] 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, wherein:

[0321] (i) at least about 50% to 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry;

[0322] (ii) the iPSCs comprising a 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 a reference iPSC in which the B2M gene is not disrupted.

[0323] (iii) generating the disrupted B2M gene by contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene, wherein:

[0324] a. the RNA-guided endonuclease is MAD7; and

[0325] b. The gRNA binds to the complementary sequence of SEQ ID NO:2.

[0326] A31. A population according to embodiment A30, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 (Cas12a).

[0327] A32. A population according to embodiment A30 or A31, wherein at least about 70% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0328] A33. A population according to any one of embodiments A30 to A32, wherein at least about 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0329] A34. 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, wherein:

[0330] (i) at least about 50% to 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry;

[0331] (ii) said iPSCs comprising a disrupted B2M gene exhibit reduced expression of HLA-A, HLA-B and / or HLA-C compared to said HLA-A, HLA-B and / or HLA-C expression in a reference iPSC in which the B2M gene is not disrupted;

[0332] (iii) generating the disrupted B2M gene by contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene, wherein:

[0333] a. the RNA-guided endonuclease is Cas12a (Cpf1); and

[0334] b. The gRNA binds to the complementary sequence of SEQ ID NO:3.

[0335] A35. A population according to embodiment A34, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 (Cas12a).

[0336] A36. A population according to embodiment A34 or A35, wherein at least about 70% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0337] A37. A 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 when measured by flow cytometry.

[0338] 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, wherein:

[0339] (i) at least about 50% to 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry;

[0340] (ii) said iPSCs comprising a disrupted B2M gene exhibit reduced expression of HLA-A, HLA-B and / or HLA-C compared to said HLA-A, HLA-B and / or HLA-C expression in a reference iPSC in which the B2M gene is not disrupted;

[0341] (iii) generating the disrupted B2M gene by contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene, wherein:

[0342] a. the RNA-guided endonuclease is MAD7; and

[0343] b. The gRNA binds to the complementary sequence of SEQ ID NO:3.

[0344] A39. A population according to embodiment A38, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 (Cas12a).

[0345] A40. A population according to embodiment A38 or A39, wherein at least about 70% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0346] A41. A population according to any one of embodiments A38 to 40, wherein at least about 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0347] 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, wherein:

[0348] (i) at least about 50% to 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry;

[0349] (ii) the iPSCs comprising a 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 a reference iPSC in which the B2M gene is not disrupted.

[0350] (iii) generating the disrupted B2M gene by contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene, wherein:

[0351] a. the RNA-guided endonuclease is Cas12a (Cpf1); and

[0352] b. The gRNA binds to the complementary sequence of SEQ ID NO:4.

[0353] A43. A population according to embodiment A38, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 (Cas12a).

[0354] A44. A population according to embodiment A42 or A43, wherein at least about 70% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0355] A45. A population according to any one of embodiments A42 to A44, wherein at least about 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0356] 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, wherein:

[0357] (i) at least about 50% to 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry;

[0358] (ii) the iPSCs comprising a 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 a reference iPSC in which the B2M gene is not disrupted.

[0359] (iii) generating the disrupted B2M gene by contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene, wherein:

[0360] a. the RNA-guided endonuclease is MAD7; and

[0361] b. The gRNA binds to the complementary sequence of SEQ ID NO:4.

[0362] A47. A population according to embodiment A46, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 (Cas12a).

[0363] A48. A 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.

[0364] A49. A population according to any one of embodiments A46 to A48, wherein at least about 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0365] A50. 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, wherein:

[0366] (iv) at least about 50% to 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry;

[0367] (v) the iPSCs comprising a 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 a reference iPSC in which the B2M gene is not disrupted.

[0368] (vi) generating the disrupted B2M gene by contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene, wherein:

[0369] a. the RNA-guided endonuclease is Cas12a (Cpf1); and

[0370] b. The gRNA binds to the complementary sequence of SEQ ID NO:5.

[0371] A51. The induced pluripotent stem cell (iPSC) population of embodiment A46, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1).

[0372] A52. A population according to embodiment A50 or A51, wherein at least about 70% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0373] A53. A population according to any one of embodiments A50 to A52, wherein at least about 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0374] A54. 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, wherein:

[0375] (i) at least about 50% to 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry;

[0376] (ii) the iPSCs comprising a 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 a reference iPSC in which the B2M gene is not disrupted.

[0377] (iii) generating the disrupted B2M gene by contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene, wherein:

[0378] a. the RNA-guided endonuclease is MAD7; and

[0379] b. The gRNA binds to the complementary sequence of SEQ ID NO:5.

[0380] A55. The induced pluripotent stem cell (iPSC) population of embodiment A50, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1).

[0381] A56. 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.

[0382] A57. A population according to any one of embodiments A54 to A56, wherein at least about 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0383] 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, wherein:

[0384] (i) at least about 50% to 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry;

[0385] (ii) the iPSCs comprising a 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 a reference iPSC in which the B2M gene is not disrupted.

[0386] (iii) generating the disrupted B2M gene by contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene, wherein:

[0387] a. the RNA-guided endonuclease is Cas12a (Cpf1); and

[0388] b. The gRNA binds to the complementary sequence of SEQ ID NO:6.

[0389] A59. The induced pluripotent stem cell (iPSC) population of embodiment A54, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1).

[0390] 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.

[0391] A61. A population according to any one of embodiments A58 to A60, wherein at least about 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0392] A62. 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, wherein:

[0393] (i) at least about 50% to 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry;

[0394] (ii) the iPSCs comprising a 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 a reference iPSC in which the B2M gene is not disrupted.

[0395] (iii) generating the disrupted B2M gene by contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene, wherein:

[0396] a. the RNA-guided endonuclease is MAD7; and

[0397] b. The gRNA binds to the complementary sequence of SEQ ID NO:6.

[0398] A63. The induced pluripotent stem cell (iPSC) population of embodiment A58, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1).

[0399] A64. 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, wherein:

[0400] (i) at least about 50% to 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry;

[0401] (ii) the iPSCs comprising a 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 a reference iPSC in which the B2M gene is not disrupted.

[0402] (iii) generating the disrupted B2M gene by contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene, wherein:

[0403] a. The RNA-guided nuclease is MAD7; and the gRNA binds to the complementary sequence of SEQ ID NO: 17.

[0404] A65. A population according to embodiment A62 or A63, wherein at least about 70% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0405] A66. A population according to any one of embodiments A62 to A65, wherein at least about 100% of the iPSCs do not express detectable levels of B2M protein when measured by flow cytometry.

[0406] A67. A population according to any of the preceding embodiments, wherein the disruption comprises an insertion of at least one nucleotide base pair, wherein the insertion is an expression cassette, wherein the expression cassette encodes a therapeutic cargo, a therapeutic protein, a TCR, or a chimeric antigen receptor.

[0407] A68. A population according to any of the preceding embodiments, wherein the complementary sequence comprises any one of SEQ ID NOs: 19-23.

[0408] In another group of embodiments (Group B embodiments), there is provided:

[0409] B1. A method for producing induced pluripotent stem cells (iPSCs), wherein the method comprises:

[0410] (a) contacting an isolated population of cells with an activation culture; wherein the activation culture comprises IL-15 and zoledronic acid;

[0411] (b) culturing the isolated population of cells in the activated culture to enrich and / or activate γδ T cells in the isolated population of cells;

[0412] (c) transducing the γδ T cells with a viral vector encoding one or more reprogramming factors;

[0413] (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state, thereby generating a population of iPSCs; and

[0414] (e) contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA);

[0415] wherein the gRNA binds to a target motif of a beta-2-microglobulin (B2M) polynucleotide sequence in the iPSC population; and

[0416] wherein said contacting results in cleavage of said B2M polynucleotide sequence.

[0417] B2. A method according to embodiment B1, wherein the RNA-guided nuclease is selected from 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, Csm 6. The group consisting of 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.

[0418] B3. A method according to embodiment B2, wherein the RNA-guided nuclease is Cas12a (Cpf1).

[0419] B4. A method according to embodiment B2, wherein the RNA-guided nuclease is MAD7.

[0420] B5. The method according to any one of embodiments B1 to B4, wherein the target motif comprises a portion of SEQ ID NO: 1.

[0421] B6. A method according to any one of embodiments B1 to B4, wherein the target motif comprises the sequence of any one of SEQ ID NO: 2-6 or 17.

[0422] B7. A method according to any one of embodiments B1 to B4, wherein the target motif consists of the sequence of any one of SEQ ID NO: 2-6 or 17.

[0423] B8. A method according to any one of embodiments B1 to B7, wherein the gRNA comprises the sequence of any one of SEQ ID NO:7-11 or 18.

[0424] B9. A method according to any one of embodiments B1 to B7, wherein the gRNA comprises SEQ ID NO:18.

[0425] B10. A method according to any one of embodiments B1 to B7, wherein the gRNA consists of the sequence of any one of SEQ ID NO: 7-11 or 18.

[0426] B12. The method according to any one of embodiments B1 to B7, wherein the gRNA consists of SEQ ID NO: 18.

[0427] B13. The method of any one of embodiments B1 to B12, wherein the cleavage of the B2M polynucleotide sequence results in reduced B2M expression in the iPSCs compared to B2M expression in a reference.

[0428] B14. A 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% compared to the B2M expression in a reference.

[0429] B15. The method according to embodiment B13 or B14, wherein the reduced B2M expression is reduced by about or at least about 70% compared to the B2M expression in a reference.

[0430] B16. A method according to any one of embodiments B1 to B15, wherein the cleavage of the B2M polynucleotide sequence results in 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 a reference.

[0431] B17. A 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% compared to the HLA-A, HLA-B and / or HLA-C expression in a reference.

[0432] B18. The method according to any one of embodiments B13 to B17, wherein the reference is an iPSC or an iPSC population that has not cut the B2M polynucleotide sequence.

[0433] B19. The method of any one of embodiments B1 to B18, wherein the activation culture further comprises IL-2.

[0434] B20. The method according to any one of embodiments B1 to B19, wherein the viral vector is a Sendai virus (SeV) vector.

[0435] B21. A method according to any one of embodiments B1 to B20, wherein the method further comprises obtaining an isolated population of the cells from a subject.

[0436] B22. The method of any one of embodiments B1 to B21, wherein the cells in the isolated population of cells are peripheral blood mononuclear cells (PBMCs).

[0437] B23. A method according to any one of embodiments B1 to B22, wherein the cells in the isolated population of cells are terminally differentiated cells.

[0438] B24. A method according to any one of embodiments B1 to B23, wherein the cells in the isolated population of cells are mammalian cells.

[0439] B25. A method according to any one of embodiments B1 to B24, wherein the cells in the isolated population of cells are human cells.

[0440] B26. A method according to any one of embodiments B22 to B25, wherein the isolated population of cells is cultured in the activated culture for at most 13 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days or at most 1 day.

[0441] B27. A method according to embodiment B26, wherein the isolated population of cells is cultured in the activated culture for up to 3 days.

[0442] B28. A method according to embodiment B26, wherein the isolated population of cells is cultured in the activated culture for 3 days.

[0443] B29. A method according to any one of embodiments B1 to B28, wherein after culturing in the activated 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.

[0444] B30. A method according to embodiment B29, wherein after culturing in the activated culture, the isolated population of cells comprises less than 35% γδ T cells.

[0445] B31. The method according to any one of embodiments B1 to B30, further comprising enriching the γδ T cells in the isolated population of cells after step (b).

[0446] B32. A method according to embodiment B31, wherein the γδ T cells are enriched by cell-cell aggregate enrichment.

[0447] B33. The method according to any one of embodiments B1 to B32, wherein at least a portion of the γδ T cells are activated in step (b) to become Vγ9 + γδ T cells.

[0448] B34. A method according to any one of embodiments B1 to B32, wherein at least a portion of the γδ T cells are activated into Vγ9δ2+γδ T cells in step (b).

[0449] B35. The method according to any one of embodiments B1 to B34, wherein the one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28 and c-Myc.

[0450] B36. The method according to any one of embodiments B1 to B35, wherein in step (d), the transduced γδ T cells are cultured in the presence of one or more feeder layers.

[0451] B37. A method according to embodiment B36, wherein in step (d), the transduced γδ T cells are cultured in the presence of a monolayer feeder layer.

[0452] B38. A method according to embodiment B36 or B37, wherein the feeder layer comprises mouse embryonic fibroblasts (MEF).

[0453] B39. The method according to any one of embodiments B1 to B38, further comprising isolating and / or purifying the generated iPSCs.

[0454] B40. The method according to any one of embodiments B1 to B39, further comprising differentiating the iPSCs into cells of a desired cell type ex vivo, thereby generating differentiated IPSCs.

[0455] B41. The method according to any one of embodiments B1 to B40, wherein the generated iPSCs are negative for Sendai virus (SeV) vectors.

[0456] B42. The method according to any one of embodiments B1 to B41, wherein the iPSCs produced are derived from γδ T cells.

[0457] B43. The method according to any one of embodiments B1 to B41, wherein the iPSCs produced have rearranged genes of the TRG and TRD loci; and wherein optionally the iPSCs produced have Vγ9 and Vδ2 gene arrangements.

[0458] B44. The method according to any one of embodiments B1 to B41, wherein the iPSCs generated are not derived from αβ T cells.

[0459] B45. A method according to any one of embodiments B1 to B44, wherein the iPSCs generated do not produce or express TCRA and / or TCRB or fragments thereof, such that there is no detectable or otherwise detectable surface expression of TCRA and TCRB.

[0460] B46. The method according to any one of embodiments B1 to B45, wherein the generated iPSCs are genomically stable without chromosome loss.

[0461] B47. A method according to embodiment B46, wherein the genomic stability of the generated iPSCs is determined by karyotyping.

[0462] B48. The method according to any one of embodiments B1 to B47, wherein the generated iPSCs are capable of growing in feeder-free medium after adoptive transfer.

[0463] B49. A method according to any one of embodiments B1 to B48, wherein the RNA-guided nuclease is Cas12a (Cpf1).

[0464] B50. A method for producing induced pluripotent stem cells (iPSCs), wherein the method comprises:

[0465] (a) contacting an isolated population of human PBMCs with an activation culture; wherein the activation culture comprises IL-15, IL-2, and zoledronic acid;

[0466] (b) culturing the isolated population of cells in the activated culture to enrich and / or activate γδT cells in the isolated population of cells, wherein at least a portion of the γδT cells are activated to Vγ9δ2 + γδT cells;

[0467] (c) transducing the γδT cells with a Sendai virus vector encoding one or more reprogramming factors;

[0468] (d) culturing the transduced γδ T cells under conditions suitable for reprogramming mammalian somatic cells to a pluripotent state, thereby generating a population of iPSCs; and

[0469] (e) contacting the iPSC population with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA), wherein the RNA-guided endonuclease is Cas12a or MAD7;

[0470] wherein the gRNA binds to a target motif of a beta-2-microglobulin (B2M) polynucleotide sequence in the iPSC population; and

[0471] wherein said contacting results in cleavage of said B2M polynucleotide sequence, wherein said cleavage of said B2M polynucleotide sequence results in reduced expression of B2M in said iPSCs compared to B2M expression in a population of iPSCs in which said B2M polynucleotide sequence has not been cleaved;

[0472] iPSCs generated from:

[0473] Negative for Sendai virus (SeV) vector; and

[0474] TCRA and / or TCRB or fragments thereof are not produced or expressed such that there is no detectable or otherwise detectable surface expression of TCRA and TCRB.

[0475] B51. A method according to any one of the preceding embodiments B1 to B48, wherein the method further comprises selecting B2M-negative cells from the cell population.

[0476] B52. A method according to embodiment B51, wherein the selection is performed by flow cytometric cell sorting, dilution plating of single cells per well of a multi-well plate, magnetic bead enrichment, or a box-based cell sorter.

[0477] B53. A method according to any one of embodiments B1 to B52, wherein the method further comprises introducing a vector encoding a knock-in box for inserting a B2M polynucleotide sequence.

[0478] B54. A method according to embodiment B53, wherein the method further comprises introducing a vector encoding a transposase, a recombinase or an integrase, wherein the vector encoding the transposase, the recombinase or the integrase is used to insert the knock-in box.

[0479] B55. A method according to embodiment B54, wherein the transposase, recombinase or integrase is conjugated to a targeting moiety, wherein the moiety comprises a zinc finger, a transcription activator-like effector or a nuclease-deficient CRISPR / CAS molecule.

[0480] B56. A method according to any one of embodiments B53 to B55, wherein the knock-in box encodes a therapeutic cargo, a therapeutic protein, a TCR, or a chimeric antigen receptor.

[0481] B57. An induced pluripotent stem cell (iPSC), which is produced according to the method of any one of embodiments B1 to B56.

[0482] B58. The iPSC population according to embodiment B1, wherein the iPSCs are generated according to the method of any one of embodiments B1 to B57.

[0483] B58. A composition comprising the iPSC according to embodiment B57.

[0484] B56. A differentiated IPSC produced according to the method of embodiment B39.

[0485] B57. A B2M-negative cell produced by any one of embodiments B1 to B56.

[0486] B58. A B2M-negative induced pluripotent stem cell for use in the manufacture of a composition for use in treating a subject in need thereof.

[0487] B59. A B2M-negative induced pluripotent stem cell for use in treating a subject in need thereof.

[0488] B60. The B2M-negative induced pluripotent stem cell for use according to embodiment B58 or embodiment 59, wherein the cell further comprises a therapeutic cargo, a therapeutic protein, a TCR or a chimeric antigen receptor.

[0489] B61. A method according to any one of embodiments B1 to B56, wherein the gRNA binds to a complementary sequence.

[0490] B62. A method according to embodiment B61, wherein the complementary sequence comprises any one of SEQ ID NOs: 19-24.

[0491] In another group of embodiments (Group C embodiments), there is provided:

[0492] C1. A method for producing induced pluripotent stem cells (iPSCs), the method comprising:

[0493] (a) performing a step of enriching and / or activating the function of γδ T cells in the isolated population of cells;

[0494] (b) performing a step of reprogramming the γδ T cells to a pluripotent state to generate iPSCs; and

[0495] (c) contacting the iPSC with an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease and a guide RNA (gRNA);

[0496] wherein the gRNA binds to a target motif of a beta-2-microglobulin (B2M) polynucleotide sequence in the iPSC; and

[0497] wherein the contacting step results in cleavage of the B2M polynucleotide sequence.

[0498] C2. An induced pluripotent stem cell (iPSC), which is produced according to the method described in embodiment C1.

[0499] C3. An isolated population of induced pluripotent stem cells (iPSCs), the isolated population of iPSCs comprising pluripotent cells, wherein the pluripotent cells comprise tools for expressing one or more reprogramming factors, and / or wherein the pluripotent cells comprise tools for rearrangement of genes encoding TRG and TRD, and wherein the pluripotent cells comprise tools for cutting the B2M gene.

[0500] C4. An isolated population of induced pluripotent stem cells (iPSCs), wherein the pluripotent cells comprise means for expressing one or more reprogramming factors, and / or wherein the cells comprise means for rearrangement of genes encoding TRG and TRD, and wherein the cells comprise means for cleaving the B2M gene.

[0501] 7. Examples

[0502] The following is a description of the various methods and materials used in the study. They are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of the invention that the inventor believes, nor are they intended to represent that the following experiments are performed and are all experiments that can be performed. It should be understood that the exemplary descriptions written in the present tense do not necessarily have to be performed, but these descriptions can be performed to generate data associated with the teachings of the present invention, etc. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be considered.

[0503] 7.1. Example 1: Generation and characterization of γδ iPSC cells with β2m knockout

[0504] This example describes the use of nucleases to produce a γδ iPSC cell line with knockout β2m. The method for producing γδ iPSC cells is disclosed in WO2021 / 257679 (PCT / US2021 / 037594), which is incorporated herein by reference in its entirety. Specifically, the γδ iPSC cells used herein correspond to clone B described in WO2021 / 257679.

[0505] iMatrix-511 coated 6-well plate

[0506] The 6-well plate was 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, about 1 mL to about 1.5 mL of the diluted iMatrix-511 laminin was added to each well of the 6-well plate and incubated at 37°C, 5% CO2 for 1 hour.

[0507] 7.1.1. Preparation of reagents

[0508] The reagents used in this example are provided in Table 2.

[0509] Table 2: Reagent List

[0510]

[0511] Preparation of crRNA

[0512] First, the vial containing the Alt-R crRNA was centrifuged. Then each Alt-R crRNA was dissolved to 100 μM using nuclease-free IDTE buffer (Table 3).

[0513] Table 3: Preparation of crRNA

[0514]

[0515] The vial was vortexed to provide a homogenous 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. The crRNA was aliquoted in 5 μL portions and stored at -20 °C. Each crRNA was used for no more than 3 freeze-thaw cycles.

[0516] Preparation of enhancer

[0517] Resuspend Alt-R Nuclease Electroporation Enhancer to 100 μM in IDTE buffer to prepare a stock solution, as provided in Table 4.

[0518] Table 4: Preparation of enhancers

[0519] Electroporation Enhancer Amount (nmol) Concentration (μM) Volume (μL) Alt-R Nuclease Electroporation Enhancer 2 100 20

[0520] Transfection

[0521] RNP assembly

[0522] For each crRNA, the crRNA was combined with a nuclease as provided in Table 5. The transfection mixture was incubated at room temperature for 20 to 30 minutes.

[0523] Table 5: Assembly of ribonucleoprotein (RNP)

[0524]

[0525] Cell harvesting

[0526] The culture medium composition was prepared as shown in Table 6.

[0527] Table 6: Culture medium composition

[0528] Reagents volume StemFit Basic 04 40mL bFGF (0.3 mg / mL) 2.66μL Pen / Strep 400μL

[0529] Harvest cells when they reach approximately 70% to 80% confluence. Aspirate spent media from the wells and discard. Then rinse cells with 1 mL of DPBS per well and add 1 mL of ReLeSR to each well of a 6-well tissue culture treated plate. Incubate the plate in an incubator with 5% CO2 at 37°C for 45 to 60 seconds. After incubation, aspirate ReLeSR and discard. The 6-well tissue culture treated plate containing cells is further incubated with 5% CO2 at 37°C for 5 minutes.

[0530] The cells were rinsed with about 1 mL of fresh StemFit Basic 04 complete growth medium and the separated cells were collected in a 15 mL centrifuge tube containing 3 mL of fresh complete growth medium (StemFit Basic 04). The cells were then 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.

[0531] Then use trypan blue and a hemocytometer to count the cells. The formula for cell counting is: total number of cells per mL = (total number of cells counted in 4 squares / 4) × dilution factor × 10 4 Cells / mL. Typically, 50,000 cells were collected per tube, pooled together, and centrifuged at 800 RPM for 2 minutes at room temperature. The supernatant was then removed without disturbing the pellet. The cells were then resuspended by adding 10 μL of resuspension buffer T per tube (containing 50,000 cells) for one electroporation.

[0532] About 8 μL of cell suspension was mixed with 2 μL of electroporation enhancer and 2 μL of RNP complex. For untransfected controls, only 2 μL of enhancer was added to the cell suspension.

[0533] Neon transfection

[0534] For neon transfection, the electroporation settings of the input system are as follows: 1400V, 20 milliseconds, and 1 pulse. The neon pipette station is established by the following method: fill the neon tube with 3mL electrolysis buffer (E2) and insert it into the station. Insert the 10μL neon tip into the neon pipette and move the 10μL mixture into the neon tip to avoid bubbles. Then insert the 10μL neon tip containing the mixture into the station and start electroporation. After electroporation, the cells are transferred to a well containing 2mL of StemFit Basic 04 complete growth medium containing ROCK inhibitor (final concentration of 10μM). Process 6-well plates as shown in Table 7.

[0535] Table 7: 6-well plate diagram

[0536]

[0537] The cells were incubated overnight at 37°C, 5% CO2. The morphology of the cells was observed under a microscope every day, the spent medium was discarded, and the wells were replenished with fresh complete medium. Once the cells reached confluence, the cells were harvested using ReLeSR for further downstream applications.

[0538] Results

[0539] γδ iPSC cells were transfected with the following substances: 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 gRNA sequences and target sequences. The gRNA sequence can be modified to increase the stability of short RNA (e.g., gRNA). In some embodiments, these modifications are AltR1 and / or AltR2. AltR1 and / or AltR2 modifications are end blocking, modifications at both ends of the gRNA sequence, or modifications at one end of the gRNA sequence.

[0540] Table 8: gRNA sequences and target sequences

[0541]

[0542] Five days after electroporation, phase contrast images of cells were recorded, as Figure 1 γδ iPSC cells were electroporated with crRNA knocking out β2m and nuclease and imaged at 5x and 10x magnification 5 days after electroporation ( Figure 1 ). Seven days after electroporation, the cells were examined for β2m expression by flow cytometry ( Figure 2 ). Cells were gated based on FSC-H and SSC-H (Gated E1). Gated E1 cells were further selected for the live population based on Pacific Blue (live / dead) staining. Live cells were then singlet gated based on FSC-H and FSC-A. The β2m-specific population was gated using singlets based on their respective FMOs. Cells transfected with β2m_nuclease_gRNA4 and β2m_nuclease_gRNA5 did not show any significant reduction in β2m expression ( Figure 2 However, cells transfected with β2m_nuclease_gRNA1, β2m_nuclease_gRNA2, and β2m_nuclease_gRNA3 showed a significant decrease in β2m expression 7 days after electroporation ( Figure 2 ). Figure 3Shown are 5x, 10x and 20x magnified γδ iPSC images 7 days after FACS sorting. The knockout (KO) efficiency observed after 7 days of treatment with each crRNA is summarized in Table 9.

[0543] Table 9: gRNA and knockout percentage

[0544]

[0545] In addition, cells were expanded and cell sorting was performed for the double negative population (β2m and HLA-A, HLA-B and HLA-C) in the total electroporated pool 14 days after electroporation ( Figure 4 ). After FACS sorting, the cells were expanded and β2m and HLA-A, HLA-B, HLA-C expression was checked again by flow cytometry 22 days after electroporation. The cells were gated based on FSC-H and SSC-H (gated E1). Based on Pacific Blue (live / dead) staining, gated E1 cells were further selected for live populations. The live cells were then singlet gated 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 cytometric analysis showed that β2m and HLA-A, HLA-B, HLA-C protein levels of cells electroporated with crRNA 1, 2, and 3 showed a 93.2%, 96.6%, and 91.1% decrease, respectively ( Figure 5 ).

[0546] This embodiment illustrates the successful production of clones of knockout β2m genes produced from γδiPSC 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 produced with >90% KO efficiency in 22 days after electroporation in γδiPSC cells using CRISPR nuclease technology. The time spent producing β2m knockouts in γδiPSC cells using CRISPR nuclease technology is about 4 weeks.

[0547] Table 10: Sequence

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562] 7.2 Example 2 Transgene insertion in human γδ T cell-derived iPSCs

[0563] A. Preparation of RNP complexes

[0564] β2m_nuclease_gRNA6Alt-R crRNA (SEQ ID NO: 18) was synthesized (IDT) and dissolved in nuclease-free duplex buffer (IDT#11-05-01-12) at 200 μM. As, the RNP complex was freshly prepared on the day of nuclear transfection. Cas12a (Cpf1) V3 (IDT#1081068), 200 pmol crRNA (1 μl containing 200 μM) dissolved in P3 buffer (Lonza #V4SP-3096) and 110 μg PGA (Sigma #P4761) were incubated at room temperature for 30 minutes. After incubation, 3 μM electroporation enhancer (IDT #1076301) was added to a total volume of 10 μL.

[0565] B. Generation of edited human iPSCs

[0566] Human γδT cell-derived iPS cells (referred to herein as "iPSCs") were pretreated with 10 μM Y-27632ROCK inhibitor (STEMCELL Technologies #72302) in Stemfit Basic 04 complete type medium (Ajinomoto #Basic04CT). iPSCs (0.5e6 cells / reactant) were collected and resuspended in 10 μL P3 buffer containing 3 μM electroporation enhancer. 2 μg HDR template (PCR amplicon with GFP) was added to the cells and incubated at room temperature for 1 minute. The cells were merged with 10 μL RNP complexes and nuclear transfection was performed in a 96-well cuvette (Lonza #V4SP-3096) using the CA-137 program on the Lonza 4D system. The iPSCs were then transferred to a well of a 24-well plate coated with 0.5 μg / cm2 of iMatrix-511 (Takara #T304) containing 0.5 mL of Stemfit Basic04 medium containing 10 μM Y-27632 ROCK inhibitor. iPSCs were expanded into 6-well plates two days after nucleofection, and the medium was changed every day until day 5, when pluripotency markers, surface expression of HLA class I, and expression of GFP were measured by flow cytometry. Figure 6 As shown, 12.7% of human γδ T cell-derived iPSC cells were positive for GFP and negative for B2M protein after electroporation with B2M-targeted RNPs combined with a DNA plasmid repair template encoding GFP.

[0567] Thus, targeted insertion of transgenes into B2M in human γδ T cell-derived iPSCs was achieved.

[0568] 7.3 Example 3 Production of γδT cell-derived iPSCs

[0569] A. Preparation of RNP complexes

[0570] β2m_nuclease_gRNA6 Alt-R crRNA (SEQ ID NO: 18) was synthesized (IDT) and dissolved in nuclease-free duplex buffer (IDT#11-05-01-12) at 200 μM. As, RNP complexes were freshly prepared on the day of nuclear transfection. Cas12a (Cpf1) V3 (IDT#1081068) or MAD7 (Aldevron Eureca-VMAD7), 200 pmol crRNA (1 μl containing 200 μM) dissolved in P3 buffer (Lonza #V4SP-3096) and 110 μg PGA (Sigma #P4761) were incubated at room temperature for 30 minutes. After incubation, 3 μM electroporation enhancer (IDT #1076301) was added to a total volume of 10 μL.

[0571] B. Generation of edited human iPSCs

[0572] Human γδT cell-derived iPS cells (referred to herein as "iPSCs") were pretreated with 10 μM Y-27632ROCK inhibitor (STEMCELL Technologies #72302) in Stemfit Basic 04 complete type medium (Ajinomoto #Basic04CT). iPSCs (0.5e6 cells / reactant) were collected and resuspended in 10 μL P3 buffer containing 3 μM electroporation enhancer. The cells were merged with 10 μL RNP complexes and nuclear transfection was performed in a 96-well cuvette (Lonza #V4SP-3096) using the CA-137 program on the Lonza 4D system. The iPSCs were then transferred to a 50 μL PBS containing 0.5 μg / cm 2 iPSCs were expanded into 6-well plates two 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. Figure 7 As shown, 83.2% and 87.9% of human γδ T cell-derived iPSC cells were negative for B2M protein after electroporation with Cas12a- or MAD7-generated RNPs targeting B2M, respectively.

[0573] Therefore, after electroporation with RNPs targeting B2M, human γδ T cell-derived iPSC cells that were negative for B2M protein were obtained.

[0574] 7.4 Example 4. Human γδT cell-derived iPSCs with CAR

[0575] A. Preparation of RNP complexes

[0576] β2m_nuclease_gRNA6 Alt-R crRNA (SEQ ID NO: 18) (Table 10) (IDT) was synthesized and dissolved in nuclease-free duplex buffer (IDT#11-05-01-12) at 200 μM. As, the RNP complex was freshly prepared on the day of nuclear transfection. Cas12a (Cpf1) V3 (IDT # 1081068) was incubated with 200 pmol crRNA (1 μl containing 200 μM) and 110 μg PGA (Sigma # P4761) dissolved in P3 buffer (Lonza # V4SP-3096) at room temperature for 30 minutes. After incubation, 3 μM electroporation enhancer (IDT # 1076301) was added to a total volume of 10 μL.

[0577] B. Generation of edited human iPSCs

[0578] Human γδT cell-derived iPS cells (referred to herein as "iPSC") were pretreated with 10 μM Y-27632ROCK inhibitor (STEMCELL Technologies #72302) in Stemfit Basic 04 complete type medium (Ajinomoto #Basic04CT). iPSCs (0.5e6 cells / reactant) were collected and resuspended in 10 μL P3 buffer containing 3 μM electroporation enhancer. 3.5 μg HDR template (DNA plasmid with GFP) was added to the cells and incubated at room temperature for 1 minute. The cells were merged with 10 μL Cas12a:crRNA RNP complexes and nuclear transfection was performed in a 96-well cuvette (Lonza #V4SP-3096) using the CA-137 program on the Lonza 4D system. The iPSCs were then transferred to a 4D plate with 0.5 μg / cm 2 iPSCs were cultured in one well of a 24-well plate coated with iMatrix-511 (Takara #T304) containing 0.5 mL of Stemfit Basic 04 medium containing 10 μM Y-27632 ROCK inhibitor. iPSCs were expanded into 6-well plates two days after nucleofection, and the medium was changed every day until day 5, when pluripotency markers, surface expression of HLA class I, and expression of BCMACAR were measured by flow cytometry. Figure 8 As shown, 2.6% of human γδ T cell-derived iPSC cells were positive for the CAR transgene and negative for B2M protein after electroporation with B2M-targeting RNPs combined with a DNA plasmid repair template encoding the CAR.

[0579] Thus, human γδ T cell-derived iPSC cells that were positive for the CAR transgene and negative for the B2M protein were obtained after treatment with B2M-targeting RNPs combined with a CAR-encoding DNA plasmid repair template.

Claims

1. An induced pluripotent stem cell (iPSC) population, wherein the iPSCs are generated by reprogramming γδ T cells, and the population comprises iPSCs containing a disrupted β-2-microglobulin (B2M) gene.

2. The iPSC population of claim 3, wherein the disrupted B2M gene comprises a deletion of at least a portion of the nucleotide sequence of SEQ ID NO: 1 or about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 90%, 95%, 96%, 97%, 98%, 99% or 100% missing.

3. The iPSC population of claim 3, wherein the nucleotide sequence of the B2M gene encodes a nucleotide sequence that is about, at least about, or at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.

4. The iPSC population of any one of claims 3 to 3, 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.

5. The iPSC population according to any one of claims 3 to 5, wherein the disruption comprises a deletion of at least one nucleotide base pair.

6. The iPSC population according to any one of claims 3 to 5, wherein the disruption comprises an insertion of at least one nucleotide base pair.

7. The iPSC population according to any one of claims 3 to 6, wherein the disrupted B2M gene exhibits reduced B2M expression relative to a non-disrupted B2M gene.

8. The iPSC population of claim 7, wherein the decreased B2M expression is decreased by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 11 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

9. The iPSC population of any one of claims 3 to 8, wherein the iPSCs comprising a disrupted B2M gene exhibit reduced HLA-A, HLA-B and / or HLA-C expression compared to HLA-A, HLA-B and / or HLA-C expression in a reference iPSC.

10. The iPSC population of claim 9, wherein the decreased HLA-A, HLA-B and / or HLA-C expression is decreased by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 80%, 90%, 90%, 100%, 110%, 111%, 120%, 125%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 250%, 251%, 252%, 253%, 254%, 255%, 256%, 257%, 258%, 259%, 260%, 261%, 262%, 263%, 264%, 265%, 266%, 267%, 268%, 269%, 270%, 271%, 272%, 273%, 274%, 275%, 276%, 277%, 278%, 279%, 280%, 281%, 282%, 283%, 284%, 285%, 286%, 287%, 288%, 289%, 290%, 291%, 292%, 293%, 294%, 295%, 301%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. 11 . The iPSC population according to any one of claims 8 to 10 , wherein the reference iPSC is an iPSC population in which the B2M gene is not disrupted.

12. The iPSC population according to any one of claims 1 to 11, wherein the disrupted B2M gene is produced by contacting the iPSC population with an RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease and a guide RNA (gRNA), and wherein the gRNA binds to a target motif of the B2M gene.

13. The iPSC population of claim 12, 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, C The group consisting of sm6, 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.

14. The iPSC population of claim 13, wherein the RNA-guided endonuclease is Cas12a (Cpf1).

15. The iPSC population of claim 13 or 14, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1).

16. The iPSC population of claim 13, wherein the RNA-guided endonuclease is MAD7.

17. The iPSC population according to any one of claims 12 to 16, wherein the gRNA binds to at least a portion of the complementary sequence of SEQ ID NO:

1.

18. The iPSC population according to any one of claims 12 to 16, wherein the gRNA binds to the complementary sequence of any one of SEQ ID NOs: 2-6 or 17.

19. The iPSC population according to any one of claims 12 to 18, wherein the gRNA comprises the sequence of any one of SEQ ID NOs: 7-11 or 18.

20. The iPSC population according to any one of claims 12 to 18, wherein the gRNA consists of the sequence of any one of SEQ ID NOs: 7-11 or 18.

21. The iPSC population of any one of claims 12 to 18, wherein the gRNA comprises SEQ ID NO:

18.

22. The iPSC population of any one of claims 12 to 18, wherein the gRNA consists of SEQ ID NO:

18.

23. A method for producing induced pluripotent stem cells (iPSCs), wherein the method comprises: (a) contacting an isolated population of cells with an activation culture; wherein the activated culture comprises IL-15 and zoledronic acid; (b) culturing the isolated population of cells in the activated culture to enrich and / or activate γδ T cells in the isolated population of 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 generating a population of iPSCs; and (e) contacting the iPSC population 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 complementary sequence in a target motif of a beta-2-microglobulin (B2M) polynucleotide sequence in the iPSC population; and wherein said contacting results in cleavage of said B2M polynucleotide sequence.

24. The method of claim 23, 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, Csm 6. The group consisting of 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.

25. The method of claim 24, wherein the RNA-guided endonuclease is Cas12a (Cpf1).

26. The method of claim 25, wherein the RNA-guided endonuclease is Acidaminococcus sp. BV3L6 Cas12a (Cpf1).

27. The method of claim 24, wherein the RNA-guided endonuclease is MAD7.

28. The method according to any one of claims 23 to 27, wherein the target motif comprises a portion of SEQ ID NO:

1.

29. The method of any one of claims 23 to 27, wherein the target motif comprises the sequence of any one of SEQ ID NO: 2-6 or 17.

30. The method according to any one of claims 23 to 27, wherein the target motif consists of the sequence of any one of SEQ ID NO: 2-6 or 17.

31. The method of any one of claims 23 to 27, wherein the gRNA comprises the sequence of any one of SEQ ID NOs: 7-11 or 18.

32. The method of any one of claims 23 to 27, wherein the gRNA comprises SEQ ID NO:

18.

33. The method according to any one of claims 23 to 27, wherein the gRNA consists of the sequence of any one of SEQ ID NO: 7-11 or 18.

34. The method of any one of claims 23 to 27, wherein the gRNA consists of SEQ ID NO:

18.

35. The method of any one of claims 23 to 34, wherein the cleavage of the B2M polynucleotide sequence results in reduced expression of B2M in the iPSCs compared to B2M expression in a reference.

36. The method of claim 35, 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% compared to the B2M expression in a reference.

37. The method of any one of claims 23 to 36, wherein said cleavage of said B2M polynucleotide sequence results in decreased expression of HLA-A, HLA-B and / or HLA-C compared to the expression of HLA-A, HLA-B and / or HLA-C in a reference.

38. The method of claim 37, wherein the decreased HLA-A, HLA-B and / or HLA-C expression is decreased by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

39. The method according to any one of claims 35 to 38, wherein the reference is an iPSC or an iPSC population in which the B2M polynucleotide sequence is not cleaved.

40. The method of any one of claims 23 to 39, wherein the activation culture further comprises IL-2.

41. The method according to any one of claims 23 to 40, wherein the viral vector is a Sendai virus (SeV) vector.

42. The method of any one of claims 23 to 41, wherein the method further comprises obtaining an isolated population of the cells from a subject.

43. The method of any one of claims 23 to 42, wherein the cells in the isolated population of cells are peripheral blood mononuclear cells (PBMCs).

44. The method of any one of claims 23 to 43, wherein the cells in the isolated population of cells are terminally differentiated cells.

45. The method of any one of claims 23 to 44, wherein the cells in the isolated population of cells are mammalian cells.

46. ​​The method of any one of claims 23 to 45, wherein the cells in the isolated population of cells are human cells.

47. The method of any one of claims 23 to 46, wherein the isolated population of cells is cultured in the activated culture for at most 13 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days, or at most 1 day.

48. The method of claim 47, wherein the isolated population of cells is cultured in the activated culture for up to 3 days.

49. The method of claim 48, wherein the isolated population of cells is cultured in the activated culture for 3 days.

50. The method of any one of claims 23 to 49, wherein after culturing in the activated culture, the isolated population of 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.

51. The method of claim 50, wherein after culturing in the activation culture, the isolated population of cells comprises less than 35% γδ T cells.

52. The method of any one of claims 23 to 51, further comprising enriching the isolated population of cells for the γδ T cells after step (b).

53. The method of claim 52, wherein the γδ T cells are enriched by cell-cell aggregate enrichment.

54. The method according to any one of claims 23 to 53, wherein at least a portion of the γδ T cells are activated in step (b) to become Vγ9 + γδ T cells.

55. The method according to any one of claims 23 to 54, wherein at least a portion of the γδ T cells are activated in step (b) to become Vγ9δ2 + γδ T cells.

56. The method of any one of claims 23 to 55, wherein the one or more reprogramming factors are selected from the group consisting of OCT3 / 4, SOX2, KLF4, LIN28 and c-Myc.

57. The method of any one of claims 23 to 56, wherein in step (d), the transduced γδ T cells are cultured in the presence of one or more feeder layers.

58. The method of claim 57, wherein in step (d), the transduced γδ T cells are cultured in the presence of a feeder monolayer.

59. The method of claim 57 or 58, wherein the feeder layer comprises mouse embryonic fibroblasts (MEFs).

60. The method of any one of claims 23 to 59, further comprising isolating and / or purifying the generated iPSCs.

61. The method of any one of claims 23 to 60, further comprising differentiating the iPSCs ex vivo into cells of a desired cell type, thereby generating differentiated IPSCs.

62. The method of any one of claims 23 to 61, wherein the generated iPSCs are negative for Sendai virus (SeV) vectors.

63. The method of any one of claims 23 to 62, wherein the iPSCs produced are derived from γδ T cells.

64. The method of any one of claims 23 to 62, wherein the iPSCs produced have rearranged genes of the TRG and TRD loci; and wherein optionally the iPSCs produced have Vγ9 and Vδ2 gene arrangements.

65. The method of any one of claims 23 to 62, wherein the iPSCs produced are not derived from αβ T cells.

66. The method of any one of claims 23 to 62, wherein the iPSCs generated do not produce or express TCRA and / or TCRB or fragments thereof such that there is no detectable or otherwise detectable surface expression of TCRA and TCRB.

67. The method of any one of claims 23 to 62, wherein the iPSCs generated are genomically stable without chromosome loss.

68. The method of claim 67, wherein the genomic stability of the generated iPSCs is determined by karyotyping.

69. The method of any one of claims 23 to 68, wherein the generated iPSCs are capable of growing in feeder-free medium after adoptive transfer.

70. An induced pluripotent stem cell (iPSC) produced according to the method of any one of claims 23 to 69.

71. The iPSC population of claim 1, wherein the iPSCs are generated according to the method of any one of claims 23 to 69.

72. A composition comprising the iPSCs according to claim 70.

73. A differentiated IPSC produced according to the method of claim 61.

74. A method for producing induced pluripotent stem cells (iPSCs), the method comprising: (a) performing a step of enriching and / or activating the function of γδ T cells in the isolated population of cells; (b) performing a step of reprogramming the γδ T cells to a pluripotent state to generate iPSCs; and (c) contacting the iPSC 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 iPSC; and wherein the contacting step results in cleavage of the B2M polynucleotide sequence.

75. An induced pluripotent stem cell (iPSC) produced according to the method of claim 74.

76. An isolated population of induced pluripotent stem cells (iPSCs), the isolated population of iPSCs comprising pluripotent cells, wherein the pluripotent cells comprise tools for expressing one or more reprogramming factors, and / or wherein the pluripotent cells comprise tools for encoding TRG and TRD gene rearrangements, and wherein the pluripotent cells comprise tools for cutting the B2M gene.

77. The iPSC population of any one of claims 1 to 22, wherein the disruption further comprises knocking in a polynucleotide encoding a transgene, wherein the polynucleotide is inserted into the B2M gene.

78. The iPSC population of claim 77, wherein the transgene encodes a chimeric antigen receptor, a TCR, a therapeutic payload, or a therapeutic protein.

79. The iPSC population of claim 77, wherein the polynucleotide is inserted into SEQ ID NO:

1.

80. The method of any one of claims 23 to 69, wherein the method further comprises introducing a vector comprising a gene encoding a transgene, wherein the gene is integrated into the target gene.

81. The method of claim 80, wherein the target gene comprises SEQ ID NO:1.

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