Gamma delta T cell composition and method of use thereof

Abstract

This disclosure provides genetically modified iPSC-derived γδT cells and their progenitor cells. It provides double genomic disruption in the cytokine signaling suppressor 1 (SOCS1) gene and the cytokine-inducible SH2-containing protein (CISH) gene, as well as triple genomic disruption in the SOCS1, CISH, and Bcl-2 interacting cell death mediator (BIM) genes, quadruple genomic disruption in the SOCS1, CISH, BIM, and cell surface cell death receptor (FAS) genes, quintuple genomic disruption in the SOCS1, CISH, BIM, β-2-microglobulin (B2M), and class II transactivator (CIITA) genes, and hexagram genomic disruption in the SOCS1, CISH, BIM, B2M, CIITA, and FAS genes. It also provides genetically modified iPSC-derived γδT cells and their progenitor cells with improved proliferation and tumor-killing activity. Furthermore, it provides genetically modified iPSC-derived γδT cells and their progenitor cells further containing CD19 CAR. This disclosure further provides methods for producing and using such cells, as well as gene editing systems.

Description

[Technical Field]

[0001] Cross-reference of related applications

[0002] This application claims priority and benefits under U.S. Patent Application No. 63 / 565,614 filed on 15 March 2024, U.S. Patent Application No. 63 / 675,543 filed on 25 July 2024, and PCT Application No. PCT / CN2024 / 125782 filed on 18 October 2024, which are incorporated herein by reference in their entirety. [Background technology]

[0003] This disclosure relates to genetically modified gamma delta T cells and their progenitor cells. Gamma delta T cells (γδT cells) are a type of T cell that has a T cell receptor (TCR) on its cell surface composed of γ glycoprotein chains and δ glycoprotein chains. Unlike alpha-beta T cells (αβT cells), which have α glycoprotein chains and β glycoprotein chains and are activated in a major histocompatibility complex-dependent manner, γδT cells do not have such activation limitations. Furthermore, γδT cells can recognize all proteins and can distinguish between normal cells and abnormal cells (e.g., infected cells or cancer cells). γδT cells can be genetically modified to adapt them to specific roles in diagnostic and therapeutic situations.

[0004] The human BIM gene (gene ID: 10018), also known as BCL2L11 or "Bcl-2 Interacting Mediator of cell death," is a BIM gene. EL BIM L , and BIM SIt is transcribed as three splice variants. BIM is a member of the BH3 (Bcl-2 homology 3)-only protein family that can directly activate the apoptosis-promoting effector proteins BAX and BAK. On the other hand, BIM can also indirectly activate BAX and BAK by binding to members of the anti-apoptotic BCL-2 family (BCL-2, BCL-xL, MCL-1, and A1).

[0005] Cytokine-inducible SH2-containing protein (CISH) / (CIS1) (gene ID: 1154, NM_013324.7, NP_037456.5) is a member of the cytokine signaling suppressor (SOCS) protein family. Members of the SOCS family are negative regulators of cytokine signaling that inhibit the JAK / STAT pathway. Each SOCS family member has a central SH2 domain and a conserved carboxy-terminal motif called the SOCS box. These proteins are important regulators of cytokine signaling, proliferation, differentiation, and immune responses.

[0006] The SOCS family consists of at least eight members, including the first identified CIS1, as well as SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, and SOCS7. SOCS1 (Suppressor of cytokine signaling 1), also known as JAB (Janus kinase-binding protein), SSI-1 (Stat-inducing Stat inhibitor-1), gene ID: 8651, NM_003745.2, NP_003736.1, and TIP3 (Tec-interacting protein 3), is a cytokine-regulated member of the SOCS family that directly inhibits each member of the JAK family through interactions within the kinase activation loop. In addition to inhibiting JAK / STAT signaling, SOCS1 can also negatively regulate Toll-like receptors that contribute to innate immunity.

[0007] The human FAS cell surface cell death receptor protein (gene ID: 355, NM_000043.6, NP_000034.1), also known as "FAS" or "CD95," is one of the five tumor necrosis factor (TNF) superfamily cell death receptors. Human FAS can induce caspase-dependent apoptosis after binding to its extracellular ligand (FAS ligand (FASL)). The apoptotic pathway induced by FASL-FAS signaling plays a crucial role in immunomodulation, cancer development, and cancer progression. Furthermore, FASL-FAS signaling also plays a role in the autoregulatory circuitry for the persistence of CAR-engineered lymphocytes.

[0008] β-2-microglobulin (gene ID: 567, NM_004048, NP_004039), also known as "β2M" or "B2M," is a component of human leukocyte antigen (HLA) class I molecules found on the surface of almost all nucleated cells. The B2M gene, which encodes β2M, is located on chromosome 15, part 15 (15q21.1), and consists of four exons. This gene encodes a 12kDa protein. β2M associates with the HLA class I heavy chain non-covalently, enabling proper folding and stabilization of HLA class I. HLA class I molecules without β2M are ultimately degraded by the proteasome in the cytosol. Removal of the β2M gene results in the loss of surface HLA class I expression and a decrease in CD8+ T cell-mediated allogeneic response.

[0009] The class II transactivator gene (gene ID: 4261, NM_001286402.1, NP_001273331.1), also known as "CIITA," encodes a class II transactivator protein. The CIITA gene is located on chromosome 16 (16p13.13) and has 19 exons. This gene encodes a 130 kDa protein. CIITA functions as a master regulator of HLA class II expression by recruiting and promoting the assembly of DNA-binding proteins, including the regulatory factor X (RFX) complex, cAMP response element-binding protein (CREB), and nuclear factor (NF-Y). This complex together enables HLA class II expression. Removal of the CIITA gene results in the loss of surface HLA class II expression and CD4 + T cell-mediated allogeneic responses are reduced.

[0010] Alloimmune recognition and rejection reactions remain obstacles in the development of cell transplantation and cell therapies. The main cause of graft rejection is HLA incompatibility. To suppress these HLA incompatibility-mediated responses, HLA gene removal can be performed in iPSC-derived γδT cells.

[0011] The interferon-gamma receptor (IFNGR) is the cytokine receptor for interferon-gamma (IFN-γ). IFNGR includes a heterodimer of interferon-gamma receptor 1 (IFNGR1) and interferon-gamma receptor 2 (IFNGR2). IFN-γ can signal IFNGR-expressing cells, enabling its immunomodulatory effects, including enhancing antigen presentation via both MHC class I and class II pathways. CD132, also known as the common γ chain (γc) of interleukin-2 receptor or IL2RG, is a cytokine receptor subunit common to at least the cytokine receptor complexes of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The interleukin-2 receptor β subunit, also known as "CD122" or "IL2RB," is the cytokine receptor subunit of the IL-2 cytokine receptor complex (IL2R). IL2R is a protein complex found on the surface of cells, including T cells, NK cells, and other immune cells. IL-2-mediated IL2R signaling promotes the differentiation of T cells into effector T cells and memory T cells. IL-2-mediated IL2R signaling enables downstream signaling pathways via the Janus kinase (JAK) / signal transducer and activator of transcription (STAT) pathway (e.g., STAT1, STAT5). Modulating these signaling pathways may be useful in treating diseases such as cancer, for example, by helping to promote the killing of tumor cells by immune cells.

[0012] CD19 is a protein primarily expressed on the surface of B cells, a type of white blood cell involved in the immune response. CD19 plays a crucial role in the regulation and activation of B cells. It is a co-receptor of the B cell receptor (BCR) and helps enhance signaling pathways that promote B cell activation, survival, and differentiation. Because CD19 is specifically expressed on B cells, it has become a target for certain therapeutic approaches.

[0013] Therapeutic γδT cells remain in demand. Such modified cells are useful in a variety of diagnostic and therapeutic situations, including the treatment of human diseases and disorders. [Overview of the Initiative]

[0014] The inventors hereby demonstrate that genomic disruption in γδT cells (e.g., iPSC-derived γδT) by disrupting 1) the endogenous cytokine signaling suppressor 1 (SOCS1) gene, 2) the endogenous cytokine-inducible SH2-containing protein (CISH) gene, and optionally 3) the endogenous Bcl-2 cell death interaction mediator (BIM) gene, optionally 4) the endogenous FAS cell surface cell death receptor (FAS) gene, optionally 5) the endogenous β-2-microglobulin (B2M) gene, and optionally 6) the endogenous class II transactivator (CIITA) gene confers enhanced cytotoxicity and persistence to γδT cells and / or increases their viability. Such modified cells can be used for a variety of therapeutic and diagnostic purposes.

[0015] In some embodiments, the present disclosure provides modified γδT cells (e.g., derived from primary γδT cells or differentiated from modified iPSCs), comprising: a genomic disruption within an endogenous SOCS1 gene that suppresses or eliminates expression of a functional suppressor of cytokine signaling 1 (SOCS1) protein; and a genomic disruption within an endogenous CISH gene that suppresses or eliminates expression of a functional cytokine-inducible SH2-containing protein (CISH), wherein the modified γδT cells exhibit a synergistic effect in promoting enhanced persistence and / or enhanced cytotoxicity of γδT cells.

[0016] In some embodiments, the present disclosure provides modified γδT cells (e.g., derived from primary γδT cells or differentiated from modified iPSCs), comprising a genomic disruption within exon 2C of an endogenous BIM gene, wherein BIM EL and BIM L the expression level and / or function of splice variants are reduced in the modified cells compared to an unmodified control, and BIM S the expression level and / or function of splice variants are retained in the modified cells, and the modified γδT cells exhibit high survival rate of γδT cells.

[0017] In some embodiments, the present disclosure provides modified γδT cells (e.g., derived from primary γδT cells or differentiated from modified iPSCs), comprising: (1) a genomic disruption within an endogenous SOCS1 gene that suppresses or eliminates expression of a functional suppressor of cytokine signaling 1 (SOCS1) protein; (2) a genomic disruption within an endogenous CISH gene that suppresses or eliminates expression of a functional cytokine-inducible SH2-containing protein (CISH) protein; and (3) a genomic disruption within exon 2C of an endogenous BIM gene, wherein BIM EL and BIM L the expression level and / or function of splice variants are reduced in the modified cells compared to an unmodified control, and BIM SThis invention provides modified γδT cells in which the expression level and / or function of splice variants are retained, resulting in enhanced persistence of γδT, enhanced cytotoxicity of γδT, and good viability of γδT cells.

[0018] In some embodiments, the present disclosure relates to modified γδT cells (e.g., derived from primary γδT cells or differentiated from modified iPSCs) comprising (1) genomic disruption within the endogenous SOCS1 gene to eliminate the expression of functional cytokine signaling repressor 1 (SOCS1) protein, and genomic disruption within the endogenous CISH gene to suppress or eliminate the expression of functional cytokine-inducible SH2-containing protein (CISH), and / or (2) genomic disruption within exon 2C of the endogenous BIM gene. EL and BIM L The expression level and / or function of splice variants are reduced in modified cells compared to unmodified controls, and / or BIM S The expression level and / or function of the splice variant are preserved in the modified cells and, optionally, further (a) genomic disruption in the endogenous FAS cell surface cell death receptor (FAS) gene, optionally, further (b) genomic disruption in the endogenous β-2-microglobulin (B2M) gene, optionally, further (c) genomic disruption in the endogenous class II transactivator (CIITA) gene, and / or optionally, further (d) one or more exogenous polynucleotides encoding an IFNγ signaling factor, characterized in the following: (1) The persistence and / or cytotoxicity of enhanced γδT, (2) Enhanced γδT survival ability, (3) Enhanced sustained lethality (for example, lethality of about 20 rounds in one embodiment), (4) The ability to secrete a larger amount of effector molecules, (5) Provide modified γδT cells that exhibit one or more of the following: (6) improved cell proliferation rate.

[0019] This disclosure also relates to modified iPSCs or intermediate cells differentiated from iPSCs, comprising (1) genomic disruption within the endogenous SOCS1 gene to eliminate the expression of cytokine signaling suppressor 1 (SOCS1), and genomic disruption within the endogenous CISH gene to suppress or eliminate the expression of cytokine-induced SH2-containing protein (CISH), and / or (2) genomic disruption within exon 2C of the endogenous BIM gene. EL and BIM L The expression level and / or function of splice variants are reduced in modified cells compared to unmodified controls, and / or BIM S The invention also provides modified iPSCs or intermediate cells differentiated from iPSCs in which the expression level and / or function of splice variants are preserved in the modified cells and optionally further comprises (a) genomic disruption in the endogenous FAS cell surface cell death receptor (FAS) gene, optionally further (b) genomic disruption in the endogenous β-2-microglobulin (B2M) gene, optionally further (c) genomic disruption in the endogenous class II transactivator (CIITA) gene, and / or optionally further (d) one or more exogenous polynucleotides encoding an IFNγ signaling factor, and which can be used for differentiation into modified γδT cells.

[0020] The modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs described above may further contain exogenous polynucleotides encoding CARs (anti-CD19 CARs) that specifically bind to human CD19.

[0021] In some embodiments, the present disclosure includes modified γδT cells (e.g., derived from primary γδT cells or differentiated from modified iPSCs) comprising an exogenous polynucleotide encoding a CAR (anti-CD19 CAR) that specifically binds to human CD19, and comprising (1) genomic disruption within the endogenous SOCS1 gene to eliminate the expression of functional cytokine signaling repressor 1 (SOCS1) protein, and genomic disruption within the endogenous CISH gene to suppress or eliminate the expression of functional cytokine-inducible SH2-containing protein (CISH) protein, and / or (2) genomic disruption within exon 2C of the endogenous BIM gene, and BIM EL and BIM L The expression level and / or function of splice variants are reduced in modified cells compared to unmodified controls, and / or BIM S The expression level and / or function of the splice variant are preserved in the modified cells and, optionally, further (a) genomic disruption in the endogenous FAS cell surface cell death receptor (FAS) gene, optionally, further (b) genomic disruption in the endogenous β-2-microglobulin (B2M) gene, optionally, further (c) genomic disruption in the endogenous class II transactivator (CIITA) gene, and / or optionally, further (d) one or more exogenous polynucleotides encoding an IFNγ signaling factor, characterized in the following: (1) Specific cytotoxicity against CD19-positive B cells (especially CD19-positive abnormal B cells), (2) Excellent cytotoxicity against CD19-positive B cells (especially CD19-positive abnormal B cells) equivalent to or greater than that of CD19-CARαβ T cells, (3) The ability to secrete a larger amount of effector molecules and / or a smaller amount of inflammatory or immunomodulatory cytokines compared to, for example, CD19-CARαβ T cells. (4) Improved cell proliferation rate, (5) A modified γδT cell is provided that exhibits one or more of the following: enhanced sustained killing ability against CD19-positive B cells (especially CD19-positive abnormal B cells) (for example, killing ability exceeding 35 rounds in one embodiment).

[0022] The disclosure also provides modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, comprising one or more exogenous polynucleotides encoding an IFNγ signaling converter, wherein the IFNγ signaling converter comprises a first IFNγ fusion protein subunit and a second IFNγ fusion protein subunit, the first IFNγ fusion protein subunit comprising the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the intracellular domain of interleukin-2 receptor subunit gamma (IL2RG), the second IFNγ fusion protein subunit comprising the ECD of interferon-gamma receptor 2 (IFNGR2) and the intracellular domain of interleukin-2 receptor subunit beta (IL2RB), and further comprising an exogenous polynucleotide encoding a chimeric antigen receptor (CAR), exhibiting superior sustained killing power and superior cell proliferation (e.g., synergistic effect).

[0023] This disclosure includes, in particular, the following embodiments among those disclosed herein.

[0024] 1. Modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, comprising: genomic disruption within the endogenous SOCS1 gene that suppresses or eliminates the expression of functional cytokine signaling suppressor 1 (SOCS1) protein; and genomic disruption within the endogenous cytokine-induced SH2-containing protein (CISH) gene that suppresses or eliminates the expression of functional CISH protein.

[0025] 2. The modified cell according to Embodiment 1, wherein the genome disruption is located in exon 2 of the SOCS1 gene, and optionally the genome disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

[0026] 3. The modified cell according to Embodiment 1 or Embodiment 2, wherein the genome disruption is located in exon 3 or exon 4 of the CISH gene, and optionally the genome disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

[0027] 4. A modified cell according to any one of Embodiments 1 to 3, wherein the genome disruption is located within a target sequence in SOCS1 including SEQ ID NO: 17 or SEQ ID NO: 23, and / or within a target sequence in CISH including SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 24.

[0028] 5. Modified cells according to any one of Embodiments 1 to 4, wherein the genomic disruption in SOCS1 and CISH is carried out by administering a guide RNA to the cells, and optionally the guide RNA is a single guide RNA (sgRNA) or crRNA, and an endonuclease or nucleic acid encoding the endonuclease is also administered to the cells.

[0029] 6. The modified cell according to Embodiment 5, wherein the guide RNA contains one or more guide sequences from sequence numbers 5, 11, 3, 4, and 12, or one or more sgRNA or crRNA sequences from sequence numbers 29, 37, 27, 28, and 38.

[0030] 7. Modified cells according to any one of Embodiments 1 to 6, further comprising genomic disruption within an endogenous Bcl-2-interacting cell death mediator (BIM) gene.

[0031] 8. The modified cell according to Embodiment 7, wherein the genome disruption within the BIM gene is located within exon 2C of the BIM gene.

[0032] 9. BIM EL and BIM L The expression level and / or function of the splice variant were reduced in the modified cells compared to the unmodified control, and BIM SThe modified cell according to Embodiment 7 or Embodiment 8, wherein the expression level and / or function of the splice variant are retained in the modified cell.

[0033] 10. A modified cell according to any one of Embodiments 7 to 9, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 2C of the endogenous BIM gene, and the mutation may be an insertion, deletion, or substitution.

[0034] 11. A modified cell according to any one of Embodiments 7 to 10, wherein the genome disruption is located within a target sequence in the BIM containing any one of Sequence IDs 14, 18, 19, 20, 21, and 22.

[0035] 12. Modified cells according to any one of Embodiments 7 to 11, wherein the genome disruption within the BIM is carried out by administering a guide RNA to the cells, wherein the guide RNA is optionally a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0036] 13. The modified cell according to Embodiment 12, wherein the guide RNA contains one of the guide sequences among Sequence IDs 2, 6, 7, 8, 9, and 10, or one or more sgRNA or crRNA sequences among Sequence IDs 26, 32, 33, 34, 35, and 36.

[0037] 14. Modified cells according to any one of Embodiments 1 to 13, further comprising genomic disruption within the endogenous FAS gene that suppresses or eliminates the expression of the functional FAS cell surface cell death receptor (FAS) protein.

[0038] 15. The modified cell according to Embodiment 14, wherein the genomic disruption within the FAS gene is located within exon 1 of the FAS gene.

[0039] 16. The modified cell according to Embodiment 14 or Embodiment 15, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 1 of the endogenous FAS gene, and the mutation may be an insertion, deletion, or substitution.

[0040] 17. A modified cell according to any one of embodiments 14 to 16, wherein the genome disruption is located within a target sequence in the FAS containing Sequence ID No. 62.

[0041] 18. Modified cells according to any one of Embodiments 14 to 17, wherein the genome disruption within the FAS is carried out by administering a guide RNA to the cells, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0042] 19. The modified cell according to Embodiment 18, wherein the guide RNA contains the guide sequence of SEQ ID NO: 61 or the sgRNA of SEQ ID NO: 60.

[0043] 20. Modified cells according to any one of Embodiments 1 to 19, further comprising genomic disruption within the endogenous β-2-microglobulin (B2M) gene that suppresses or eliminates the expression of a functional B2M protein.

[0044] 21. The modified cell according to Embodiment 20, wherein the genomic disruption within the B2M gene is located within exon 2 of the B2M gene.

[0045] 22. The modified cell according to Embodiment 20 or Embodiment 21, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 2 of the endogenous B2M gene, and the mutation may be an insertion, deletion, or substitution.

[0046] 23. A modified cell according to any one of embodiments 20 to 22, wherein the genome disruption is located within a target sequence in B2M containing Sequence ID No. 56.

[0047] 24. Modified cells according to any one of Embodiments 20 to 23, wherein the genome disruption within B2M is carried out by administering a guide RNA to the cells, wherein the guide RNA is optionally a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0048] 25. The modified cell according to Embodiment 24, wherein the guide RNA contains the guide sequence of SEQ ID NO: 55 or the sgRNA of SEQ ID NO: 54.

[0049] 26. Modified cells according to any one of Embodiments 1 to 25, further comprising genomic disruption within the endogenous class II transactivator (CIITA) gene that suppresses or eliminates the expression of a functional B2M protein.

[0050] 27. The modified cell according to Embodiment 26, wherein the genome disruption within the CIITA gene is located within exon 3 of the CIITA gene.

[0051] 28. The modified cell according to Embodiment 26 or Embodiment 27, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 3 of the endogenous CIITA gene, and the mutation may be an insertion, deletion, or substitution.

[0052] 29. A modified cell according to any one of embodiments 26 to 28, wherein the genome disruption is located within a target sequence in CIITA containing Sequence ID No. 59.

[0053] 30. Modified cells according to any one of embodiments 26 to 29, wherein the genome disruption within the CIITA is carried out by administering a guide RNA to the cells, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0054] 31. The modified cell according to Embodiment 30, wherein the guide RNA includes the guide sequence of SEQ ID NO: 58 or the sgRNA of SEQ ID NO: 57.

[0055] 32. A modified cell according to any one of Embodiments 1 to 31, further comprising genomic disruption within the endogenous T cell receptor α constant region (TRAC) gene that suppresses or eliminates the expression of a functional TRAC protein.

[0056] 33. A modified cell according to any one of Embodiments 1 to 32, wherein the genome disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

[0057] 34. A modified cell according to any one of Embodiments 1 to 33, further comprising one or more exogenous polynucleotides encoding an IFNγ signaling factor, wherein the IFNγ signaling factor comprises a first IFNγ fusion protein subunit and a second IFNγ fusion protein subunit, the first IFNγ fusion protein subunit comprising the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the intracellular domain of interleukin-2 receptor subunit gamma (IL2RG), and the second IFNγ fusion protein subunit comprising the ECD of interferon-gamma receptor 2 (IFNGR2) and the intracellular domain of interleukin-2 receptor subunit beta (IL2RB).

[0058] 35. The modified cell according to Embodiment 34, wherein the amino acid sequence of the ECD of the first IFNγ fusion protein subunit is SEQ ID NO: 63, and the amino acid sequence of the ECD of the second IFNγ fusion protein subunit is SEQ ID NO: 64.

[0059] 36. A modified cell according to Embodiment 34 or Embodiment 35, wherein the amino acid sequence of the intracellular domain of the first IFNγ fusion protein subunit is SEQ ID NO: 69, and the amino acid sequence of the intracellular domain of the second IFNγ fusion protein subunit is SEQ ID NO: 70.

[0060] 37. A modified cell according to any one of embodiments 34 to 36, wherein the first IFNγ fusion protein subunit further comprises a transmembrane domain between the ECD and its intracellular domain, and the second IFNγ fusion protein subunit further comprises a transmembrane domain between the ECD and its intracellular domain.

[0061] 38. The modified cell according to Embodiment 37, wherein the transmembrane domain of the first IFNγ fusion protein subunit is that of the interleukin-2 receptor subunit gamma, and the transmembrane domain of the second IFNγ fusion protein subunit is that of the interleukin-2 receptor subunit beta.

[0062] 39. The modified cell according to Embodiment 38, wherein the amino acid sequence of the transmembrane domain of the first IFNγ fusion protein subunit is SEQ ID NO: 67, and the amino acid sequence of the transmembrane domain of the second IFNγ fusion protein subunit is SEQ ID NO: 68.

[0063] 40. A modified cell according to any one of Embodiments 34 to 39, wherein the first IFNγ fusion protein subunit further comprises a membrane-proximal region between the ECD and its intracellular domain, and the second IFNγ fusion protein subunit further comprises a membrane-proximal region between the ECD and its intracellular domain.

[0064] 41. The modified cell according to Embodiment 40, wherein the membrane proximal region of the first IFNγ fusion protein subunit is that of the interleukin-2 receptor subunit gamma, and the membrane proximal region of the second IFNγ fusion protein subunit is that of the interleukin-2 receptor subunit beta.

[0065] 42. The modified cell according to Embodiment 41, wherein the amino acid sequence of the membrane proximal region of the first IFNγ fusion protein subunit is Sequence ID No. 65, and the amino acid sequence of the membrane proximal region of the second IFNγ fusion protein subunit is Sequence ID No. 66.

[0066] 43. A modified cell according to any one of embodiments 34 to 42, wherein a first exogenous polynucleotide encoding the first IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the SOCS1 locus.

[0067] 44. A modified cell according to any one of embodiments 34 to 43, wherein a second exogenous polynucleotide encoding the second IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the ROSA26 locus.

[0068] 45. A modified cell according to any one of embodiments 34 to 44, wherein the first IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 71, the second IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 72, and optionally the first exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 81, and the second exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 82.

[0069] 46. ​​A modified cell according to any one of embodiments 34 to 45, wherein the amino acid sequence of the first IFNγ fusion protein subunit is SEQ ID NO: 71, and the amino acid sequence of the second IFNγ fusion protein subunit is SEQ ID NO: 72.

[0070] 47. A modified cell according to any one of Embodiments 1 to 46, further comprising an exogenous polynucleotide encoding a B2M-HLA-E fusion protein (B2M-HLA-E) which includes at least a portion of B2M fused with at least a portion of HLA-E.

[0071] 48. The modified cell according to Embodiment 47, wherein the exogenous polynucleotide encoding B2M-HLA-E is inserted into the B2M locus.

[0072] 49. The modified cell according to Embodiment 47 or Embodiment 48, wherein the B2M-HLA-E contains the amino acid sequence of Sequence ID No. 85.

[0073] 50. A modified cell according to any one of Embodiments 1 to 49, further comprising an exogenous polynucleotide encoding a chimeric antigen receptor (CAR).

[0074] 51. The modified cell according to Embodiment 50, wherein the exogenous polynucleotide encoding the CAR is inserted into the AAVS1 locus.

[0075] 52. The modified cell according to Embodiment 50 or Embodiment 51, wherein the exogenous polynucleotide encodes a CAR that specifically binds to human CD19.

[0076] 53. The modified cell according to Embodiment 52, wherein the CAR that specifically binds to human CD19 contains the amino acid of SEQ ID NO: 83 or SEQ ID NO: 84.

[0077] 54. A modified cell according to any one of Embodiments 1 to 53, wherein the iPSC is derived from a primary T cell (T-iPSC).

[0078] 55. A modified cell according to any one of Embodiments 1 to 54, wherein the iPSC is derived from a primary γδT cell (γδT-iPSC).

[0079] 56. A modified cell according to any one of Embodiments 1 to 55, wherein the γδT cell is a Vd2γδT cell.

[0080] 57. A modified cell according to any one of Embodiments 1 to 56, wherein the γδT cells are iPSC-derived γδT (iγδT) cells, and optionally the γδT cells are T-iPSC-derived γδT cells (T-iγδT).

[0081] 58. The modified cell according to any one of Embodiments 1 to 57, wherein the γδT cells are γδT cells (γδT-iγδT) differentiated from γδT-iPSC cells.

[0082] 59. A modified cell according to any one of Embodiments 1 to 58, wherein the intermediate cell differentiated from the iPSC is a γδT-iHSC, a γδT-iCLP, and / or an immature T-iγδT cell.

[0083] 60. A population of cells comprising the modified cells described in any one of Embodiments 1 to 59.

[0084] 61. A population of cells according to Embodiment 60, which have been cryopreserved at approximately -196°C.

[0085] 62. A method for producing modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, comprising contacting the cells with a guide RNA and a nuclease or a nucleic acid encoding the nuclease, wherein the contact results in genomic disruption within the endogenous SOCS1 gene that suppresses or eliminates the expression of functional SOCS1 protein, and genomic disruption within the endogenous CISH gene that suppresses or eliminates the expression of functional CISH protein.

[0086] 63. The method according to Embodiment 62, wherein the iPSC is derived from a primary T cell (T-iPSC).

[0087] 64. The method according to Embodiment 62 or Embodiment 63, wherein the iPSC is derived from a primary γδT cell (γδT-iPSC).

[0088] 65. A modified cell according to any one of embodiments 62 to 64, wherein the γδT cell is a Vd2γδT cell.

[0089] 66. The method according to any one of Embodiments 62 to 65, wherein the γδT cells are iPSC-derived γδT (iγδT) cells, and optionally the γδT cells are T-iPSC-derived γδT cells (T-iγδT).

[0090] 67. The method according to any one of embodiments 62 to 66, wherein the γδT cells are γδT cells (γδT-iγδT) differentiated from γδT-iPSC cells.

[0091] 68. The method according to any one of embodiments 62 to 67, wherein the intermediate cells differentiated from the iPSC are γδT-iHSC, γδT-iCLP, and / or immature T-iγδT cells.

[0092] 69. The method according to any one of embodiments 62 to 68, wherein the genome disruption is located in exon 2 of the SOCS1 gene, optionally the genome disruption is a loss-of-function nucleic acid mutation, optionally the mutation is an insertion, deletion, or substitution.

[0093] 70. The method according to any one of Embodiments 62 to 69, wherein the genome disruption is located in exon 3 or exon 4 of the CISH gene, and optionally the genome disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

[0094] 71. The method according to any one of embodiments 62 to 70, wherein the genome disruption is located within a target sequence in SOCS1 including SEQ ID NO: 17 or SEQ ID NO: 23, and / or the genome disruption is located within a target sequence in CISH including SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 24.

[0095] 72. The method according to any one of Embodiments 62 to 71, wherein the SOCS1 and genomic disruption within CISH is carried out by administering a guide RNA to the cells, wherein the guide RNA is optionally a single guide RNA (sgRNA) or crRNA, and optionally a Cas endonuclease or a nucleic acid encoding the Cas endonuclease is also administered to the cells.

[0096] 73. The method according to Embodiment 72, wherein the guide RNA includes one or more guide sequences from SEQ ID NOs. 5, 11, 3, 4, and 12, or one or more sgRNA or crRNA sequences from SEQ ID NOs. 29, 37, 27, 28, and 38.

[0097] 74. The method according to any one of embodiments 62 to 73, further comprising inhibiting the expression of an endogenous BIM gene by causing genomic disruption within the endogenous BIM gene.

[0098] 75. The method according to Embodiment 74, wherein the genome disruption within the BIM gene is located within exon 2C of the BIM gene.

[0099] 76. BIM EL and BIM L The expression level and / or function of the splice variant were reduced in the cells compared to the unmodified control, BIM S The method according to Embodiment 74 or Embodiment 75, wherein the expression level and / or function of the splice variant are maintained in the cells.

[0100] 77. The method according to any one of embodiments 74 to 76, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 2C of the endogenous BIM gene, and optionally the mutation is an insertion, deletion, or substitution.

[0101] 78. The method according to any one of embodiments 74 to 77, wherein the genome disruption is located within a target sequence in a BIM containing any one of sequence numbers 14, 18, 19, 20, 21, and 22.

[0102] 79. The method according to any one of embodiments 74 to 78, wherein the genome disruption within the BIM is carried out by administering a guide RNA to the cells, wherein the guide RNA is optionally a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0103] 80. The method according to Embodiment 79, wherein the guide RNA includes one of the guide sequences among SEQ ID NOs: 2, 6, 7, 8, 9, and 10, or one or more sgRNA or crRNA sequences among SEQ ID NOs: 26, 32, 33, 34, 35, and 36.

[0104] 81. The method according to any one of Embodiments 62 to 80, further comprising inhibiting the expression of an endogenous FAS gene by causing genomic disruption within the endogenous FAS gene that suppresses or eliminates the expression of a functional FAS protein.

[0105] 82. The method according to Embodiment 81, wherein the genomic disruption within the FAS gene is located within exon 1 of the FAS gene.

[0106] 83. The method according to Embodiment 81 or Embodiment 82, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 1 of the endogenous FAS gene, and the mutation may be an insertion, deletion, or substitution.

[0107] 84. The method according to any one of embodiments 81 to 83, wherein the genome disruption is located within a target sequence in the FAS containing SEQ ID NO: 62.

[0108] 85. The method according to any one of Embodiments 81 to 84, wherein the genome disruption within the FAS is carried out by administering a guide RNA to the cells, wherein the guide RNA is optionally a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0109] 86. The method according to Embodiment 85, wherein the guide RNA includes the guide sequence of SEQ ID NO: 61 or the sgRNA of SEQ ID NO: 60.

[0110] 87. The method according to any one of Embodiments 62 to 86, further comprising inhibiting the expression of an endogenous B2M gene by causing genomic disruption within the endogenous B2M gene that suppresses or eliminates the expression of a functional B2M protein.

[0111] 88. The method according to Embodiment 87, wherein the genome disruption within the B2M gene is located within exon 2 of the B2M gene.

[0112] 89. The method according to Embodiment 87 or Embodiment 88, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 2 of the endogenous B2M gene, and the mutation may be an insertion, deletion, or substitution.

[0113] 90. The method according to any one of embodiments 87 to 89, wherein the genome disruption is located within a target sequence in B2M containing SEQ ID NO: 56.

[0114] 91. The method according to any one of Embodiments 87 to 90, wherein the genomic disruption within B2M is carried out by administering a guide RNA to the cells, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0115] 92. The method according to Embodiment 91, wherein the guide RNA includes the guide sequence of SEQ ID NO: 55 or the sgRNA of SEQ ID NO: 54.

[0116] 93. The method according to any one of Embodiments 62 to 92, further comprising inhibiting the expression of an endogenous CIITA gene by causing genomic disruption within the endogenous CIITA gene that suppresses or eliminates the expression of a functional CIITA protein.

[0117] 94. The method according to Embodiment 93, wherein the genome disruption within the CIITA gene is located within exon 3 of the CIITA gene.

[0118] 95. The method according to Embodiment 93 or Embodiment 94, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 3 of the endogenous CIITA gene, and the mutation may be an insertion, deletion, or substitution.

[0119] 96. The method according to any one of embodiments 93 to 95, wherein the genome disruption is located within a target sequence in CIITA containing sequence number 59.

[0120] 97. The method according to any one of embodiments 93 to 96, wherein the genome disruption within the CIITA is carried out by administering a guide RNA to the cells, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0121] 98. The method according to Embodiment 97, wherein the guide RNA includes the guide sequence of SEQ ID NO: 58 or the sgRNA of SEQ ID NO: 57.

[0122] 99. The method according to any one of embodiments 62 to 98, further comprising inhibiting the expression of an endogenous TRAC gene by causing genomic disruption within the endogenous TRAC gene that suppresses or eliminates the expression of a functional TRAC protein.

[0123] 100. The method according to any one of Embodiments 62 to 99, wherein the genome disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

[0124] 101. A modified cell according to any one of Embodiments 62 to 100, further comprising introducing one or more exogenous polynucleotides encoding an IFNγ signaling factor into the cell, wherein the IFNγ signaling factor comprises a first IFNγ fusion protein subunit and a second IFNγ fusion protein subunit, the first IFNγ fusion protein subunit comprising the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the intracellular domain of interleukin-2 receptor subunit gamma (IL2RG), and the second IFNγ fusion protein subunit comprising the ECD of interferon-gamma receptor 2 (IFNGR2) and the intracellular domain of interleukin-2 receptor subunit beta (IL2RB).

[0125] 102. The method according to Embodiment 101, wherein a first exogenous polynucleotide encoding the first IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted into the SOCS1 locus.

[0126] 103. The method according to Embodiment 101 or 102, wherein a second exogenous polynucleotide encoding the second IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the ROSA26 locus.

[0127] 104. The method according to any one of embodiments 101 to 103, wherein the first IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 71, the second IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 72, and optionally the first exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 81, and the second exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 82.

[0128] 105. The method according to any one of Embodiments 62 to 104, further comprising introducing an exogenous polynucleotide encoding a B2M-HLA-E fusion protein (B2M-HLA-E) comprising at least a portion of B2M fused with at least a portion of HLA-E into the cells.

[0129] 106. The method according to Embodiment 105, wherein the exogenous polynucleotide encoding B2M-HLA-E is inserted into the B2M locus.

[0130] 107. The method according to any one of embodiments 105 to 106, wherein the B2M-HLA-E comprises the amino acid sequence of SEQ ID NO: 85.

[0131] 108. The method according to any one of embodiments 62 to 107, further comprising introducing an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) into the cells.

[0132] 109. The method according to Embodiment 108, wherein the exogenous polynucleotide encoding the CAR is inserted into the AAVS1 locus.

[0133] 110. The method according to Embodiment 108 or Embodiment 109, wherein the exogenous polynucleotide encodes a CAR that specifically binds to human CD19.

[0134] 111. The method according to Embodiment 110, wherein the CAR that specifically binds to human CD19 comprises the amino acid of SEQ ID NO: 83 or SEQ ID NO: 84.

[0135] 112. Modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, which include genomic disruption within exon 2C of the endogenous BIM gene, and BIM EL and BIM L The expression level and / or function of the splice variant are reduced in the modified cells compared to the unmodified control, BIM S Modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, wherein the expression level and / or function of the splice variant are preserved in the modified cells.

[0136] 113. The modified cell according to Embodiment 112, wherein the genome disruption is a loss-of-function nucleic acid mutation within exon 2C, and the mutation may be an insertion, deletion, or substitution.

[0137] 114. The modified cell according to Embodiment 112 or Embodiment 113, wherein the genome disruption is located within a target sequence in the BIM containing any one of Sequence IDs 14, 20, 21, and 22.

[0138] 115. Modified cells according to any one of embodiments 112 to 114, wherein the genome disruption within the BIM is carried out by administering a guide RNA to the cells, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0139] 116. The modified cell according to Embodiment 115, wherein the guide RNA includes a guide sequence that differs by three or fewer nucleotides from any one of SEQ ID NOs: 26, 34, 35, and 36, or any one of SEQ ID NOs: 2, 8, 9, and 10.

[0140] 117. A modified cell according to Embodiment 115 or Embodiment 116, wherein the guide RNA comprises a guide sequence that differs from Sequence ID No. 36 or Sequence ID No. 10 by three or fewer nucleotides, and the endonuclease is a MAD7 endonuclease.

[0141] 118. A modified cell according to Embodiment 115 or Embodiment 116, wherein the guide RNA comprises a guide sequence that differs by three or fewer nucleotides from any one of SEQ ID NOs: 26, 34, and 35, or any one of SEQ ID NOs: 2, 8, and 9, and the endonuclease is a Cas9 endonuclease.

[0142] 119. A method for inhibiting the expression of the endogenous BIM gene in iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, comprising contacting the cells with a guide RNA and an endonuclease or a nucleic acid encoding the endonuclease, wherein the contact results in genomic disruption within exon 2C of the endogenous BIM gene, thereby inhibiting BIM EL and BIM L The expression level and / or function of the splice variant are reduced in the modified cells compared to the unmodified control, BIM S The method wherein the expression level and / or function of the splice variant are maintained in the modified cells.

[0143] 120. The method according to Embodiment 119, wherein the genome disruption is a loss-of-function nucleic acid mutation within exon 2C, and the mutation is optionally an insertion, deletion, or substitution.

[0144] 121. The method according to Embodiment 119 or Embodiment 120, wherein the genome disruption is located within a target sequence in BIM containing any one of Sequence IDs 14, 20, 21, and 22.

[0145] 122. The method according to any one of Embodiments 119 to 121, wherein the guide RNA includes one of the guide sequences among Sequence IDs 2, 8, 9, and 10, or one or more sgRNA or crRNA sequences among Sequence IDs 26, 34, 35, and 36, and optionally the guide RNA is a single guide RNA (sgRNA) or crRNA.

[0146] 123. The method according to any one of Embodiments 119 to 122, wherein the γδT cells are γδT cells (γδT-iγδT) differentiated from γδT-iPSC cells.

[0147] 124. The method according to any one of embodiments 119 to 123, wherein the cells are prepared from the method according to any one of embodiments 62 to 111.

[0148] 125. Modified cells prepared by any one of embodiments 62-111 and 119-124.

[0149] 126. Modified iPSC, γδT cell, or intermediate cell differentiated from iPSC, comprising one or more exogenous polynucleotides encoding an IFNγ signaling factor, wherein the IFNγ signaling factor comprises a first IFNγ fusion protein subunit and a second IFNγ fusion protein subunit, wherein the first IFNγ fusion protein subunit comprises the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the intracellular domain of interleukin-2 receptor subunit gamma (IL2RG), and the second IFNγ fusion protein subunit comprises interferon gamma The modified iPSC, γδT cell, or intermediate cell differentiated from an iPSC comprises the intracellular domain of the γ receptor 2 (IFNGR2) and the interleukin-2 receptor subunit beta (IL2RB), and optionally further comprises an exogenous polynucleotide encoding a chimeric antigen receptor (CAR), optionally having the exogenous polynucleotide encoding the CAR inserted into the AAVS1 locus, optionally encoding the CAR that specifically binds to human CD19, and optionally the CAR that specifically binds to human CD19 comprises SEQ ID NO: 83 or SEQ ID NO: 84.

[0150] 127. The modified cell according to Embodiment 126, wherein the amino acid sequence of the ECD of the first IFNγ fusion protein subunit is SEQ ID NO: 63, and the amino acid sequence of the ECD of the second IFNγ fusion protein subunit is SEQ ID NO: 64.

[0151] 128. A modified cell according to Embodiment 126 or Embodiment 127, wherein the amino acid sequence of the intracellular domain of the first IFNγ fusion protein subunit is SEQ ID NO: 69, and the amino acid sequence of the intracellular domain of the second IFNγ fusion protein subunit is SEQ ID NO: 70.

[0152] 129. The modified cell according to any one of embodiments 126 to 128, wherein said first IFNγ fusion protein subunit further comprises a transmembrane domain between said ECD and its intracellular domain, and said second IFNγ fusion protein subunit further comprises a transmembrane domain between said ECD and its intracellular domain.

[0153] 130. The modified cell according to embodiment 129, wherein said transmembrane domain of said first IFNγ fusion protein subunit is that of interleukin-2 receptor subunit gamma, and said transmembrane domain of said second IFNγ fusion protein subunit is that of interleukin-2 receptor subunit beta.

[0154] 131. The modified cell according to embodiment 130, wherein the amino acid sequence of the transmembrane domain of said first IFNγ fusion protein subunit is SEQ ID NO: 67, and the amino acid sequence of the transmembrane domain of said second IFNγ fusion protein subunit is SEQ ID NO: 68.

[0155] 132. The modified cell according to any one of embodiments 126 to 131, wherein said first IFNγ fusion protein subunit further comprises a juxtamembrane region between said ECD and its intracellular domain, and said second IFNγ fusion protein subunit further comprises a juxtamembrane region between said ECD and its intracellular domain.

[0156] 133. The modified cell according to embodiment 132, wherein said juxtamembrane region of said first IFNγ fusion protein subunit is that of interleukin-2 receptor subunit gamma, and said juxtamembrane region of said second IFNγ fusion protein subunit is that of interleukin-2 receptor subunit beta.

[0157] 134. The modified cell according to Embodiment 133, wherein the amino acid sequence of the membrane proximal region of the first IFNγ fusion protein subunit is Sequence ID No. 65, and the amino acid sequence of the membrane proximal region of the second IFNγ fusion protein subunit is Sequence ID No. 66.

[0158] 135. A modified cell according to any one of embodiments 126 to 134, wherein a first exogenous polynucleotide encoding the first IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the SOCS1 locus.

[0159] 136. A modified cell according to any one of embodiments 126 to 135, wherein a second exogenous polynucleotide encoding the second IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the ROSA26 locus.

[0160] 137. A modified cell according to any one of Embodiments 126 to 136, wherein the first IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 71, the second IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 72, and optionally the first exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 81, and the second exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 82.

[0161] 138. A modified cell according to any one of embodiments 126 to 137, wherein the amino acid sequence of the first IFNγ fusion protein subunit is SEQ ID NO: 71, and the amino acid sequence of the second IFNγ fusion protein subunit is SEQ ID NO: 72.

[0162] 139. A method for enhancing the proliferation and killing capacity of modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, comprising introducing one or more exogenous polynucleotides encoding an IFNγ signaling factor into the iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, wherein the IFNγ signaling factor comprises a first IFNγ fusion protein subunit and a second IFNγ fusion protein subunit, the first IFNγ fusion protein subunit comprising the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the intracellular domain of interleukin-2 receptor subunit gamma (IL2RG) The method comprising, wherein the second IFNγ fusion protein subunit comprises the ECD of interferon-gamma receptor 2 (IFNGR2) and the intracellular domain of interleukin-2 receptor subunit beta (IL2RB), and optionally further comprising introducing an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) into the cell, optionally the exogenous polynucleotide encoding the CAR being inserted into the AAVS1 locus, optionally the exogenous polynucleotide encoding the CAR that specifically binds to human CD19, and optionally the CAR that specifically binds to human CD19 comprising SEQ ID NO: 83 or SEQ ID NO: 84.

[0163] 140. The method according to Embodiment 139, wherein the first exogenous polynucleotide encoding the first IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted into the SOCS1 locus.

[0164] 141. The method according to Embodiment 139 or 140, wherein a second exogenous polynucleotide encoding the second IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the ROSA26 locus.

[0165] 142. The method according to any one of Embodiments 139 to 141, wherein the first IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 71, the second IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 72, and optionally the first exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 81, and the second exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 82.

[0166] 143. The method according to any one of embodiments 139 to 142, wherein the cells are prepared from the method according to any one of embodiments 62 to 111.

[0167] 144. Modified cells prepared by the method described in any one of embodiments 139 to 143.

[0168] 145. A modified cell or method according to any one of the prior embodiments, wherein the cell is a human cell.

[0169] 146. Modified cells or methods according to any one of the prior embodiments, wherein the cells are in vivo, in vitro, ex vivo, or for human subjects.

[0170] 147. A gene editing system comprising one or more guide RNAs and an endonuclease or a nucleic acid encoding the endonuclease, wherein the one or more guide RNAs comprise one or more single guide RNAs (sgRNAs) or CRISPR RNAs (crRNAs), and the one or more sgRNAs or crRNAs a. A guide sequence that binds to a target sequence containing one of sequence numbers 17, 23, 15, 16, and 24. b. A guide sequence that binds to a target sequence containing any one of sequence numbers 14, 18, 19, 20, 21, and 22. c. A first guide sequence that binds to a target sequence containing one of sequence numbers 17 and 23, and a second guide sequence that binds to a target sequence containing one of sequence numbers 15, 16, and 24. d. A first guide sequence that binds to a target sequence containing one of sequence numbers 17 and 23, a second guide sequence that binds to a target sequence containing one of sequence numbers 15, 16, and 24, and a third guide sequence that binds to a target sequence containing one of sequence numbers 14, 18, 19, 20, 21, and 22. e. A first guide sequence that binds to a target sequence containing any one of sequence numbers 17 and 23; a second guide sequence that binds to a target sequence containing any one of sequence numbers 15, 16, and 24; a third guide sequence that binds to a target sequence containing any one of sequence numbers 14, 18, 19, 20, 21, and 22; a fourth guide sequence that binds to a target sequence containing sequence number 56; and a fifth guide sequence that binds to a target sequence containing sequence number 59. f. A first guide sequence that binds to a target sequence containing one of sequence numbers 17 and 23; a second guide sequence that binds to a target sequence containing one of sequence numbers 15, 16, and 24; a third guide sequence that binds to a target sequence containing one of sequence numbers 14, 18, 19, 20, 21, and 22; a sixth guide sequence that binds to a target sequence containing sequence number 62; a fourth guide sequence that binds to a target sequence containing sequence number 56; and a fifth guide sequence that binds to a target sequence containing sequence number 59. g. A guide sequence containing either sequence number 5 or 11. h. sgRNA or crRNA sequences including sequence numbers 29 and 37, i. A guide sequence containing one of sequence numbers 3, 4, and 12, j. An sgRNA or crRNA sequence containing any one of sequence numbers 27, 28, and 38, k. A guide sequence containing one of sequence numbers 2, 6, 7, 8, 9, or 10. l. An sgRNA or crRNA sequence containing any one of sequence numbers 26, 32, 33, 34, 35, and 36. m. A first guide sequence containing one of sequence numbers 5 and 11, and a second guide sequence containing one of sequence numbers 3, 4, and 12. n. A first sgRNA or crRNA sequence containing one of sequence numbers 29 and 37, and a second sgRNA or crRNA sequence containing one of sequence numbers 27, 28, and 38. o. A first guide sequence containing one of sequence numbers 5 and 11, a second guide sequence containing one of sequence numbers 3, 4, and 12, and a third guide sequence containing one of sequence numbers 2, 6, 7, 8, 9, and 10. p. A first sgRNA or crRNA sequence containing one of sequence numbers 29 and 37, a second sgRNA or crRNA sequence containing one of sequence numbers 27, 28, and 38, and a third sgRNA or crRNA sequence containing one of sequence numbers 26, 32, 33, 34, 35, and 36, q. A first guide sequence containing either sequence number 5 or 11, a second guide sequence containing either sequence number 3, 4, or 12, a third guide sequence containing either sequence number 2, 6, 7, 8, 9, or 10, a fourth guide sequence containing sequence number 55, and a fifth guide sequence containing sequence number 58, r. A first sgRNA or crRNA guide sequence containing one of sequence numbers 29 and 37, a second sgRNA or crRNA sequence containing one of sequence numbers 27, 28, and 38, a third sgRNA or crRNA sequence containing one of sequence numbers 26, 32, 33, 34, 35, and 36, a fourth sgRNA or crRNA sequence containing sequence number 54, and a fifth sgRNA or crRNA sequence containing sequence number 57. s. a first guide sequence comprising any one of SEQ ID NO: 5 and 11, a second guide sequence comprising any one of SEQ ID NO: 3, 4, and 12, a third guide sequence comprising any one of SEQ ID NO: 2, 6, 7, 8, 9, and 10, a sixth guide sequence comprising SEQ ID NO: 61, a fourth guide sequence comprising SEQ ID NO: 55, and a fifth guide sequence comprising SEQ ID NO: 58, or t. the gene editing system, comprising a first sgRNA or crRNA guide sequence comprising any one of SEQ ID NO: 29 and 37, a second sgRNA or crRNA sequence comprising any one of SEQ ID NO: 27, 28, and 38, a third sgRNA or crRNA sequence comprising any one of SEQ ID NO: 26, 32, 33, 34, 35, and 36, a sixth sgRNA or crRNA sequence comprising SEQ ID NO: 60, a fourth sgRNA or crRNA sequence comprising SEQ ID NO: 54, and a fifth sgRNA or crRNA sequence comprising SEQ ID NO: 57.

[0171] 148. The gene editing system according to embodiment 147, wherein the endonuclease is a Cas endonuclease.

[0172] 149. The gene editing system according to embodiment 148, wherein the endonuclease is an MAD7 endonuclease.

[0173] 150. a. the target sequence comprises SEQ ID NO: 22, and / or b. the gene editing system according to embodiment 149, wherein the crRNA comprises a guide sequence that differs from SEQ ID NO: 10 by no more than 3 nucleotides.

[0174] 151. a. the target sequence comprises SEQ ID NO: 23, and / or b. the gene editing system according to embodiment 149, wherein the crRNA comprises a guide sequence that differs from SEQ ID NO: 11 by no more than 3 nucleotides.

[0175] 152. a. the target sequence comprises SEQ ID NO: 24, and / or b. The gene editing system according to Embodiment 149, wherein the crRNA includes a guide sequence that differs from Sequence ID No. 12 by three or fewer nucleotides.

[0176] 153. The gene editing system according to Embodiment 148, wherein the Cas endonuclease is a Cas9 endonuclease.

[0177] 154. a. The target sequence includes any one of sequence numbers 14, 18, 19, 20, and 21, and / or b. The gene editing system according to Embodiment 153, wherein the sgRNA includes a guide sequence that differs from any one of sequence numbers 2, 6, 7, 8, and 9 by three or fewer nucleotides.

[0178] 155. a. The target sequence includes sequence number 17, and / or b. The gene editing system according to Embodiment 153, wherein the sgRNA includes a guide sequence that differs from Sequence ID No. 5 by three or fewer nucleotides.

[0179] 156. a. The target sequence includes sequence number 15 or 16, and / or b. The gene editing system according to Embodiment 153, wherein the sgRNA includes a guide sequence that differs from sequence number 3 or 4 by three or fewer nucleotides.

[0180] 157. a. The target sequence includes sequence number 62, and / or b. The gene editing system according to Embodiment 153, wherein the sgRNA includes a guide sequence that differs from Sequence ID No. 61 by three or fewer nucleotides.

[0181] 158. a. The target sequence includes sequence number 56, and / or b. The gene editing system according to Embodiment 153, wherein the sgRNA includes a guide sequence that differs from Sequence ID No. 55 by three or fewer nucleotides.

[0182] 159. a. The target sequence includes sequence number 59, and / or b. The gene editing system according to Embodiment 153, wherein the sgRNA includes a guide sequence that differs from Sequence ID No. 58 by three or fewer nucleotides.

[0183] 160. Guide RNA containing SEQ ID NO: 5 (SOCS1), SEQ ID NO: 29 (SOCS1), SEQ ID NO: 11 (SOCS1), SEQ ID NO: 37 (SOCS1), SEQ ID NO: 27 (CISH), SEQ ID NO: 28 (CISH), SEQ ID NO: 12 (CISH), SEQ ID NO: 38 (CISH), SEQ ID NO: 6 (BIM), SEQ ID NO: 32 (BIM), SEQ ID NO: 7 (BIM), SEQ ID NO: 33 (BIM), SEQ ID NO: 8 (BIM), SEQ ID NO: 34 (BIM), SEQ ID NO: 9 (BIM), SEQ ID NO: 35 (BIM), SEQ ID NO: 10 (BIM), or SEQ ID NO: 36 (BIM).

[0184] 161. A guide RNA comprising a CRISPR RNA (crRNA) sequence, wherein the crRNA sequence comprises 2'-O-methyl modifications at each of the first three positions of the 5' end of the crRNA sequence and at each of the fourth, third, and second to last positions of the 3' end, and comprises phosphorothioate bonds between each base at the first three positions of the 5' end of the crRNA sequence and phosphorothioate bonds between each base at the last three positions of the 3' end.

[0185] 162. A guide RNA according to embodiment 161, which functions in conjunction with the MAD7 Cas endonuclease.

[0186] 163. The guide RNA according to Embodiment 162, wherein the crRNA is approximately 56 nucleotides long.

[0187] 164. The guide RNA according to Embodiment 163, wherein the crRNA sequence includes 2'-O-methyl modifications at positions 1, 2, 3, 53, 54, and 55 of the 3' end of the crRNA sequence.

[0188] 165. A guide RNA according to any one of embodiments 161 to 164, wherein the crRNA includes SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, which includes the modification thereof.

[0189] 166. A guide RNA according to any one of embodiments 161 to 165, wherein the last base of the crRNA is modified, but not 2'-O-methylated.

[0190] 167. The guide RNA according to Embodiment 166, wherein the modification of the last base of the crRNA is a pseudoknot.

[0191] 168. The guide RNA according to Embodiment 166, wherein the modification of the last base of the crRNA is a modification that facilitates use with MAD7.

[0192] 169. Modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, produced by any of the following: i) Contacting the cells with the gene editing system described in any one of embodiments 147 to 159, wherein the contact results in genomic disruption in a target sequence that binds to the single guide RNA (sgRNA) of the gene editing system, or ii) Contacting the cells with the guide RNA and endonuclease described in any one of Embodiments 160 to 168 or the nucleic acid encoding the endonuclease, The contact described above results in genomic disruption within a target sequence that binds to the guide RNA described in Embodiment 160, wherein the genomic disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

[0193] 170. Modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, which contain genomic disruption within exon 2C of the endogenous BIM gene, and BIM EL and BIM LThe expression level and / or function of the splice variant are reduced in the modified cells compared to the unmodified control, BIM S The modified iPSC, γδT cell, or intermediate cell differentiated from an iPSC, wherein the expression level and / or function of the splice variant are preserved in the modified cell, and the genome disruption is located within a target sequence in the BIM containing any one of sequence numbers 14, 20, 21, and 22.

[0194] 171. The modified cell according to Embodiment 170, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 2C, and the mutation may be an insertion, deletion, or substitution.

[0195] 172. Modified cells according to any one of embodiments 170 to 171, wherein the genome disruption within the BIM is carried out by administering a guide RNA to the cells, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0196] 173. The modified cell according to Embodiment 172, wherein the guide RNA includes one of sequence numbers 26, 34, 35, and 36, or includes a guide sequence that differs from one of sequence numbers 2, 8, 9, and 10 by three or fewer nucleotides.

[0197] 174. A modified cell according to Embodiment 172 or Embodiment 173, wherein the guide RNA includes SEQ ID NO: 36 or a guide sequence that differs from SEQ ID NO: 10 by three or fewer nucleotides, and the endonuclease is MAD7 endonuclease.

[0198] 175. A modified cell according to Embodiment 172 or Embodiment 173, wherein the guide RNA includes one of SEQ ID NOs: 26, 34, and 35, or a guide sequence that differs from one of SEQ ID NOs: 2, 8, and 9 by three or fewer nucleotides, and the endonuclease is a Cas9 endonuclease.

[0199] 176. Modified iPSC, γδT cell, or intermediate cell differentiated from iPSC, comprising genomic disruption within a target sequence of the endogenous SOCS1 gene that suppresses or eliminates the expression of functional SOCS1 protein, wherein the target sequence includes SEQ ID NO: 17 or SEQ ID NO: 23.

[0200] 177. The modified cell according to Embodiment 176, wherein the genome disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

[0201] 178. Embodiment 177 or the modified cell according to Embodiment 177, wherein the genomic disruption within SOCS1 is carried out by administering a guide RNA to the cell, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cell.

[0202] 179. The modified cell according to Embodiment 178, wherein the guide RNA contains either one of sequence numbers 29 and 37, or a guide sequence that differs from sequence number 5 or 11 by three or fewer nucleotides.

[0203] 180. A modified cell according to Embodiment 178 or Embodiment 179, wherein the guide RNA includes SEQ ID NO: 37 or a guide sequence that differs from SEQ ID NO: 11 by three or fewer nucleotides, and the endonuclease is MAD7 endonuclease.

[0204] 181. A modified cell according to Embodiment 178 or Embodiment 179, wherein the guide RNA includes SEQ ID NO: 29 or a guide sequence that differs from SEQ ID NO: 5 by three or fewer nucleotides, and the endonuclease is a Cas9 endonuclease.

[0205] 182. Modified iPSC, γδT cell, or intermediate cell differentiated from iPSC, comprising genomic disruption within a target sequence of an endogenous CISH gene that suppresses or eliminates CISH expression, wherein the target sequence includes Sequence ID No. 24.

[0206] 183. The modified cell according to Embodiment 182, wherein the genome disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

[0207] 184. Modified cells according to Embodiment 182 or Embodiment 183, wherein the genome disruption within the CISH is carried out by administering a guide RNA to the cells, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0208] 185. The modified cell according to Embodiment 184, wherein the guide RNA includes SEQ ID NO: 38 or a guide sequence that differs from SEQ ID NO: 12 by three or fewer nucleotides.

[0209] 186. A modified cell according to Embodiment 184 or Embodiment 185, wherein the guide RNA includes SEQ ID NO: 38 or a guide sequence that differs from SEQ ID NO: 12 by three or fewer nucleotides, and the endonuclease is MAD7 endonuclease.

[0210] 187. A modified cell according to any one of embodiments 169 to 186, wherein the iPSC is derived from a primary T cell (T-iPSC).

[0211] 188. A modified cell according to any one of embodiments 169 to 187, wherein the iPSC is derived from a primary γδT cell (γδT-iPSC).

[0212] 189. A modified cell according to any one of embodiments 169 to 188, wherein the γδT cell is a Vd2γδT cell.

[0213] 190. A modified cell according to any one of Embodiments 169 to 189, wherein the γδT cells are iPSC-derived γδT (iγδT) cells, and in some cases the γδT cells are γδT cells differentiated from T-iPSCs (T-iγδT).

[0214] 191. The modified cell according to any one of Embodiments 169 to 190, wherein the γδT cells are γδT cells (γδT-iγδT) differentiated from γδT-iPSC cells.

[0215] 192. A modified cell according to any one of Embodiments 169 to 191, wherein the intermediate cell differentiated from the iPSC is a γδT-iHSC, a γδT-iCLP, and / or an immature T-iγδT cell.

[0216] 193. A modified cell according to any one of embodiments 169 to 192, wherein the cell is a human cell.

[0217] 194. Modified cells according to any one of embodiments 169 to 193, wherein the cells are in vivo, in vitro, ex vivo, or for human subjects.

[0218] 195. Modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, comprising: 1) an exogenous polynucleotide encoding a CAR that specifically binds to human CD19; 2) a genomic disruption within the endogenous SOCS1 gene that suppresses or eliminates the expression of functional cytokine signaling suppressor 1 (SOCS1) protein; and 3) a genomic disruption within the endogenous CISH gene that suppresses or eliminates the expression of functional cytokine-inducible SH2-containing protein (CISH) protein.

[0219] 196. Modified cells according to any one of Embodiments 195, further comprising genomic disruption within an endogenous Bcl-2-interacting cell death mediator (BIM) gene.

[0220] 197. The modified cell according to Embodiment 196, wherein the genomic disruption within the BIM gene is located within exon 2C of the BIM gene.

[0221] 198. BIM EL and BIM L The expression level and / or function of the splice variant were reduced in the modified cells compared to the unmodified control, and BIM S The modified cell according to Embodiment 197, wherein the expression level and / or function of the splice variant are retained in the modified cell.

[0222] 199. A modified cell according to any one of Embodiments 196 to 198, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 2C of the endogenous BIM gene, and optionally the mutation is an insertion, deletion, or substitution.

[0223] 200. A modified cell according to any one of embodiments 196 to 199, wherein the genome disruption is located within a target sequence in the BIM containing any one of sequence numbers 14, 18, 19, 20, 21, and 22.

[0224] 201. Modified cells according to any one of Embodiments 196 to 200, wherein the genome disruption within the BIM is carried out by administering a guide RNA to the cells, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0225] 202. The modified cell according to Embodiment 201, wherein the guide RNA includes one of the guide sequences among Sequence IDs 2, 6, 7, 8, 9, and 10, or one or more sgRNA or crRNA sequences among Sequence IDs 26, 32, 33, 34, 35, and 36.

[0226] 203. Modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, comprising: 1) an exogenous polynucleotide encoding a CAR that specifically binds to human CD19; 2) a genomic disruption within the endogenous SOCS1 gene that suppresses or eliminates the expression of functional cytokine signaling suppressor 1 (SOCS1) protein; 3) a genomic disruption within the endogenous cytokine-induced SH2-containing protein (CISH) gene that suppresses or eliminates the expression of functional CISH protein; and 4) a genomic disruption within exon 2C of the endogenous BIM gene. EL and BIM L The expression level and / or function of the splice variant are reduced in the modified cells compared to the unmodified control, BIM S Modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, wherein the expression level and / or function of the splice variant are maintained in the modified cells.

[0227] 204. The modified cell according to Embodiment 203, wherein the genome disruption is a loss-of-function nucleic acid mutation within exon 2C, and the mutation may be an insertion, deletion, or substitution.

[0228] 205. The modified cell according to Embodiment 203 or Embodiment 204, wherein the genome disruption is located within a target sequence in the BIM containing any one of Sequence IDs 14, 20, 21, and 22.

[0229] 206. Modified cells according to any one of embodiments 203 to 205, wherein the genome disruption within the BIM is carried out by administering a guide RNA to the cells, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0230] 207. The modified cell according to Embodiment 206, wherein the guide RNA includes one of sequence numbers 26, 34, 35, and 36, or one of sequence numbers 2, 8, 9, and 10.

[0231] 208. A modified cell according to Embodiment 206 or Embodiment 207, wherein the guide RNA includes SEQ ID NO: 36 or the guide sequence of SEQ ID NO: 10, and the endonuclease is MAD7 endonuclease.

[0232] 209. A modified cell according to Embodiment 206 or Embodiment 207, wherein the guide RNA comprises one of sequence numbers 26, 34, and 35, or one of sequence numbers 2, 8, and 9, and the endonuclease is a Cas9 endonuclease.

[0233] 210. A modified cell according to any one of embodiments 195 to 209, wherein the exogenous polynucleotide encoding the CAR is inserted into the AAVS1 locus.

[0234] 211. A modified cell according to any one of Embodiments 195 to 210, wherein the CAR that specifically binds to human CD19 contains the amino acid of SEQ ID NO: 83 or SEQ ID NO: 84.

[0235] 212. A modified cell according to any one of Embodiments 195 to 211, wherein the genome disruption is located in exon 2 of the SOCS1 gene, optionally the genome disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

[0236] 213. A modified cell according to any one of Embodiments 195 to 212, wherein the genome disruption is located in exon 3 or exon 4 of the CISH gene, and optionally the genome disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

[0237] 214. A modified cell according to any one of embodiments 195 to 213, wherein the genome disruption is located within a target sequence in SOCS1 including SEQ ID NO: 17 or SEQ ID NO: 23, and / or the genome disruption is located within a target sequence in CISH including SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 24.

[0238] 215. Modified cells according to any one of Embodiments 195 to 214, wherein the SOCS1 and genomic disruption within CISH is carried out by administering a guide RNA to the cells, and optionally the guide RNA is a single guide RNA (sgRNA), and an endonuclease, or a nucleic acid encoding the endonuclease, is also administered to the cells.

[0239] 216. The modified cell according to Embodiment 215, wherein the guide RNA includes one or more of SEQ ID NOs: 29, 37, 27, 28, and 38, or one or more of the guide sequences: 5, 11, 3, 4, and 12.

[0240] 217. Modified cells according to any one of embodiments 195 to 216, further comprising genomic disruption within the endogenous FAS gene that suppresses or eliminates the expression of the functional FAS cell surface cell death receptor (FAS) protein.

[0241] 218. The modified cell according to Embodiment 217, wherein the genomic disruption within the FAS gene is located within exon 1 of the FAS gene.

[0242] 219. The modified cell according to Embodiment 217 or Embodiment 218, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 1 of the endogenous FAS gene, and the mutation may be an insertion, deletion, or substitution.

[0243] 220. A modified cell according to any one of embodiments 217 to 219, wherein the genome disruption is located within a target sequence in the FAS containing Sequence ID No. 62.

[0244] 221. Modified cells according to any one of Embodiments 217 to 220, wherein the genome disruption within the FAS is carried out by administering a guide RNA to the cells, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0245] 222. The modified cell according to Embodiment 221, wherein the guide RNA includes SEQ ID NO: 60 or the guide sequence of SEQ ID NO: 61.

[0246] 223. Modified cells according to any one of Embodiments 195 to 222, further comprising genomic disruption within the endogenous B2M gene that suppresses or eliminates the expression of functional β-2-microglobulin (B2M) protein.

[0247] 224. The modified cell according to Embodiment 223, wherein the genomic disruption within the B2M gene is located within exon 2 of the B2M gene.

[0248] 225. The modified cell according to Embodiment 223 or Embodiment 224, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 2 of the endogenous B2M gene, and the mutation may be an insertion, deletion, or substitution.

[0249] 226. A modified cell according to any one of embodiments 223 to 225, wherein the genome disruption is located within a target sequence in B2M containing Sequence ID No. 56.

[0250] 227. Modified cells according to any one of embodiments 223 to 226, wherein the genome disruption within B2M is carried out by administering a guide RNA to the cells, wherein the guide RNA is optionally a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0251] 228. The modified cell according to Embodiment 227, wherein the guide RNA includes SEQ ID NO: 54 or the guide sequence of SEQ ID NO: 55.

[0252] 229. Modified cells according to any one of Embodiments 195 to 228, further comprising genomic disruption within the endogenous class II transactivator (CIITA) gene that suppresses or eliminates the expression of functional B2M protein.

[0253] 230. The modified cell according to Embodiment 229, wherein the genome disruption within the CIITA gene is located within exon 3 of the CIITA gene.

[0254] 231. The modified cell according to Embodiment 229 or Embodiment 230, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 3 of the endogenous CIITA gene, and the mutation may be an insertion, deletion, or substitution.

[0255] 232. A modified cell according to any one of embodiments 229 to 231, wherein the genome disruption is located within a target sequence in CIITA containing Sequence ID No. 59.

[0256] 233. Modified cells according to any one of embodiments 229 to 232, wherein the genome disruption within the CIITA is carried out by administering a guide RNA to the cells, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cells.

[0257] 234. The modified cell according to Embodiment 233, wherein the guide RNA includes SEQ ID NO: 57 or the guide sequence of SEQ ID NO: 58.

[0258] 235. Modified cells according to any one of embodiments 195 to 234, further comprising genomic disruption within the endogenous T cell receptor α constant region (TRAC) gene that suppresses or eliminates the expression of a functional TRAC protein.

[0259] 236. A modified cell according to any one of Embodiments 195 to 235, wherein the genome disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

[0260] 237. A modified cell according to any one of Embodiments 195 to 236, further comprising one or more exogenous polynucleotides encoding an IFNγ signaling factor, wherein the IFNγ signaling factor comprises a first IFNγ fusion protein subunit and a second IFNγ fusion protein subunit, the first IFNγ fusion protein subunit comprising the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the intracellular domain of interleukin-2 receptor subunit gamma (IL2RG), and the second IFNγ fusion protein subunit comprising the ECD of interferon-gamma receptor 2 (IFNGR2) and the intracellular domain of interleukin-2 receptor subunit beta (IL2RB).

[0261] 238. A modified cell according to Embodiment 237, wherein a first exogenous polynucleotide encoding the first IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the SOCS1 locus.

[0262] 239. Modified cells according to Embodiment 237 or Embodiment 238, wherein a second exogenous polynucleotide encoding the second IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the ROSA26 locus.

[0263] 240. A modified cell according to any one of embodiments 237 to 239, wherein the first IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 71, the second IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 72, and optionally the first exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 81, and the second exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 82.

[0264] 241. A modified cell according to any one of embodiments 237 to 240, wherein the amino acid sequence of the first IFNγ fusion protein subunit is SEQ ID NO: 71, and the amino acid sequence of the second IFNγ fusion protein subunit is SEQ ID NO: 72.

[0265] 242. A modified cell according to any one of Embodiments 195 to 241, further comprising an exogenous polynucleotide encoding a B2M-HLA-E fusion protein (B2M-HLA-E) which comprises at least a portion of B2M fused with at least a portion of HLA-E.

[0266] 243. The modified cell according to Embodiment 242, wherein the exogenous polynucleotide encoding B2M-HLA-E is inserted into the B2M locus.

[0267] 244. The modified cell according to Embodiment 242 or Embodiment 243, wherein the B2M-HLA-E contains the amino acid sequence of Sequence ID No. 85.

[0268] 245. A modified cell according to any one of Embodiments 195 to 244, wherein the iPSC is derived from a primary T cell (T-iPSC).

[0269] 246. A modified cell according to any one of Embodiments 195 to 245, wherein the iPSC is derived from a primary γδT cell (γδT-iPSC).

[0270] 247. A modified cell according to any one of embodiments 195 to 246, wherein the γδT cell is a Vd2γδT cell.

[0271] 248. A modified cell according to any one of Embodiments 195 to 247, wherein the γδT cells are iPSC-derived γδT (iγδT) cells, and optionally the γδT cells are T-iPSC-derived γδT cells (T-iγδT).

[0272] 249. The modified cell according to any one of Embodiments 195 to 248, wherein the γδT cells are γδT cells (γδT-iγδT) differentiated from γδT-iPSC cells.

[0273] 250. A modified cell according to any one of Embodiments 195 to 249, wherein the intermediate cell differentiated from the iPSC is a γδT-iHSC, a γδT-iCLP, and / or an immature T-iγδT cell.

[0274] 251. A pharmaceutical composition comprising modified γδT cells according to any one of Embodiments 195 to 250 and a pharmaceutically acceptable carrier.

[0275] 252. A method for killing CD19-positive B cells having abnormal B cell function, comprising administering a therapeutically effective amount of modified γδ T cells described in any one of embodiments 195 to 250 or a pharmaceutical composition described in embodiment 251 to a target subject.

[0276] 253. The method according to Embodiment 252, wherein the B cells are CD19-positive B cells from a patient with diffuse large B-cell lymphoma (DLBCL) or systemic lupus erythematosus (SLE).

[0277] 254. A method for treating DLBCL or SLE, comprising administering a therapeutically effective amount of modified γδT cells according to any one of embodiments 195 to 250 or a pharmaceutical composition according to embodiment 251 to a subject of interest.

[0278] 255. Modified γδT cells according to any one of Embodiments 195 to 250 or a pharmaceutical composition according to Embodiment 251, for use in therapeutic purposes.

[0279] 256. Modified γδ T cells according to any one of Embodiments 195 to 250 or a pharmaceutical composition according to Embodiment 251 for use in the treatment of DLBCL or SLE.

[0280] 257. Use of modified cells according to any one of Embodiments 195 to 250 or the pharmaceutical composition according to Embodiment 251 in the manufacture of a drug for the treatment of DLBCL or SLE.

[0281] 258. A method for producing modified γδT cells according to any one of Embodiments 195 to 250, comprising: (i) optionally producing modified iPSC cells according to any one of Embodiments 195 to 250 according to the method described in any one of Embodiments 62 to 111; and (ii) differentiating the modified iPSC cells into modified γδT cells.

[0282] 259. A method for producing modified γδT cells according to any one of Embodiments 1 to 59, comprising: (i) optionally producing modified iPSC cells according to any one of Embodiments 1 to 59 according to the method described in any one of Embodiments 62 to 111; and (ii) differentiating the modified iPSC cells into modified γδT cells.

[0283] 260. The method according to any one of Embodiments 62-111, 119-124, and 139-143, further comprising differentiating the iPSC cells produced by the method according to any one of Embodiments 62-111, 119-124, and 139-143 into the modified γδT cells.

[0284] Furthermore, this disclosure also includes, in particular, the following embodiments among those disclosed herein.

[0285] Embodiment 1102. A modified iPSC, γδT cell, or intermediate cell differentiated from an iPSC, comprising: 1) genomic disruption within the endogenous SOCS1 gene to suppress or eliminate SOCS1 expression; 2) genomic disruption within the endogenous CISH gene to suppress or eliminate CISH expression; and 3) genomic disruption within exon 2C of the endogenous BIM gene. EL and BIM L The expression level and / or function of the splice variant are reduced in the modified cells compared to the unmodified control, BIM S Modified iPSCs, γδT cells, or intermediate cells differentiated from iPSCs, wherein the expression level and / or function of the splice variant are maintained in the modified cells.

[0286] Embodiment 1103. Modified iPSC, γδT cell, or intermediate cell differentiated from iPSC, comprising: 1) genomic disruption within the endogenous SOCS1 gene that suppresses or eliminates SOCS1 expression; 2) genomic disruption within the endogenous CISH gene that suppresses or eliminates CISH expression; and 3) BIM EL and BIM L The expression level and / or function of the splice variant are reduced in the modified cells compared to the unmodified control, BIM SA modified iPSC, γδT cell, or intermediate cell differentiated from an iPSC, comprising: 4) genomic disruption in exon 2C of the endogenous BIM gene, which preserves the expression level and / or function of the splice variant in the modified cell; 4) genomic disruption in the endogenous B2M gene, which suppresses or eliminates the expression of B2M; and 5) genomic disruption in the endogenous CIITA gene, which suppresses or eliminates the expression of CIITA.

[0287] Embodiment 1104. Modified iPSC, γδT cell, or intermediate cell differentiated from iPSC, comprising: 1) genomic disruption within the endogenous SOCS1 gene that suppresses or eliminates SOCS1 expression; 2) genomic disruption within the endogenous CISH gene that suppresses or eliminates CISH expression; and 3) BIM EL and BIM L The expression level and / or function of the splice variant are reduced in the modified cells compared to the unmodified control, BIM S A modified iPSC, γδT cell, or intermediate cell differentiated from an iPSC, comprising: 4) genomic disruption in exon 2C of the endogenous BIM gene, which preserves the expression level and / or function of the splice variant in the modified cell; 4) genomic disruption in the endogenous B2M gene, which suppresses or eliminates the expression of B2M; 5) genomic disruption in the endogenous CIITA gene, which suppresses or eliminates the expression of CIITA; and 6) genomic disruption in the endogenous FAS gene, which suppresses or eliminates the expression of FAS.

[0288] Embodiment 1105. A modified cell according to any one of Embodiments 1102 to 1104, wherein the iPSC is derived from a primary T cell (T-iPSC).

[0289] Embodiment 1106. A modified cell according to any one of Embodiments 1102 to 1105, wherein the iPSC is derived from a primary γδT cell (γδT-iPSC).

[0290] Embodiment 1107. A modified cell according to any one of Embodiments 1102 to 1106, wherein the γδT cell is a Vd2γδT cell.

[0291] Embodiment 1108. A modified cell according to any one of Embodiments 1102 to 1107, wherein the γδT cells are iPSC-derived γδT (iγδT) cells, and optionally the γδT cells are T-iPSC-derived γδT cells (T-iγδT).

[0292] Embodiment 1109. A modified cell according to any one of Embodiments 1102 to 1108, wherein the γδT cells are γδT cells (γδT-iγδT) differentiated from γδT-iPSC cells.

[0293] Embodiment 1110. A modified cell according to any one of Embodiments 1102 to 1109, wherein the intermediate cell differentiated from the iPSC is a γδT-iHSC, a γδT-iCLP, and / or an immature T-iγδT cell.

[0294] Embodiment 1111. A modified cell according to any one of Embodiments 1102 to 1110, wherein the genomic disruption in SOCS1 is located in exon 2 of the SOCS1 gene, and the genomic disruption in CISH is located in exon 3 or 4 of the CISH gene.

[0295] Embodiment 1112. A modified cell according to any one of Embodiments 1102 to 1111, wherein the genome disruption is located within a target sequence in SOCS1 including SEQ ID NO: 17 or SEQ ID NO: 23, and / or within a target sequence in CISH including SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 24.

[0296] Embodiment 1113. A modified cell according to any one of Embodiments 1102 to 1112, wherein the SOCS1 and genome disruption within CISH are performed by administering a guide RNA to the cells, wherein the guide RNA is a single guide RNA (sgRNA), and optionally an endonuclease or a nucleic acid encoding an endonuclease is also administered to the cells.

[0297] Embodiment 1114. The modified cell according to Embodiment 1113, wherein the guide RNA comprises one or more of SEQ ID NOs: 5, 29, 11, 37, 3, 27, 4, 28, 12, and 38.

[0298] Embodiment 1115. A modified cell according to any one of Embodiments 1102 to 1114, wherein the genome disruption is located within a target sequence in the BIM containing any one of Sequence IDs 14, 20, 21, and 22.

[0299] Embodiment 1116. A modified cell according to any one of Embodiments 1102 to 1115, wherein the genome disruption in the BIM is carried out by administering a guide RNA to the cell, wherein the guide RNA is optionally a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding an endonuclease is also administered to the cell.

[0300] Embodiment 1117. The modified cell according to Embodiment 1116, wherein the guide RNA comprises one of sequence numbers 2, 26, 8, 34, 9, 35, 10, and 36.

[0301] Embodiment 1118. A modified cell according to any one of Embodiments 1102 to 1117, wherein the genome disruption within FAS is located in exon 1 of the FAS gene.

[0302] Embodiment 1119. A modified cell according to any one of Embodiments 1102 to 1118, wherein the genome disruption is located within a target sequence in the FAS containing Sequence ID No. 62.

[0303] Embodiment 1120. A modified cell according to any one of Embodiments 1102 to 1119, wherein the genome disruption within the FAS is carried out by administering a guide RNA to the cell, wherein the guide RNA is optionally a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding an endonuclease is also administered to the cell.

[0304] Embodiment 1121. The modified cell according to Embodiment 1120, wherein the guide RNA comprises one of sequence numbers 60 and 61.

[0305] Embodiment 1122. A modified cell according to any one of Embodiments 1102 to 1121, wherein the genome disruption within B2M is located in exon 2 of the B2M gene.

[0306] Embodiment 1123. A modified cell according to any one of Embodiments 1102 to 1122, wherein the genome disruption is located within a target sequence in B2M containing Sequence ID No. 56.

[0307] Embodiment 1124. A modified cell according to any one of Embodiments 1102 to 1123, wherein the genome disruption within B2M is carried out by administering a guide RNA to the cell, wherein the guide RNA is optionally a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding an endonuclease is also administered to the cell.

[0308] Embodiment 1125. The modified cell according to Embodiment 1124, wherein the guide RNA comprises one of SEQ ID NOs. 54 and 55.

[0309] Embodiment 1126. A modified cell according to any one of Embodiments 1102 to 1125, wherein the genome disruption within CIITA is located in exon 3 of the CIITA gene.

[0310] Embodiment 1127. A modified cell according to any one of Embodiments 1102 to 1126, wherein the genome disruption is located within a target sequence in CIITA containing Sequence ID No. 59.

[0311] Embodiment 1128. A modified cell according to any one of Embodiments 1102 to 1127, wherein the genome disruption within the CIITA is carried out by administering a guide RNA to the cell, wherein the guide RNA is optionally a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding an endonuclease is also administered to the cell.

[0312] Embodiment 1129. The modified cell according to Embodiment 1128, wherein the guide RNA comprises one of sequence numbers 57 and 58.

[0313] Embodiment 1130. A modified cell according to any one of Embodiments 1102 to 1129, further comprising one or more exogenous polynucleotides encoding an IFNγ signaling factor, wherein the IFNγ signaling factor comprises a first IFNγ fusion protein subunit and a second IFNγ fusion protein subunit, the first IFNγ fusion protein subunit comprising the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the intracellular domain of interleukin-2 receptor subunit gamma (IL2RG), and the second IFNγ fusion protein subunit comprising the ECD of interferon-gamma receptor 2 (IFNGR2) and the intracellular domain of interleukin-2 receptor subunit beta (IL2RB).

[0314] Embodiment 1131. The modified cell according to Embodiment 1130, wherein the amino acid sequence of the first IFNγ fusion protein subunit is SEQ ID NO: 71, and the amino acid sequence of the second IFNγ fusion protein subunit is SEQ ID NO: 72.

[0315] Furthermore, this disclosure also includes, in particular, the following embodiments among those disclosed herein.

[0316] Embodiment 2001. An IFNγ signaling factor comprising a first IFNγ fusion protein subunit and a second IFNγ fusion protein subunit, wherein the first IFNγ fusion protein subunit comprises the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the intracellular domain of interleukin-2 receptor subunit gamma (IL2RG), and the second IFNγ fusion protein subunit comprises the ECD of interferon-gamma receptor 2 (IFNGR2) and the intracellular domain of interleukin-2 receptor subunit beta (IL2RB).

[0317] Embodiment 2002. The IFNγ signal-converting factor according to Embodiment 2001, wherein the amino acid sequence of the ECD of the first IFNγ fusion protein subunit is SEQ ID NO: 63, and the amino acid sequence of the ECD of the second IFNγ fusion protein subunit is SEQ ID NO: 64.

[0318] Embodiment 2003. An IFNγ signal-converting factor according to Embodiment 2001 or Embodiment 2002, wherein the amino acid sequence of the intracellular domain of the first IFNγ fusion protein subunit is SEQ ID NO: 69, and the amino acid sequence of the intracellular domain of the second IFNγ fusion protein subunit is SEQ ID NO: 70.

[0319] Embodiment 2004. An IFNγ signal-converting factor according to any one of Embodiments 2001 to 2003, wherein the first IFNγ fusion protein subunit further comprises a transmembrane domain between the ECD and its intracellular domain, and the second IFNγ fusion protein subunit further comprises a transmembrane domain between the ECD and its intracellular domain.

[0320] Embodiment 2005. The IFNγ signal-converting factor according to Embodiment 2004, wherein the transmembrane domain of the first IFNγ fusion protein subunit is that of the interleukin-2 receptor subunit gamma, and the transmembrane domain of the second IFNγ fusion protein subunit is that of the interleukin-2 receptor subunit beta.

[0321] Embodiment 2006. The IFNγ signal-converting factor according to Embodiment 2004, wherein the amino acid sequence of the transmembrane domain of the first IFNγ fusion protein subunit is SEQ ID NO: 67, and the amino acid sequence of the transmembrane domain of the second IFNγ fusion protein subunit is SEQ ID NO: 68.

[0322] Embodiment 2007. An IFNγ signal-converting factor according to any one of Embodiments 2001 to 2006, wherein the first IFNγ fusion protein subunit further comprises a membrane-proximal region between the ECD and its intracellular domain, and the second IFNγ fusion protein subunit further comprises a membrane-proximal region between the ECD and its intracellular domain.

[0323] Embodiment 2008. The IFNγ signal-converting factor according to Embodiment 2007, wherein the membrane proximal region of the first IFNγ fusion protein subunit is that of the interleukin-2 receptor subunit gamma, and the membrane proximal region of the second IFNγ fusion protein subunit is that of the interleukin-2 receptor subunit beta.

[0324] Embodiment 2009. The IFNγ signal-converting factor according to Embodiment 2008, wherein the amino acid sequence of the membrane-proximal region of the first IFNγ fusion protein subunit is SEQ ID NO: 65, and the amino acid sequence of the membrane-proximal region of the second IFNγ fusion protein subunit is SEQ ID NO: 66.

[0325] Embodiment 2010. An IFNγ signal-converting factor according to any one of Embodiments 2001 to 2009, wherein the first IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 71, the second IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 72, and optionally the first exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 81, and the second exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 82.

[0326] Embodiment 2011. An IFNγ signal-converting factor according to any one of Embodiments 2001 to 2010, wherein the amino acid sequence of the first IFNγ fusion protein subunit is SEQ ID NO: 71, and the amino acid sequence of the second IFNγ fusion protein subunit is SEQ ID NO: 72. [Brief explanation of the drawing]

[0327] [Figure 1] This shows the knockout efficiency of candidate genes (CISH, SOCS1, and BIM) in T-iγδT (iPSC-derived γδT) cells as determined by Western blotting. [Figure 2A] This study demonstrates the synergistic effect of CISH- / -SOCS1- / - double knockout (DKO) and CISH- / -SOCS1- / -BIM- / - triple knockout (TKO) T-iγδT cells in enhanced cytotoxicity and / or persistence against HepG2 cells. It also shows the sequential killing ability of CISH- / - single KO, SOCS1- / - single KO, BIM- / - single KO, CISH- / -SOCS1- / -DKO, and CISH- / -SOCS1- / -BIM- / -TKO T-iγδT cells against HepG2 cells in different effector:target (E:T) ratios, monitored by the Incucyte® SX5 live cell analysis system in the absence of zoledronic acid. [Figure 2B]This study demonstrates the synergistic effect of CISH- / -SOCS1- / - double knockout (DKO) and CISH- / -SOCS1- / -BIM- / - triple knockout (TKO) T-iγδT cells in enhanced cytotoxicity and / or persistence against HepG2 cells. It also shows the sequential killing ability of CISH- / - single KO, SOCS1- / - single KO, BIM- / - single KO, CISH- / -SOCS1- / -DKO, and CISH- / -SOCS1- / -BIM- / -TKO T-iγδT cells against HepG2 cells in different effector:target (E:T) ratios, monitored by the Incucyte® SX5 live cell analysis system in the absence of zoledronic acid. [Figure 2C] This study demonstrates the synergistic effect of CISH- / -SOCS1- / - double knockout (DKO) and CISH- / -SOCS1- / -BIM- / - triple knockout (TKO) T-iγδT cells in enhanced cytotoxicity and / or persistence against HepG2 cells. It also shows the sequential killing ability of CISH- / - single KO, SOCS1- / - single KO, BIM- / - single KO, CISH- / -SOCS1- / -DKO, and CISH- / -SOCS1- / -BIM- / -TKO T-iγδT cells against HepG2 cells at different effector:target (E:T) ratios, as monitored by the Incucyte® SX5 live cell analysis system. [Figure 2D] This study demonstrates the synergistic effect of CISH- / -SOCS1- / - double knockout (DKO) and CISH- / -SOCS1- / -BIM- / - triple knockout (TKO) T-iγδT cells in enhanced cytotoxicity and / or persistence against HepG2 cells. It also shows the sequential killing ability of CISH- / - single KO, SOCS1- / - single KO, BIM- / - single KO, CISH- / -SOCS1- / -DKO, and CISH- / -SOCS1- / -BIM- / -TKO T-iγδT cells against HepG2 cells at different effector:target (E:T) ratios, as monitored by the Incucyte® SX5 live cell analysis system. [Figure 2E]This study demonstrates the synergistic effect of CISH- / -SOCS1- / - double knockout (DKO) and CISH- / -SOCS1- / -BIM- / - triple knockout (TKO) T-iγδT cells in enhanced cytotoxicity and / or persistence against HepG2 cells. It also shows the cytotoxicity of CISH- / - single KO, SOCS1- / - single KO, BIM- / - single KO, CISH- / -SOCS1- / -DKO, and CISH- / -SOCS1- / -BIM- / -TKO T-iγδT cells against HepG2 3D spheroids at an effector:target (E:T) ratio of 10:1 in the absence of zoledronic acid, as monitored by the Incucyte® SX5 live cell analysis system. [Figure 2F] This study demonstrates the synergistic effect of CISH- / -SOCS1- / - double knockout (DKO) and CISH- / -SOCS1- / -BIM- / - triple knockout (TKO) T-iγδ T cells on enhanced cytotoxicity and / or persistence against HepG2 cells. HepG2 3D spheroid images after co-culture with CISH- / - single KO, SOCS1- / - single KO, BIM- / - single KO, CISH- / -SOCS1- / -DKO, and CISH- / -SOCS1- / -BIM- / -TKO T-iγδ T cells are shown. EGFP-positive HepG2 cells are indicated by black arrows (bright areas), and surrounded T-iγδ T cells are indicated by white arrows. [Figure 3] T-iγδT cells lacking BIMEL and / or BIML (knockout by BIMsgRNA#5 (SEQ ID NO: 26)), or CISH- / -SOCS1- / -BIM- / -TKO T-iγδT cells with BIM- / - modifications that lack BIMEL and BIML, exhibit extended survival for 2–12 days after IL-15 depletion (experimental IL-15 levels were 0 ng / mL, 0.05 ng / mL, 0.3 ng / mL, and 10 ng / mL). [Figure 4A] This shows that the BIM short-chain splice variant (BIMS) is necessary for the survival of T-iγδT cells after IL-15 depletion. The schematic diagram of the BIM gene structure shows that there are three major splice variants expressed in T-iγδT cells. [Figure 4B]This study demonstrates that BIM short splice variants (BIMS) are necessary for the survival of T-iγδT cells after IL-15 depletion. Knockout of each BIM splice variant by different BIMsgRNAs (BIM#5 (SEQ ID NO: 26), #9 (SEQ ID NO: 32), #19 (SEQ ID NO: 33), #24 (SEQ ID NO: 34), or #25 (SEQ ID NO: 35)) is shown by Western blotting. [Figure 4C] This study demonstrates that the BIM short-chain splice variant (BIMS) is necessary for the survival of T-iγδT cells after IL-15 depletion. It also shows that BIMEL and BIML splice variants (which retain BIMS) mediated by BIMsgRNA #5 (SEQ ID NO: 26) and #25 (SEQ ID NO: 35) extended the survival rate of T-iγδT cells when cultured under IL-15 concentration conditions of 0 and 0.05 ng / mL. [Figure 5A] This report shows the editing efficiency of CISHcrRNA (CRISPR RNA) using CRISPR / MAD7-mediated gene editing. The insertion-deletion (indel) rates (ICEd) for control and CISHcrRNA samples, calculated by ICE analysis, are shown. Data are expressed as mean ± standard deviation. [Figure 5B] This shows the editing efficiency of CISHcrRNA (CRISPR RNA) using CRISPR / MAD7-mediated gene editing. Representative screenshots of Sanger sequencing chromatograms showing indel formation after CRISPR / MAD7-mediated gene editing at CISH loci are shown. Shaded areas indicate targeting sites after indel formation by CISHcrRNA. [Figure 5C] This shows the editing efficiency of CISH crRNA (CRISPR RNA) using CRISPR / MAD7-mediated gene editing. A summary chart of single clone selection and analysis of CISH-edited cells is also shown. [Figure 6A] This shows the editing efficiency of CISHcrRNA using CRISPR / MAD7-mediated gene editing. The indel rate (ICEd) for control and CISHcrRNA samples, calculated by ICE analysis, is shown. Data are expressed as mean ± SD. [Figure 6B]This shows the editing efficiency of CISHcrRNA using CRISPR / MAD7-mediated gene editing. Western blot analysis of SOCS1 protein loss in two SOCS1 homozygous knockout clones (#13 and #36) is shown. [Figure 6C] This shows the editing efficiency of CISHcrRNA using CRISPR / MAD7-mediated gene editing. A representative screenshot of Sanger sequencing results showing indel formation after CRISPR / MAD7-mediated gene editing at the SOCS1 locus is shown. The shaded area indicates the targeting site after indel formation by SOCS1crRNA. [Figure 6D] This shows the editing efficiency of CISHcrRNA using CRISPR / MAD7-mediated gene editing. A summary chart of single clone selection and analysis of SOCS1-edited cells is also shown. [Figure 7A] This shows the editing efficiency of BIMcrRNA using CRISPR / MAD7-mediated gene editing. The indel rate (ICEd) for control and BIMcrRNA samples, calculated by ICE analysis, is shown. Data are expressed as mean ± SD. [Figure 7B] This paper demonstrates the editing efficiency of BIMcrRNA using CRISPR / MAD7-mediated gene editing. Western blot analysis of BIM protein loss in one heterozygous (#21) and one homozygous (#39) knockout clones is shown. The crRNA designed for BIM is intended to eliminate BIMEL and BIML splice morphologies while retaining BIMS. [Figure 7C] This shows the editing efficiency of BIMcrRNA using CRISPR / MAD7-mediated gene editing. Representative screenshots of Sanger sequencing results showing indel formation after CRISPR / MAD7-mediated gene editing at BIM loci are shown. Shaded areas indicate targeting sites after indel formation by BIMcrRNA. [Figure 7D] This shows the editing efficiency of BIMcrRNA using CRISPR / MAD7-mediated gene editing. A summary chart of single clone selection and analysis of BIM-edited cells is shown. [Figure 8] This study shows extended survival times for FAS-deficient (FAS gene knockout by FASsgRNA#1 (SEQ ID NO: 60)) T-iγδT cells and functionally enhanced 6KO (B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / - / FAS- / -) T-iγδT cells compared to 5KO (B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / -) T-iγδT cells in the presence of FAS ligand (FASL, an inducer of cell apoptosis) and in the absence of human recombinant IL-15. [Figure 9A] This study demonstrates enhanced cytotoxicity and persistence against tumor cells of FAS-deficient and functionally enhanced 6KO T-iγδT cells compared to 5KO T-iγδT cells. Live-cell imaging and analysis monitor the sequential killing ability of 5KO(B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / -) and 6KO(B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / - / FAS- / -) T-iγδT cells against HepG2 cells at different effector:target (E:T) ratios. [Figure 9B] This study demonstrates enhanced cytotoxicity and persistence against tumor cells of FAS-deficient and functionally enhanced 6KO T-iγδT cells compared to 5KO T-iγδT cells. Live-cell imaging and analysis monitor the continuous killing ability of 5KO(B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / -) and 6KO(B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / - / FAS- / -) T-iγδT cells against HepG2 cells at different effector:target (E:T) ratios, after second-round activation. [Figure 9C]This study demonstrates enhanced cytotoxicity and persistence against tumor cells of FAS-deficient and functionally enhanced 6KO T-iγδT cells compared to 5KO T-iγδT cells. Live-cell imaging and analysis monitor the continuous killing ability of 5KO(B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / -) and 6KO(B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / - / FAS- / -) T-iγδT cells against HepG2 cells at different effector:target (E:T) ratios, after second-round activation. [Figure 9D] This study demonstrates enhanced cytotoxicity and persistence against tumor cells of FAS-deficient and functionally enhanced 6KO T-iγδT cells compared to 5KO T-iγδT cells. Live-cell imaging and analysis monitor the serial killing ability of 5KO(B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / -) and 6KO(B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / - / FAS- / -)T-iγδT cells against U937 cells at an E:T ratio of 5:1. [Figure 10] This study shows an increase in effector molecules in FAS-deficient 6KO T-iγδT cells after serial killing of HepG2 cells, compared to 5KO T-iγδT cells. The concentration of effector molecules in the culture supernatant after co-culture with HepG2 cells was quantified using a bead-based multiplex assay. [Figure 11A] This shows the increased IFN-γ production rate (%) in FAS-deficient 6KO T-iγδT cells, and the enhanced IFN-γ production capacity of individual FAS-deficient 6KO T-iγδT cells compared to 5KO T-iγδT cells. It also shows the percentage (%) of TCR Vδ2+ cells and FAS+ cells in 5KO (B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / -) and 6KO (B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / - / FAS- / -) T-iγδT cells, as measured by flow cytometry. [Figure 11B]This shows the increased IFN-γ production rate (%) in FAS-deficient 6KO T-iγδT cells, and the enhanced IFN-γ production capacity of individual FAS-deficient 6KO T-iγδT cells compared to 5KO T-iγδT cells. It also shows the percentage of IFN-γ producing TCR Vδ2+ T-iγδT cells among 5KO (B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / -) and 6KO (B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / - / FAS- / -) T-iγδT cells, measured using flow cytometry, both in the absence of HepG2 cells and after 24 hours of co-culture with HepG2 cells. [Figure 11C] This shows the increased IFN-γ production rate (%) in FAS-deficient 6KO T-iγδT cells and the enhanced IFN-γ production capacity of individual FAS-deficient 6KO T-iγδT cells compared to 5KO T-iγδT cells. It also shows the mean fluorescence intensity (MFI) of IFN-γ in individual 5KO (B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / -) and 6KO (B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / - / FAS- / -) T-iγδT cells after 24 hours of co-culture with HepG2 cells, as measured using flow cytometry. [Figure 12] This shows the extracellular domain (ECD) of an interferon-gamma (IFN-γ) receptor fused to the intracellular domain (ICD) of another cytokine receptor via its transmembrane domain. [Figure 13A]This study demonstrates that the IFNγR-IL2 / IL15R signaling factor can induce phosphorylation of STAT1 and STAT5 in mature iγδT5 KO (B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / -) cells. Surface expression of IFNγR-IL2 / IL15R is shown on mature iγδT5 KO cells transduced with a lentivirus encoding the IFNγR-IL2 / IL15R signaling factor, with approximately 19% of iγδT5 KO cells expressing IFNγR-IL2 / IL15R. In contrast, no expression of IFNγR-IL2 / IL15R was observed in control cells that were not transduced with a lentivirus encoding the IFNγR-IL2 / IL15R signaling factor (0.099%). [Figure 13B] This study demonstrates that IFNγR-IL2 / IL15R signaling converter can induce phosphorylation of STAT1 and STAT5 in mature iγδT5 KO (B2M- / - / CIITA- / - / CISH- / - / SOCS1- / - / BIM- / -) cells. The results of treating mature iγδT5 KO cells expressing IFNγR-IL2 / IL15R signaling converter with 5 ng / mL of IFN-γ in a time-dependent manner (15, 60, and 120 minutes) are shown. Phosphorylation of STAT1, STAT3, and STAT5 was examined by immunoblotting. The results show that phosphorylation of STAT1 and STAT5 was significantly induced in mature iγδT5 KO cells expressing IFNγR-IL2 / IL15R signaling converter after 15 minutes of IFN-γ treatment. The increase in STAT1 and STAT5 phosphorylation persisted for 120 minutes. Phosphorylation of STAT3 was also induced after IFN-γ treatment. [Figure 14]The following conditions, namely IL-7 and IL-15 starvation, IL-7 and IL-15 starvation with 5 ng / mL IFN-γ supplementation, or 1 ng / mL IL-7 and 1 ng / mL IL-15 supplementation, were used to show the 6-day increase in the proliferation of mature iγδT 5KO cells expressing IFNγR-IL2 / IL15R signaling converter. The proliferation rate (%) was analyzed by flow cytometry and normalized to day 0. The results show that IFNγR-IL2 / IL15R signaling converter increased the cell proliferation of mature iγδT 5Ko cells by up to 42.3% over 6 days under IFN-γ treatment compared to control cells (lacking IFNγR-IL2 / IL15R signaling converter, with a cell proliferation increase of 11.3%). [Figure 15] This study, monitored using the Incucyte SX5 live cell imaging system (Sartorius), demonstrates improved serial tumor-killing ability of mature iγδT 5KO cells expressing IFNγR-IL2 / IL15R signaling converter against HepG2 cells (hepatoblastoma cell line) at an effector:target (E:T) ratio of 5:1 in the absence of cytokines. The results show that IFNγR-IL2 / IL15R signaling converter significantly enhanced the serial tumor-killing effect of mature iγδT 5KO cells against HepG2 cells, maintaining this effect for 24 rounds compared to 5 rounds of killing demonstrated by control cells. Control cells were iγδT 5KO cells lacking IFNγR-IL2 / IL15R signaling converter. [Figure 16] When co-cultured with HepG2 cells, mature iγδT 5KO cells expressing IFNγR-IL2 / IL15R signaling converters show time-dependent increases in the secretion of granzyme A (A), granzyme B (B), and perforin (C) at 24, 48, 72, and 96 hours. [Figure 17A]This study demonstrates dose-dependent and antigen-specific cytotoxicity of modified CD19-CAR iγδT cells. It also shows the cytotoxic activity of wild-type (WT, unedited) iγδT, modified CD19-CAR iγδT, donor-derived unedited αβT, and donor-derived CD19-CAR αβT cells using bioluminescence-based cytotoxicity assays. Cytotoxicity against the CD19-positive tumor cell lines Nalm-6-Luc and Raji-Luc was tested at the indicated effector:target ratio (E:T) after 24 hours and analyzed by relative bioluminescence activity. [Figure 17B] Modified CD19-CAR iγδ T cells exhibit dose-dependent and antigen-specific cytotoxicity. While modified CD19-CAR iγδ T cells showed significant killing activity against Nalm-6 using a FACS-based cytotoxicity assay, they did not show significant killing activity against Nalm-6-CD19KO (Nalm6 cells lacking CD19 expression) or several normal human primary cells, demonstrating CD19-specific killing activity of modified CD19-CAR iγδ T cells. [Figure 18A] This shows the secretion profiles of cytokines and cytolytic granules from WT iγδT, modified CD19-CAR iγδT, donor-derived unedited αβT, and donor-derived CD19-CAR αβT cells against Nalm-6-Luc over 24 hours at an E:T ratio of 2, using a bead-based multiplex method. It shows increased production of cytolytic granules, including granzyme A, granzyme B, perforin, and granulosin, from modified CD19-CAR iγδT cells. [Figure 18B]This study shows the secretion profiles of cytokines and cytolytic granules from WT iγδT, modified CD19-CAR iγδT, donor-derived unedited αβT, and donor-derived CD19-CAR αβT cells against Nalm-6-Luc over 24 hours at an E:T ratio of 2, using a bead-based multiplex method. It demonstrates relatively lower secretion of inflammatory cytokines (i.e., IL-4, IL-6, and IL-17a) and immunomodulatory cytokines (i.e., GM-CSF and IL-10) from modified CD19-CAR iγδT cells compared to donor-derived CD19-CAR αβT cells. [Figure 19A] This study demonstrates the dose-dependent and antigen-specific cytotoxicity of modified CD19-CAR iγδT cells against healthy donor cells (19A) and patient-derived PBMCs (19B). The cytotoxic activity of WTiγδT, modified CD19-CAR iγδT, donor-derived unedited αβT, and donor-derived CD19-CAR αβT cells is shown using a FACS-based cytotoxicity assay. Cytotoxicity against healthy donor PBMCs was tested using the indicated effector:PBMC ratio (E:PBMC), and the specific killing activity of modified CD19-CAR iγδT against CD19-positive PBMCs was analyzed by relative viable CD19-positive or CD19-negative cells in the test sample relative to the PBMCs. [Figure 19B] The modified CD19-CAR iγδT cells demonstrate dose-dependent and antigen-specific cytotoxicity against healthy donor (19A) and patient-derived PBMCs (19B). The cytotoxicity of modified CD19-CAR iγδT and donor-derived CD19-CAR αβT cells against PBMCs derived from DLBCL or SLE patients is shown at the effector:PBMC ratio (E:PBMC). [Figure 20A] Modified CD19-CAR iγδT cells exhibit a remarkable sustained cytotoxic response and proliferation dynamics. The extended tumor-killing capacity of modified CD19-CAR iγδT cells, observed by microwell-based live-cell imaging over multiple subsequent tumor challenges with Nalm-6-iRFP713 at an E:T ratio of 2, is also demonstrated. [Figure 20B]Modified CD19-CAR iγδ T cells exhibit a markedly persistent cytotoxic response and proliferation dynamics. The trend of cell proliferation over time in modified CD19-CAR iγδ T cells is also shown. Surviving CD3-positive T cells were quantified by FACS analysis. [Figure 21A] Microwell-based cell imaging over multiple subsequent tumor challenges with Nalm-6-iRFP713 at an E:T ratio of 4 revealed an extended tumor-killing capacity of IFNγSC+CD19-CAR 5KOiγδT cells, demonstrating a sustained cytotoxic response in CD19-CAR 5KOiγδT and IFNγSC+CD19-CAR 5KOiγδT cells. [Figure 21B] This study demonstrates the proliferative capacity of CD19-CAR 5KO iγδT and IFNγSC+CD19-CAR 5KO iγδT cells co-cultured with CD19-KO or CD19 wild-type (WT) Nalm-6 tumors over 24 hours at ET=2. This reveals the synergistic proliferative activity of IFNγSC and CD19-CAR in iγδT cells. The proliferation detection assay was based on nucleoside analog (EdU) uptake and measurement of EdU-positive cells by FACS analysis. [Modes for carrying out the invention]

[0328] Detailed explanation I. Definition Unless otherwise specified, the following terms and phrases have the meanings set forth below. These definitions are not restrictive and are intended to provide a clearer understanding of certain aspects of this disclosure.

[0329] Unless otherwise defined elsewhere in this document, all other technical and scientific terms used herein have the meanings generally understood by those skilled in the art.

[0330] As used herein, including in the appended claims, singular words such as "a," "an," and "the" refer to multiple corresponding subjects unless specifically indicated by the context.

[0331] The term "or" is used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

[0332] Induced pluripotent stem cells (iPSCs): As used herein, the terms “induced pluripotent stem cells” or “iPSCs” refer to induced stem cells produced from somatic cells (e.g., fibroblasts or T cells) using inducible pluripotency reprogramming factors such as Yamanaka factors: Oct3 / 4, Sox2, Klf4, and c-Myc (see Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006 Aug 25;126(4):663-76; further see Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007 Nov 30;131(5):861-72).

[0333] T-iPSC: As used herein, the terms “iPSC,” “T-iPSC,” “T-cell derived iPSC,” “T-derived iPSC,” “T-derived iPSC,” and similar terms refer to iPSCs produced from T cells using induced pluripotency reprogramming factor, which may be primary T cells or γδT cells.

[0334] γδT-iPSC: As used herein, the terms “γδT-iPSC,” “γδT-derived iPSC,” “γδT-derived iPSC,” and similar terms refer to iPSCs created from γδ (gamma / delta) T cells (γδT cells) using induced pluripotency reprogramming factor. The γδT cells may be Vd2 (or Vδ2)γδT cells or Vd1 (or Vδ1)γδT cells.

[0335] γδT cells: As used herein, “γδT cells,” “γδT,” “gamma / delta T,” and similar terms refer to a type of T cell having a T cell receptor (TCR) composed of γ glycoprotein chains and δ glycoprotein chains on the cell surface, regardless of how the γδT cells were produced / obtained. γδT cells may be primary γδT cells or γδT cells differentiated from iPSCs (iγδT). γδT cells may be Vd2 (or Vδ2)γδT cells or Vd1 (or Vδ1)γδT cells.

[0336] iγδT: As used herein, the terms “iPSC-derived γδT cells,” “iPSC-γδT,” “iγδT,” and similar terms refer to γδT cells differentiated from iPSCs.

[0337] T-iγδT: The terms "T-iγδT," "T-igdT," "T-derived iγδT," and similar terms refer to γδT cells differentiated from iPSCs derived from T cells (T-iPSCs).

[0338] γδT-iγδT: The terms "γδT-iγδT," "γδT-iPSC-derived γδT," and similar terms refer to γδT cells differentiated from iPSCs derived from γδT cells (γδT-iPSCs).

[0339] The terms "T-iγδT," "T-derived iγδT," "γδT-iγδT," and "γδT-iPSC-derived γδT" may be used interchangeably herein. When iPSCs are used for differentiation into T-iγδT during the reprogramming of T cells into iPSCs, γδT cells are typically selected for reprogramming into iPSCs.

[0340] As used herein, “intermediate cells differentiated from iPSCs” and similar terms refer to cells differentiated from iPSCs that have the ability to differentiate into γδT cells. Intermediate cells differentiated from iPSCs include, but are not limited to, γδT-iHSCs, γδT-iCLPs, and / or immature T-iγδT cells.

[0341] iPSC-derived hematopoietic stem cells (iHSCs): As used herein, the terms "iPSC-derived hematopoietic stem cells" or "iHSC" refer to hematopoietic stem cells derived from iPSCs.

[0342] γδT-iPSC hematopoietic stem cells (γδT-iHSC): As used herein, the terms "γδT-iPSC-derived hematopoietic stem cells" or "γδT-iHSC" refer to hematopoietic stem cells derived from γδT-iPSCs.

[0343] iPSC-derived common lymphoid progenitor cells (iCLP cells): As used herein, the terms “iPSC-derived common lymphoid progenitor cells,” “iCLP cells,” or “iCLP” refer to common lymphoid progenitor cells differentiated from iHSCs, iHSCs are derived from iPSCs.

[0344] γδT-iPSC common lymphoid progenitor cells (γδT-iCLP): As used herein, the terms “γδT-iPSC-derived common lymphoid progenitor cells” or “γδT-iCLP cells” refer to common lymphoid progenitor cells differentiated from T-iHSCs, which are derived from γδT-iPSCs.

[0345] iPSC-derived immature T cells (immature iT cells): As used herein, the term “iPSC-derived immature T cells” or “immature iT cells” refers to immature T cells differentiated from iCLP cells, iCLP cells differentiate from iHSCs, and iHSCs are produced from iPSCs.

[0346] γδ-TiPSC-derived immature T cells (immature T-iγδT cells): As used herein, the term "γδT cell-immature T cell" or "immature T-iγδT cell" refers to immature γδT cells differentiated from T-iCLP cells, which are differentiated from T-iHSCs, and which are produced from γδT-iPSCs.

[0347] Genome disruption: As used herein, the term “genome disruption” refers to any genetic alteration of a gene sequence (e.g., substitution, deletion, and / or insertion) or epigenetic modification that does not involve alteration of the gene sequence (e.g., DNA methylation, histone modification). Genome disruption can result in alterations to gene transcription, mRNA translation, and / or protein function. Genome disruption may include alterations to coding sequences, non-coding sequences (e.g., promoters, enhancers, insulators, operons, or silencers), or combinations thereof.

[0348] Splice Variant: As used herein, the term “splice variant” refers to the mature mRNA and its coding protein produced by alternative splicing of a gene (e.g., a BIM gene) in which each exon of the mRNA precursor is rejoined in at least two different forms during the RNA splicing process. EL (Including coding exons E1, E2A, E2B, E2C, E4, and E5), BIM L It is transcribed as three splice variants: (including coding exons E1, E2A, E2C, E4, and E5) and BIMs (including coding E1, E2A, E4, and E5).

[0349] Identity: As used herein, the term “identity” in the context of sequence comparison refers to the number of exact matches between two different sequences in a sequence alignment. Examples of sequence alignment methods and software include the Basic Local Alignment Search Tool (BLAST, which includes, for example, BLASTP for protein sequences and BLASTN for nucleic acid sequences), ClustalOmega, MUSCLE, and MAFFT.

[0350] Bcl-2 Interacting Mediator of cell death (BIM): As used herein, the terms “Bcl-2 Interacting Mediator of cell death,” “BIM,” “Human BIM gene,” “Genes ID: 10018,” or “BCL2L11” refer to a member of the BH3 (Bcl-2 homology 3)-only protein family that can directly activate the pro-apoptotic effector proteins BAX and BAK. BIM can also indirectly activate BAX and BAK by binding to members of the anti-apoptotic BCL-2 family (BCL-2, BCL-xL, MCL-1, and A1). EL (NM_138621.5; NP_619527.1, including coding exons E1, E2A, E2B, E2C, E4 and E5), BIM L It is transcribed as three splice variants: (NM_006538.5; NP_006529.1, including coding exons E1, E2A, E2C, E4 and E5) and BIMs (NM_001204106.1; NP_001191035.1, including coding E1, E2A, E4 and E5).

[0351] Cytokine-inducible SH2-containing protein (CISH): As used herein, the terms “CISH,” “cytokine-inducible SH2-containing protein,” “CIS1,” “gene ID: 1154,” “NM_013324.7,” or “NP_037456.5” refer to members of the cytokine signaling suppressor (SOCS) protein family. Members of the SOCS family are negative regulators of cytokine signaling that inhibit the JAK / STAT pathway. Each SOCS family member has a central SH2 domain and a conserved carboxy-terminal motif called the SOCS box. These proteins are important regulators of cytokine signaling, proliferation, differentiation, and immune responses.

[0352] Cytokine signaling suppressor 1 (SOCS1): As used herein, the terms “cytokine signaling suppressor 1,” “SOCS1,” “gene ID: 8651,” “NM_003745.2,” “NP_003736.1,” “Janus kinase-binding protein,” “JAB,” “Stat-inducible Stat inhibitor 1,” “SSI-1,” “Tec-interacting protein 3,” or “TIP3” refer to cytokine-regulating SOCS family members that directly inhibit JAK family members through interactions within their kinase activation loops. In addition to inhibiting JAK / STAT signaling, SOCS1 can also negatively regulate Toll-like receptors that contribute to innate immunity.

[0353] FAS cell surface cell death receptor: As used herein, the terms “FAS cell surface cell death receptor,” “FAS,” “gene ID:355,” “NM_000043.6,” and “NP_000034.1” refer to tumor necrosis factor (TNF) superfamily cell death receptors that induce caspase-dependent apoptosis after binding to their extracellular ligand (FAS ligand (FASL)).

[0354] β2-microglobulin: As used herein, the terms “β2-microglobulin,” “beta-2-microglobulin,” “B2M,” “gene ID: 567,” “NM_004048,” and “NP_004039” refer to components of human leukocyte antigen (HLA) class I molecules found on the surface of virtually all nucleated cells. Furthermore, β2M binds to the HLA class I heavy chain non-covalently, enabling proper folding and stabilization of HLA class I.

[0355] Class II transactivator (CIITA): As used herein, the terms "Class II transactivator," "CIITA," "gene ID: 4261," "NM_001286402.1," and "NP_001273331.1" refer to a master regulator that controls HLA class II expression by recruiting and promoting the assembly of DNA-binding proteins, including regulatory factor X (RFX) complex, cAMP response element-binding protein (CREB), and nuclear factor (NF-Y).

[0356] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR): As used herein, the terms “CRISPR” refer to a gene editing system that uses a guide sequence and a nuclease to modify the cellular genome at a specific location. The nuclease cuts the cellular genome at a specific location targeted by the guide sequence, allowing for the removal of the target sequence, the addition of a sequence, or a combination thereof.

[0357] Programmable Addition Via Site-Specific Target Elements (PASTE): As used herein, the terms “Programmable Addition Via Site-Specific Target Elements” or “PASTE” refer to a gene editing system that uses a guide sequence and CRISPR-Cas9 nickase and serine integrase fused to a reverse transcriptase to modify the cell’s genome at a specific location. The nickase cleaves one strand of the double-stranded DNA in the cell’s genome at a specific location targeted by the guide sequence, allowing for the removal of the target sequence, the addition of the sequence using serine integrase, or a combination thereof.

[0358] Prime editing: As used herein, the term “prime editing” refers to a gene editing system that uses a guide sequence and an imperfectly catalytic Cas nickase (e.g., Cas9 nickase or Cas12 nickase) fused to a reverse transcriptase to modify the cell’s genome at a specific location. The nickase cleaves one strand of the double-stranded DNA in the cell’s genome at a specific target location of the guide sequence, allowing for the removal of the target sequence, the addition of the sequence using reverse transcriptase, or a combination thereof.

[0359] Base editing: As used herein, the term “base editing” refers to a gene editing system that uses a guide sequence and a base editor, which is a Cas nickase (e.g., Cas9 nickase or Cas12 nickase) and a nucleoside deaminase, such as cytosine deaminase or adenine deaminase, to modify the cellular genome at a specific location. The nickase cleaves one strand of double-stranded DNA in the cellular genome at a specific target location of the guide sequence, allowing the nucleoside deaminase to remove an amino group from a specific type of nucleoside (e.g., cytosine or adenine) at that specific location.

[0360] Transcription Activator-like Effector Nuclease (TALEN): As used herein, the terms “Transcription Activator-like Effector Nuclease” or “TALEN” refer to a gene editing system that uses an endonuclease (e.g., Fokl) fused to a transcription activator-like effector (TALE) that has been engineered to bind to a target DNA sequence in order to facilitate endonuclease-mediated DNA cleavage at a specific site designated by the TALE.

[0361] Zinc finger nuclease (ZFN): As used herein, the terms “zinc finger nuclease” or “ZFN” refer to a gene editing system that uses an endonuclease (e.g., Fokl) fused to multiple zinc fingers. Each zinc finger targets a specific nucleotide sequence in which the endonuclease forms a double-strand DNA break.

[0362] Endonucleases: As used herein, the term “endonuclease” refers to an enzyme that cleaves phosphodiester bonds in polynucleotide molecules. Endonucleases that target specific nucleotide sequences are restriction endonucleases. As used herein, the terms “homing endonucleases” and “meganucleases” refer to endonucleases that target specific asymmetric sequences of about 12– and 45 base pairs in length, typically about 14–25 base pairs in length.

[0363] Transfection: As used herein, the term “transfection” refers to a variety of methods for introducing foreign nucleic acid molecules (e.g., DNA) into host cells, including electroporation, physical transfection (e.g., microinjection, particle bombardment, or phototransfection), lipid-mediated transfection, diethylaminoethyl (DEAE)-dextran transfection, calcium phosphate precipitation, cationic polymer transfection, or viral transfection.

[0364] Interferon-gamma receptor: As used herein, the terms “interferon-gamma receptor,” “IFNGR,” and “IFN-γ receptor” refer to cytokine receptors that include heterodimers. The heterodimer of IFNGR includes interferon-gamma receptor 1 (IFNGR1) and interferon-gamma receptor 2 (IFNGR2). Interferon-gamma cytokines bind to IFNGR.

[0365] CD132: As used herein, “CD132,” “the common γ chain (γc) of interleukin-2 receptor,” and “IL2RG” refer to cytokine receptor subunits common to at least the cytokine receptor complexes of IL-2, IL-15, IL-4, IL-7, IL-9, and IL-21. CD132 includes an intracellular domain, a transmembrane domain, and a proximal membrane domain.

[0366] Interleukin-2 receptor beta subunit: As used herein, the terms “interleukin-2 receptor beta subunit,” “interleukin-2 receptor subunit beta,” “CD122,” and “IL2RB” refer to the cytokine receptor subunit of the IL-2 cytokine receptor complex.

[0367] As used herein, the terms “IFNγR-IL2 / IL15R signaling factor,” “IFNγR signaling factor,” “IFNγ signaling factor,” “IFN-γ signaling factor,” “IFNg signaling factor,” and “IFNγSC” refer to signaling factors comprising a chimeric heterodimer. The IFNγ signaling factor comprises a first IFNγ fusion protein subunit and a second IFNγ fusion protein subunit, the first IFNγ fusion protein subunit comprising the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the intracellular domain of interleukin-2 receptor subunit gamma (IL2RG), and the second IFNγ fusion protein subunit comprising the ECD of interferon-gamma receptor 2 (IFNGR2) and the intracellular domain of interleukin-2 receptor subunit beta (IL2RB).

[0368] As used herein, “unmodified control” and “unmodified cell” refer to the control cell being compared. In some embodiments, the unmodified control is identical to the modified cell herein, except that it expresses each splice variant of BIM at the same endogenous levels as wild-type cells (e.g., γδT cells). In some embodiments, the unmodified control is identical to the modified cell herein, except that it expresses the same endogenous levels as wild-type cells (e.g., γδT cells) of the specific protein or combination of proteins being compared. The unmodified control may also have further modifications (other than the protein(s) being compared) as those present in the modified cell being compared.

[0369] As used herein, “suppress or eliminate” means any reduction in the level of a functional protein expressed from a gene compared to an unmodified control.

[0370] As used herein, “loss-of-function nucleic acid mutation” means any nucleic acid mutation that results in any reduction in the level of a functional protein expressed from a gene compared to an unmodified control.

[0371] B2M-HLA-E: As used herein, the terms “B2M-HLA-E,” “B2M-HLAE,” and “B2M-HLA-E fusion protein” refer to a fusion protein comprising at least a portion of B2M and a portion of HLA-E, wherein the portion of B2M and the portion of HLA-E are linked directly or indirectly via a linker. In some embodiments, B2M-HLA-E can bind to inhibitory receptors on the surface of NK cells. In some embodiments, B2M-HLA-E comprises the amino acid sequence of SEQ ID NO: 85.

[0372] TRAC: As used herein, the term "TRAC" refers to the T cell receptor alpha constant region gene. TRAC is part of the T cell receptor (TCR) complex and is involved in antigen recognition in association with the major histocompatibility complex (MHC) on antigen-presenting cells.

[0373] The terms "CAR" and "chimeric antigen receptor" refer to chimeric receptors that have been engineered to confer antigen specificity to them while retaining or enhancing their ability to recognize target cells, trigger signaling pathways, and kill target cells. A chimeric antigen receptor may include, for example, (i) an extracellular binding domain containing an antigen-specific element (e.g., scFv that binds to a specific antigen) and optionally a hinge region, (ii) a transmembrane domain, and (iii) an intracellular signaling domain, such as one or more costimulatory domains and one or more activating domains. Each domain may be heterogeneous (i.e., composed of sequences derived from (or corresponding to) different protein chains).

[0374] As used herein, the terms “administer,” “administer,” “treat,” and “therapeutic” mean, when applied to animals, humans, subjects, cells, tissues, organs, or biological fluids, the contact of an exogenous medicamental, therapeutic, or diagnostic agent or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of cells includes the contact of a reagent with a cell, and, if a fluid is in contact with a cell, the contact of a reagent with such fluid. The terms “administer” and “therapeutic” also mean in vitro and ex vivo treatment of cells, for example, with a reagent, diagnostic, conjugate compound, or another cell. In one embodiment, treating any disease or disorder means alleviating the disease or disorder (i.e., delaying, preventing, or reducing the onset of at least one of the disease or its clinical symptoms). In another embodiment, “treat,” “treat,” or “therapeutic” means alleviating or improving at least one physical parameter, including those that may not be identifiable by the patient. In yet another embodiment, “to treat,” “to treat,” or “treatment” means to modulate a disease or disorder either or both physically (e.g., stabilization of identifiable symptoms) or physiologically (e.g., stabilization of physical parameters). In yet another embodiment, “to treat,” “to treat,” or “treatment” means to prevent or delay the onset, development, or progression of a disease or disorder.

[0375] In connection with this disclosure, the term “Subject” means mammals, for example, primates, preferably higher primates, for example, humans (e.g., patients having or at risk of having any of the diseases described herein).

[0376] As used herein, "specifically binds" an antibody to a target protein means that such an antibody exhibits selective binding to its target compared to binding to other proteins, but this specificity does not necessarily require absolute binding specificity. "Specifically binding" or "selectively binding" of an antibody is used in the context of describing an interaction between an antigen (e.g., a protein) and an antibody or antigen-binding antibody fragment, referring to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biologics, e.g., a biological sample, blood, serum, plasma, or tissue sample. Therefore, under specific, designated immunoassay conditions, the antibody or its antigen-binding fragment binds specifically to a particular antigen at least twice as much as the background level, and does not specifically bind to other antigens present in the sample in significant amounts. In one embodiment, under designated immunoassay conditions, the antibody or its antigen-binding fragment binds specifically to a particular antigen at least ten times as much as the background level of binding, and does not specifically bind to other antigens present in the sample in significant amounts.

[0377] In this specification, the terms “cancer” or “tumor” have the broadest meaning as understood in the art and refer to a physiological condition in mammals typically characterized by uncontrolled cell proliferation. In connection with this disclosure, cancer is not limited to any particular type or location.

[0378] The term “nucleic acid” is used herein in the same sense as the term “polynucleotide” and refers to single-stranded or double-stranded deoxyribonucleotides or ribonucleotides and polymers thereof. This term encompasses nucleic acids containing known nucleotide analogs or modified skeletal residues or bonds, which include synthetic nucleic acids, naturally occurring nucleic acids, and naturally occurring nucleic acids, which have similar binding properties to the reference nucleic acid and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs, but not limited to, include phosphorothioates, phosphoramides, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0379] As used herein, the term “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, isotonic agent, and absorption retarder. The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or dermal administration (e.g., by injection or infusion).

[0380] As used herein, the term “therapeutic effective dose” means the amount of a therapeutic agent sufficient to produce such treatment for a disease, disorder, or symptom when administered to a subject to treat the disease, disorder, or symptom of the disease or disorder. “Therapeutic effective dose” may vary depending on the therapeutic agent, the disease, disorder, and / or the symptoms of the disease or disorder, the severity of the symptoms of the disease, disorder, and / or the age of the subject being treated, and / or the weight of the subject being treated. The appropriate dose in any particular case may be obvious to those skilled in the art, or it may be determined by common practice. In the case of combination therapy, “therapeutic effective dose” means the total amount of the combination agent for the effective treatment of the disease, disorder, or condition.

[0381] II. Modified Cells This specification discloses modified γδT cells, such as iPSC-derived γδT cells, and their precursor cells (e.g., iPSCs (e.g., γδT-iPSCs)), or intermediate cells differentiated from iPSCs (e.g., iHSCs, iCLPs, and immature iγδT cells).

[0382] Modified cells with one or more genome disruptions In one embodiment, such modified cells include one or more genome disruptions selected from the following: 1) genome disruption in the SOCS1 gene, 2) genome disruption in the CISH gene, 3) genome disruption in the BIM gene, 4) genome disruption in the B2M gene, 5) genome disruption in the CIITA gene, 6) genome disruption in the FAS gene, and 7) genome disruption in the TRAC gene.

[0383] In another embodiment, such modified cells (e.g., iPSCs or γδT cells) include at least two genomic disruptions: 1) within the cytokine signaling suppressor 1 (SOCS1) gene, and 2) within the cytokine-inducible sh2-containing protein (CISH) gene. In some embodiments, the modified cells include at least three genomic disruptions: 1) within the SOCS1 gene, 2) within the CISH gene, 3) within the BIM gene, and 3) within the Bcl-2 interacting cell death mediator (BIM) gene. In some embodiments, the modified cells include at least four genomic disruptions: 1) within the SOCS1 gene, 2) within the CISH gene, 3) within the BIM gene, and, for example, 4) within the FAS cell surface cell death receptor (FAS) gene. In some embodiments, the modified cells include at least five genomic disruptions: 1) within the SOCS1 gene, 2) within the CISH gene, 3) within the BIM gene, and, for example, 4) within the β2-microglobulin (B2M) gene, and 5) within the class II transactivator (CIITA) gene. In some embodiments, the modified cells include at least six genomic disruptions within 1) the SOCS1 gene, 2) the CISH gene, 3) the BIM gene, and, for example, 4) the B2M gene, 5) the CIITA gene, and 6) the FAS gene. In some embodiments, the modified cells include at least seven genomic disruptions within 1) the SOCS1 gene, 2) the CISH gene, 3) the BIM gene, and, for example, 4) the B2M gene, 5) the CIITA gene, 6) the FAS gene, and 7) the FAS gene.

[0384] In another embodiment, the modified cell (e.g., iPSC or γδT cell) includes 1) a genomic disruption within the SOCS1 gene (e.g., within exon 2 of the SOCS1 gene), and 2) at least two genomic disruptions within the CISH gene (e.g., within exon 3 of the CISH gene). In some embodiments, the modified cell (e.g., iPSC or γδT cell) includes at least three genomic disruptions: 1) a genomic disruption within the SOCS1 gene (e.g., within exon 2 of the SOCS1 gene), 2) a genomic disruption within the CISH gene (e.g., within exon 3 of the CISH gene), and 3) a genomic disruption within the BIM gene (e.g., within exon 2C of the BIM gene). In some embodiments, the modified cells (e.g., iPSCs or γδT cells) include at least four genomic disruptions: 1) genomic disruption within the SOCS1 gene (e.g., within exon 2 of the SOCS1 gene), 2) genomic disruption within the CISH gene (e.g., within exon 3 of the CISH gene), 3) genomic disruption within the BIM gene (e.g., within exon 2C of the BIM gene), and 4) genomic disruption within the FAS gene. In some embodiments, the modified cells (e.g., iPSCs or γδT cells) include at least five genomic disruptions: 1) genomic disruption within the SOCS1 gene (e.g., within exon 2 of the SOCS1 gene), 2) genomic disruption within the CISH gene (e.g., within exon 3 of the CISH gene), 3) genomic disruption within the BIM gene (e.g., within exon 2C of the BIM gene), 4) genomic disruption within the B2M gene, and 5) genomic disruption within the CIITA gene. In some embodiments, the modified cells (e.g., iPSCs or γδT cells) include at least six genomic disruptions: 1) genomic disruption within the SOCS1 gene (e.g., within exon 2 of the SOCS1 gene), 2) genomic disruption within the CISH gene (e.g., within exon 3 of the CISH gene), 3) genomic disruption within the BIM gene (e.g., within exon 2C of the BIM gene), 4) genomic disruption within the FAS gene, 5) genomic disruption within the B2M gene, and 6) genomic disruption within the CIITA gene.In some embodiments, the modified cells (e.g., iPSCs or γδT cells) further include at least seven genomic disruptions: 1) genomic disruption within the SOCS1 gene (e.g., within exon 2 of the SOCS1 gene), 2) genomic disruption within the CISH gene (e.g., within exon 3 of the CISH gene), 3) genomic disruption within the BIM gene (e.g., within exon 2C of the BIM gene), 4) genomic disruption within the FAS gene, 5) genomic disruption within the B2M gene, 6) genomic disruption within the CIITA gene, and 7) genomic disruption within the TRAC gene.

[0385] Modified cells involving the disruption of one or more genomes and the incorporation of one or more polynucleotides. In one embodiment, modified cells (e.g., iPSCs or γδT cells) undergo the following: 1) genomic disruption within the SOCS1 gene (e.g., within exon 2 of the SOCS1 gene), 2) genomic disruption within the CISH gene (e.g., within exon 3 of the CISH gene), 3) genomic disruption within the BIM gene (e.g., within exon 2C of the BIM gene), 4) genomic disruption within the FAS gene, 5) genomic disruption within the B2M gene, 6) genomic disruption within the CIITA gene, 7) genomic disruption within the TRAC gene, and 8) encoding B2M-HLA-E. 9) comprising one or more exogenous polynucleotides selected from an exogenous polynucleotide (e.g., inserted at the B2M locus) and one or more exogenous polynucleotides encoding a signal conversion factor (e.g., IFNγ signal conversion factor) comprising a first fusion protein subunit and a second fusion protein subunit (e.g., the first fusion protein subunit of the signal conversion factor is inserted at the SOCS1 locus and the second fusion protein subunit of the signal conversion factor is inserted at the ROSA26 locus, or vice versa).

[0386] In another embodiment, in addition to the combination of genome disruption described above, the modified cell (e.g., iPSC or γδT cell) further comprises at least one or two of the following: 1) an exogenous polynucleotide encoding B2M-HLA-E (e.g., one inserted into the B2M locus), and 2) one or more exogenous polynucleotides encoding an IFNγ signaling factor.

[0387] In another embodiment, in addition to the combination of genome disruption described above, the modified cells (e.g., iPSCs or γδT cells) include 1) an exogenous polynucleotide encoding B2M-HLA-E (e.g., one inserted at the B2M locus), and 2) one or more exogenous polynucleotides encoding an IFNγ signaling factor.

[0388] In one embodiment, the modified cells (e.g., iPSCs or γδT cells) further include all of the following modifications selected from: 1) genomic disruption within the SOCS1 gene (e.g., within exon 2 of the SOCS1 gene), 2) genomic disruption within the CISH gene (e.g., within exon 3 of the CISH gene), 3) genomic disruption within the BIM gene (e.g., within exon 2C of the BIM gene), 4) genomic disruption within the FAS gene, 5) genomic disruption within the B2M gene, 6) genomic disruption within the CIITA gene, 7) genomic disruption within the TRAC gene 8) an exogenous polynucleotide encoding B2M-HLA-E (e.g., inserted at the B2M locus), and 9) one or more exogenous polynucleotides encoding a signal-converting factor (e.g., IFNγ signal-converting factor) comprising a first fusion protein subunit and a second fusion protein subunit (e.g., the first fusion protein subunit of the signal-converting factor is inserted at the SOCS1 locus and the second fusion protein subunit of the signal-converting factor is inserted at the ROSA26 locus, or vice versa).

[0389] In some embodiments, the modified cells are γδT cells. These γδT cells may be Vd2 (or Vδ2)γδT cells or Vd1 (or Vδ1)γδT cells. In some embodiments, the γδT cells can be modified before reprogramming into iPSCs. In some embodiments, the modified cells are iPSC-derived γδT cells (iγδT). In some embodiments, the γδT cells may be primary cells. In some embodiments, the modified γδT cells are differentiated from modified iPSCs.

[0390] In some embodiments, the modified cells are iPSCs. In some embodiments, somatic cells (e.g., T cells or PBMCs) can be modified before being reprogrammed into iPSCs. iPSCs are PSCs created from somatic cells using induced pluripotency reprogramming factors. In some embodiments, somatic cells are T cells, including γδ (gamma / delta) T cells, peripheral blood mononuclear cells (PBMCs), fibroblasts, or umbilical cord blood cells.

[0391] In some embodiments, the modified cells are iPSCs derived from primary T cells (T-iγδT).

[0392] In some embodiments, the modified cells are γδT-iPSCs. γδT-iPSCs are iPSCs created from γδ (gamma / delta) T cells (γδT cells) using induced pluripotency reprogramming factor. In some embodiments, the γδT cells used to create iPSCs using the reprogramming factor are Vd2γδT cells.

[0393] In some embodiments, the modified cells are Vd2γδT cells. In some embodiments, the modified cells are primary γδT cells. In some embodiments, the modified cells are iPSC-derived γδT (iγδT) cells. In some embodiments, the modified cells are T-iPSC (T-iγδT)-derived iγδT cells. In some embodiments, the modified cells are γδT-iPSC-derived γδT cells (γδT-iγδT).

[0394] In some embodiments, the modified cells are iHSCs, which refer to hematopoietic stem cells derived from iPSCs. In some embodiments, the modified cells are γδT-iHSCs, which refer to hematopoietic stem cells derived from γδT-iPSCs.

[0395] In some embodiments, the modified cells are iCLP cells, which are common lymphoid progenitor cells differentiated from iHSCs. In some embodiments, the modified cells are γδT-iCLP cells, which are common lymphoid progenitor cells differentiated from γδT-iHSCs.

[0396] In some embodiments, the modified cells are immature iT cells, which are immature T cells differentiated from iHSCs and / or iCLP cells. In some embodiments, the modified cells are immature T-iγδT cells, which are immature γδT cells differentiated from iHSCs and / or γδT-iCLP cells.

[0397] In some embodiments, one or more of the SOCS1, CISH, BIM, FAS, B2M, CIITA, and TRAC genes are genomically disrupted in γδT-iPSCs, γδT-iHSCs, γδT-iCLPs, or immature T-iγδT cells before differentiation into T-iγδT cells.

[0398] In some embodiments, the SOCS1 and CISH genes are genomically disrupted in γδT-iPSCs, γδT-iHSCs, γδT-iCLPs, or immature T-iγδT cells before differentiation into T-iγδT cells. In some embodiments, the SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iPSCs, γδT-iHSCs, γδT-iCLPs, or immature T-iγδT cells before differentiation into T-iγδT cells. In some embodiments, the genomic disruption of the SOCS1 and CISH genes occurs at different cell differentiation stages; for example, the SOCS1 gene is genomically disrupted in T-iCLP cells, and the CISH gene is genomically disrupted in immature T-iγδT cells differentiated from T-iCLPs containing the genomically disrupted SOCS1 gene. A cell differentiation stage is one of the following cell types: self-renewing and pluripotent stem cells (e.g., induced pluripotent stem cells), adult stem cells differentiated from self-renewing and pluripotent stem cells (e.g., hematopoietic stem cells), progenitor cells differentiated from adult stem cells (e.g., common lymphoid progenitor cells), specific cell types differentiated from progenitor cells (e.g., immature T cells), and mature specific cell types differentiated from specific cell types (e.g., γδT cells). A cell differentiation stage is one of the following: γδT-iPSC, T-iHSC, T-iCLP, immature T-iγδT cells, or T-iγδT cells.

[0399] In some embodiments, genomic disruption of the SOCS1, CISH, and BIM genes occurs at different cell differentiation stages. For example, the SOCS1 gene is disrupted in T-iHSCs, the CISH gene is disrupted in T-iCLPs differentiated from T-iHSCs containing the disrupted SOCS1 gene, and the BIM gene is disrupted in immature T-iγδT cells differentiated from T-iCLPs containing the disrupted SOCS1 and CISH genes. In some embodiments, genomic disruption of two of the genes selected from SOCS1, CISH, and BIM occurs at a single cell differentiation stage, and a third genomic disruption of the remaining undisrupted gene occurs at a second cell differentiation stage. For example, the SOCS1 and CISH genes are disrupted in T-iHSCs, and the BIM gene is disrupted in T-iCLPs differentiated from T-iHSCs containing the disrupted SOCS1 and CISH genes. In some embodiments, genomic disruption of two of the genes selected from SOCS1, CISH, and BIM occurs at a single cell differentiation stage. In some embodiments, genomic disruption in each of three genes selected from SOCS1, CISH, and BIM occurs at a single cell differentiation stage.

[0400] In some embodiments, the cells include genomic disruption of the SOCS1, CISH, BIM, and FAS genes. In some embodiments, the genomic disruption of the SOCS1, CISH, BIM, and FAS genes occurs at different cell differentiation stages. In some embodiments, genomic disruption of three genes selected from FAS, SOCS1, CISH, and BIM occurs at a single cell differentiation stage, and a fourth genomic disruption of the remaining undisrupted gene occurs at a second cell differentiation stage. In some embodiments, genomic disruption of two genes selected from FAS, SOCS1, CISH, and BIM occurs at a single cell differentiation stage. In some embodiments, genomic disruption of one gene selected from FAS, SOCS1, CISH, and BIM occurs at a single cell differentiation stage. In some embodiments, genomic disruption in each of the four genes selected from FAS, SOCS1, CISH, and BIM occurs at a single cell differentiation stage.

[0401] In some embodiments, the cells contain genomic disruptions of the SOCS1, CISH, BIM, B2M, and CIITA genes. In some embodiments, the genomic disruptions of the SOCS1, CISH, BIM, B2M, and CIITA genes occur at different cell differentiation stages. In some embodiments, genomic disruptions of four genes selected from SOCS1, CISH, BIM, B2M, and CIITA occur at a single cell differentiation stage. In some embodiments, genomic disruptions of three genes selected from SOCS1, CISH, BIM, B2M, and CIITA occur at a single cell differentiation stage. In some embodiments, genomic disruptions of two genes selected from SOCS1, CISH, BIM, B2M, and CIITA occur at a single cell differentiation stage. In some embodiments, genomic disruptions of one gene selected from SOCS1, CISH, BIM, B2M, and CIITA occur at a single cell differentiation stage. In some embodiments, genomic disruptions in each of the genes selected from SOCS1, CISH, BIM, B2M, and CIITA occur at a single cell differentiation stage.

[0402] In some embodiments, the cells include genomic disruption of the SOCS1, CISH, BIM, B2M, CIITA, and FAS genes. In some embodiments, the genomic disruption of the SOCS1, CISH, BIM, B2M, CIITA, and FAS genes occurs at different cell differentiation stages. In some embodiments, genomic disruption of five genes selected from SOCS1, CISH, BIM, B2M, CIITA, and FAS occurs at a single cell differentiation stage. In some embodiments, genomic disruption of four genes selected from SOCS1, CISH, BIM, B2M, CIITA, and FAS occurs at a single cell differentiation stage. In some embodiments, genomic disruption of three genes selected from SOCS1, CISH, BIM, B2M, CIITA, and FAS occurs at a single cell differentiation stage. In some embodiments, genomic disruption of two genes selected from SOCS1, CISH, BIM, B2M, CIITA, and FAS occurs at a single cell differentiation stage. In some embodiments, genomic disruption of one of the genes selected from SOCS1, CISH, BIM, B2M, CIITA, and FAS occurs at a single cell differentiation stage. In some embodiments, genomic disruption in each of the genes selected from SOCS1, CISH, BIM, B2M, CIITA, and FAS occurs at a single cell differentiation stage.

[0403] In some embodiments, one or more of the SOCS1, CISH, BIM, FAS, B2M, and CIITA genes are genomically disrupted in iPSC cells. In some embodiments, the SOCS1 and CISH genes are genomically disrupted in iPSC cells. In some embodiments, the SOCS1, CISH, and BIM genes are genomically disrupted in iPSC cells. In some embodiments, the FAS, SOCS1, CISH, and BIM genes are genomically disrupted in iPSC cells. In some embodiments, the B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in iPSC cells. In some embodiments, the FAS, B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in iPSC cells.

[0404] In some embodiments, one or more of the SOCS1, CISH, BIM, FAS, B2M, and CIITA genes are genomically disrupted in γδT-iPSC cells. In some embodiments, the SOCS1 and CISH genes are genomically disrupted in γδT-iPSC cells. In some embodiments, the SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iPSC cells. In some embodiments, the FAS, SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iPSC cells. In some embodiments, the B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iPSC cells. In some embodiments, the FAS, B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iPSC cells.

[0405] In some embodiments, one or more of the SOCS1, CISH, BIM, FAS, B2M, and CIITA genes are genomically disrupted in iPSC-derived γδT cells (iγδT cells). In some embodiments, the SOCS1 and CISH genes are genomically disrupted in iPSC-derived γδT cells (iγδT cells). In some embodiments, the SOCS1, CISH, and BIM genes are genomically disrupted in iγδT cells. In some embodiments, the FAS, SOCS1, CISH, and BIM genes are genomically disrupted in iγδT cells. In some embodiments, the B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in iγδT cells. In some embodiments, the FAS, B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in iγδT cells.

[0406] In some embodiments, one or more of the SOCS1, CISH, BIM, FAS, B2M, and CIITA genes are genomically disrupted in T-iPSC-derived γδT cells (T-iγδT cells). In some embodiments, the SOCS1 and CISH genes are genomically disrupted in T-iPSC-derived γδT cells (T-iγδT cells). In some embodiments, the SOCS1, CISH, and BIM genes are genomically disrupted in T-iPSC-derived γδT cells (T-iγδT cells). In some embodiments, the FAS, SOCS1, CISH, and BIM genes are genomically disrupted in T-iPSC-derived γδT cells (T-iγδT cells). In some embodiments, the B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in T-iPSC-derived γδT cells (T-iγδT cells). In some embodiments, the FAS, B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in T-iPSC-derived γδT cells (T-iγδT cells).

[0407] In some embodiments, one or more of the SOCS1, CISH, BIM, FAS, B2M, and CIITA genes are genomically disrupted in γδT-iPSC-derived γδT cells (γδT-iγδT cells). In some embodiments, the SOCS1 and CISH genes are genomically disrupted in γδT-iPSC-derived γδT cells (γδT-iγδT cells). In some embodiments, the SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iPSC-derived γδT cells (γδT-iγδT cells). In some embodiments, the FAS, SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iPSC-derived γδT cells (γδT-iγδT cells). In some embodiments, the B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iPSC-derived γδT cells (γδT-iγδT cells). In some embodiments, the FAS, B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iPSC-derived γδT cells (γδT-iγδT cells).

[0408] In some embodiments, one or more of the SOCS1, CISH, BIM, FAS, B2M, and CIITA genes are genomically disrupted in iHSC. In some embodiments, the SOCS1 and CISH genes are genomically disrupted in iHSC. In some embodiments, the SOCS1, CISH, and BIM genes are genomically disrupted in iHSC. In some embodiments, the FAS, SOCS1, CISH, and BIM genes are genomically disrupted in iHSC. In some embodiments, the B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in iHSC. In some embodiments, the FAS, B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in iHSC.

[0409] In some embodiments, one or more of the SOCS1, CISH, BIM, FAS, B2M, and CIITA genes are genomically disrupted in γδT-iHSC. In some embodiments, the SOCS1 and CISH genes are genomically disrupted in γδT-iHSC. In some embodiments, the SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iHSC. In some embodiments, the FAS, SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iHSC. In some embodiments, the B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iHSC. In some embodiments, the FAS, B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iHSC.

[0410] In some embodiments, one or more of the SOCS1, CISH, BIM, FAS, B2M, and CIITA genes are genomically disrupted in iCLP cells. In some embodiments, the SOCS1 and CISH genes are genomically disrupted in iCLP cells. In some embodiments, the SOCS1, CISH, and BIM genes are genomically disrupted in iCLP cells. In some embodiments, the FAS, SOCS1, CISH, and BIM genes are genomically disrupted in iCLP cells. In some embodiments, the B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in iCLP cells. In some embodiments, the FAS, B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in iCLP cells.

[0411] In some embodiments, one or more of the SOCS1, CISH, BIM, FAS, B2M, and CIITA genes are genomically disrupted in γδT-iCLP cells. In some embodiments, the SOCS1 and CISH genes are genomically disrupted in γδT-iCLP cells. In some embodiments, the SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iCLP cells. In some embodiments, the FAS, SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iCLP cells. In some embodiments, the B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iCLP cells. In some embodiments, the FAS, B2M, CIITA, SOCS1, CISH, and BIM genes are genomically disrupted in γδT-iCLP cells.

[0412] In some embodiments, the SOCS1 and CISH genes are genomically disrupted in γδT-iPSC cells, and the FAS, B2M, CIITA, and / or BIM genes are genomically disrupted in any one of the following: T-iγδT, immature T-iγδT cells, γδT-iHSC, or γδT-iCLP cells.

[0413] In some embodiments, the SOCS1 and CISH genes are genomically disrupted in immature T-iγδT cells, and the FAS, B2M, CIITA, and / or BIM genes are genomically disrupted in any one of the following cells: γδT-iPSC, T-iγδT, γδT-iHSC, or γδT-iCLP cells.

[0414] In some embodiments, the SOCS1 and CISH genes are genomically disrupted in T-iγδT cells, and the FAS, B2M, CIITA, and / or BIM genes are genomically disrupted in any one of the following: γδT-iPSC, immature T-iγδT cells, γδT-iHSC, or γδT-iCLP cells.

[0415] In some embodiments, the SOCS1 and CISH genes are genomically disrupted in γδT-iHSC cells, and the FAS, B2M, CIITA, and / or BIM genes are genomically disrupted in any one of the following: γδT-iPSC, immature T-iγδT cells, T-iγδT, or γδT-iCLP cells.

[0416] In some embodiments, the SOCS1 and CISH genes are genomically disrupted in γδT-iCLP cells, and the FAS, B2M, CIITA, and / or BIM genes are genomically disrupted in any one of the following: γδT-iPSC, immature T-iγδT cells, T-iγδT, or γδT-iHSC cells.

[0417] In some embodiments, an exogenous polynucleotide encoding B2M-HLA-E is incorporated into the B2M locus of an iPSC (e.g., γδT-iPSC), and / or one or more exogenous polynucleotides encoding signal conversion factors (e.g., IFNγ signal conversion factor) are incorporated into the iPSC (e.g., γδT-iPSC).

[0418] In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells.

[0419] In some embodiments, the cells are in vivo, in vitro, ex vivo, or of human interest.

[0420] A population of cells containing one or more modified cells as described herein is also included.

[0421] In some embodiments, the cells are stored for a certain period before use. In some embodiments, this period is several weeks or several months. In some embodiments, this period is several months or several years.

[0422] In some embodiments, the cells are cryopreserved in liquid nitrogen (-196°C).

[0423] A. Gene disruption of cytokine signaling suppressor 1 (SOCS1) This disclosure provides modified γδT cells, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) comprising one, two, three, four, five, six, or seven genomic disruptions and / or the incorporation of one, two, three, or four exogenous polynucleotides, one of which is a genomic disruption of the endogenous cytokine signaling suppressor 1 (SOCS1) gene. In some embodiments, the modified cells are modified T-iγδT cells comprising a genomic disruption of the endogenous SOCS1 gene. In some embodiments, the modified cells are modified (e.g., alone or with genomic disruption in one or more of CISH, BIM, FAS, B2M, CIITA, and TRAC, and / or with the incorporation of one or more of B2M-HLA-E and signaling factors) or non-modified γδT-iPSCs, modified or non-modified T-iHSCs, modified or non-modified T-iCLPs, or γδT cells differentiated from modified or non-modified T-iγδT cells. In some embodiments, the modified cells are derived from primary γδT cells.

[0424] In some embodiments, the modified cells include genomic disruption within the endogenous SOCS1 gene that suppresses or eliminates the expression of the SOCS1 gene compared to an unmodified control. In some embodiments, the genomic disruption within the SOCS1 gene is a loss-of-function nucleic acid mutation (e.g., knockout). In some embodiments, the loss-of-function nucleic acid mutation within the SOCS1 gene is an insertion, deletion, substitution, or any combination thereof. As used herein, “suppresses or eliminates” means any reduction in the level of functional protein expressed from the gene compared to an unmodified control. As used herein, “loss-of-function nucleic acid mutation” means any nucleic acid mutation that causes any reduction in the level of functional protein expressed from the gene compared to an unmodified control. In some embodiments, the genomic disruption within the SOCS1 gene is an endonuclease-mediated insertion or deletion (indel). In some embodiments, loss-of-function nucleic acid mutations within the SOCS1 gene are endonuclease-mediated indels, such as targeted knockouts that can be obtained by a CRISPR-Cas system designed to target SOCS1 (e.g., Cas9, Cas12, or MAD7).

[0425] In some embodiments, the genomic disruption of the endogenous SOCS1 gene is located in exon 2. In some embodiments, the genomic disruption is a loss-of-function nucleic acid mutation in exon 2. In some embodiments, the genomic disruption is located in a target sequence within SOCS1 that includes any one nucleotide sequence from sequence number 17, sequence number 23, or any nucleotide sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with these.

[0426] Assays to determine the relative expression levels of genes or the function of proteins encoded by those genes in modified cells (e.g., T-iγδT cells) can be performed using a variety of methods. For example, the relative expression level of the SOCS1 gene in modified γδT cells (e.g., T-iγδT cells) compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or to original γδT cells (e.g., T-iγδT cells) can be measured using quantitative polymerase chain reaction (qPCR), digital PCR (dPCR), droplet digital PCR (ddPCR), or nucleotide sequencing (e.g., next-generation sequencing). ddPCR can be performed using appropriate nucleic acid quantification kits or instruments, such as the Qubit BR dsDNA assay (Thermo Fisher Scientific), Qubit HS dsDNA assay (Thermo Fisher Scientific), Nanodrop spectrophotometer (Thermo Fischer Scientific), Stunner spectrophotometer (Unchained Labs), and Lunatic spectrophotometer (Unchained Labs). Nucleotide sequencing can be performed using NextSeq (Ilumina), GridION (Oxford Nanopore Technologies), Revio System (Pacific Biosciences), etc.

[0427] B. Genome disruption of cytokine-induced SH2-containing proteins (CISH). This disclosure provides modified γδT cells, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) comprising one, two, three, four, five, six, or seven genomic disruptions and / or the incorporation of one, two, three, or four exogenous polynucleotides, one of which is a genomic disruption of an endogenous cytokine-induced SH2-containing protein (CISH) gene. In some embodiments, the modified cells are modified T-iγδT cells comprising a genomic disruption of an endogenous CISH gene. In some embodiments, the modified cells are modified (e.g., alone or with genomic disruption in one or more of SOCS1, BIM, FAS, B2M, CIITA, and TRAC, and / or with the incorporation of one or more of B2M-HLA-E and signaling factors) or non-modified γδT-iPSCs, modified or non-modified T-iHSCs, modified or non-modified T-iCLPs, or γδT cells differentiated from modified or non-modified T-iγδT cells. In some embodiments, the modified cells are derived from primary γδT cells.

[0428] In some embodiments, the cells include genomic disruption within the endogenous CISH gene that suppresses or eliminates the expression of the CISH gene compared to an unmodified control. In some embodiments, the genomic disruption within the CISH gene is a loss-of-function nucleic acid mutation (e.g., a knockout). In some embodiments, the loss-of-function nucleic acid mutation within the CISH gene is an insertion, deletion, substitution, or any combination thereof. In some embodiments, the genomic disruption within the CISH gene is an endonuclease-mediated insertion or deletion (indel). In some embodiments, the loss-of-function nucleic acid mutation within the CISH gene is an endonuclease-mediated indel, such as a targeted knockout that can be obtained by a CRISPR-Cas system designed to target CISH (e.g., Cas9, Cas12, or MAD7).

[0429] In some embodiments, the genomic disruption of the endogenous CISH gene is located in exon 3, exon 4, or a combination thereof. In some embodiments, the genomic disruption is a loss-of-function nucleic acid mutation located in exon 3, exon 4, or a combination thereof. In some embodiments, the genomic disruption is located in a target sequence within the CISH gene that contains one of the nucleotide sequences of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 24, or a nucleotide sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with these.

[0430] Assays to determine the relative expression levels of genes or the function of proteins encoded by those genes in modified cells (e.g., T-iγδT cells) can be performed using a variety of methods. For example, the relative expression levels of CISH genes in modified γδT cells (e.g., T-iγδT cells) compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or to original γδT cells (e.g., T-iγδT cells) can be measured using qPCR, dPCR, ddPCR, or nucleotide sequencing (e.g., next-generation sequencing). ddPCR can be performed using appropriate nucleic acid quantification kits or instruments, including, for example, the Qubit BR dsDNA assay (Thermo Fisher Scientific), the Qubit HS dsDNA assay (Thermo Fisher Scientific), the Nanodrop spectrophotometer (Thermo Fischer Scientific), the Stunner spectrophotometer (Unchained Labs), and the Lunatic spectrophotometer (Unchained Labs). Nucleotide sequencing can be performed using systems such as NextSeq (Ilumina), GridION (Oxford Nanopore Technologies), and Revio System (Pacific Biosciences).

[0431] Genome disruption of C.Bcl-2-interacting cell death mediator (BIM) This disclosure provides modified γδT cells, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) comprising one, two, three, four, five, six, or seven genomic disruptions and / or the incorporation of one, two, three, or four exogenous polynucleotides, one of which is a genomic disruption of an endogenous Bcl-2-interacting cell death mediator (BIM) gene. In some embodiments, the modified cells are modified T-iγδT cells comprising a genomic disruption of the endogenous BIM gene. In some embodiments, the modified cells are modified (e.g., alone or with genomic disruption in one or more of SOCS1, CISH, FAS, B2M, CIITA, and TRAC, and / or with the incorporation of one or more of B2M-HLA-E and signaling factors) or non-modified γδT-iPSCs, modified or non-modified T-iHSCs, modified or non-modified T-iCLPs, or γδT cells differentiated from modified or non-modified T-iγδT cells. In some embodiments, the modified cells are derived from primary γδT cells.

[0432] In some embodiments, the genomic disruption within the BIM gene is a loss-of-function nucleic acid mutation (e.g., a knockout). In some embodiments, the loss-of-function nucleic acid mutation within the BIM gene is an insertion, deletion, substitution, or any combination thereof. In some embodiments, the genomic disruption within the BIM gene is an endonuclease-mediated insertion / deletion (indel). In some embodiments, the loss-of-function nucleic acid mutation within the BIM gene is an endonuclease-mediated indel, such as a targeted knockout that can be obtained by a CRISPR-Cas system designed to target BIM (e.g., Cas9, Cas12, or MAD7).

[0433] In some embodiments, the genomic disruption of the endogenous BIM gene is located in exon 2A. In some embodiments, the genomic disruption is a loss-of-function nucleic acid mutation within exon 2A. In some embodiments, the genomic disruption of the endogenous BIM gene in modified cells is compared to the unmodified control, resulting in BIM, BIM EL BIM L , and reduces the expression and / or function of all splice variants of BIMs.

[0434] In some embodiments, the genome disruption occurs within a target sequence in the BIM that includes either one of the nucleotide sequences of SEQ ID NO: 18 and SEQ ID NO: 19, or a nucleotide sequence having at least 70% identity with them (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0435] In some embodiments, the genomic disruption of the endogenous BIM gene is located in exon 2C. In some embodiments, the genomic disruption is a loss-of-function nucleic acid mutation within exon 2C.

[0436] In some embodiments, genomic disruption of the endogenous BIM gene in modified cells compared to unmodified controls resulted in BIM being a splice variant of BIM. EL This reduces the expression level and / or function of the endogenous BIM gene. In some embodiments, genomic disruption of the endogenous BIM gene reduces the expression level and / or function of BIM, which is a splice variant of BIM, compared to an unmodified control. L This reduces the expression level and / or function of the endogenous BIM gene. In some embodiments, genomic disruption of the endogenous BIM gene reduces the expression level and / or function of BIM, which is a splice variant of BIM, compared to an unmodified control. EL and BIM L It reduces the expression level and / or function of [the substance].

[0437] In some embodiments, genomic disruption of the endogenous BIM gene in modified cells compared to unmodified controls resulted in BIM being a splice variant of BIM. s Maintains the expression level and / or function of [the substance].

[0438] In some embodiments, genomic disruption of the endogenous BIM gene is equivalent to BIM splice variants compared to an unmodified control. EL and BIM L While reducing the expression level and / or function of BIM, BIM is a splice variant of BIM. s Maintains the expression level and / or function of [the substance].

[0439] In some embodiments, the genome disruption occurs within a target sequence in the BIM that includes any one of the nucleotide sequences of SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, or a nucleotide sequence having at least 70% identity with them (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0440] Assays to determine the relative expression levels of genes or protein function in modified cells can be performed using various methods. Similarly, assays to determine the relative expression levels or function of splice variants can be performed using various methods. For example, BIM in modified cells (e.g., T-iγδT cells) compared with unmodified controls (e.g., unmodified T-iγδT cells). s BIM EL , and BIM L The relative expression levels of can be determined using qPCR, dPCR, ddPCR, or nucleotide sequencing (e.g., next-generation sequencing). Furthermore, BIM in modified γδT cells (e.g., T-iγδT cells) s BIM EL , and BIM LThe relative expression levels of these molecules in modified γδT cells (e.g., T-iγδT cells) can be determined using qPCR, dPCR, ddPCR, or nucleotide sequencing (e.g., next-generation sequencing).

[0441] D. Genomic disruption of the cell surface cell death receptor (FAS) This disclosure provides modified γδT cells, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) comprising one, two, three, four, five, six, or seven genomic disruptions and / or the incorporation of one, two, three, or four exogenous polynucleotides, one of which is a genomic disruption of the endogenous cytokine-induced FAS cell surface cell death receptor (FAS) gene. In some embodiments, the modified cells are modified T-iγδT cells comprising a genomic disruption of the endogenous FAS gene. In some embodiments, the modified cells are modified (e.g., with genomic disruption in one or more of SOCS1, CISH, BIM, B2M, CIITA, and TRAC, and / or incorporation of one or more of B2M-HLA-E and signaling factors) or non-modified γδT-iPSCs, modified or non-modified T-iHSCs, modified or non-modified T-iCLPs, or γδT cells differentiated from modified or non-modified T-iγδT cells. In some embodiments, the genomic disruption is in at least two or all three of SOCS1, CISH, and BIM, and further in FAS, and optionally further in CIITA and / or B2M. In some embodiments, the modified cells are derived from primary γδT cells.

[0442] In some embodiments, the modified cells include genomic disruption within the endogenous FAS gene that suppresses or eliminates the expression of the FAS gene compared to an unmodified control. In some embodiments, the genomic disruption within the FAS gene is a loss-of-function nucleic acid mutation (e.g., a knockout). In some embodiments, the loss-of-function nucleic acid mutation within the FAS gene is an insertion, deletion, substitution, or any combination thereof. In some embodiments, the genomic disruption within the FAS gene is an endonuclease-mediated insertion or deletion (indel). In some embodiments, the loss-of-function nucleic acid mutation within the FAS gene is an endonuclease-mediated indel, such as a targeted knockout that can be obtained by a CRISPR-Cas system designed to target FAS (e.g., Cas9, Cas12, or MAD7).

[0443] In some embodiments, the genomic disruption of the endogenous FAS gene is located in exon 1. In some embodiments, the genomic disruption is a loss-of-function nucleic acid mutation within exon 1. In some embodiments, the genomic disruption is located within a target sequence in the FAS that includes the nucleotide sequence of SEQ ID NO: 62, or a nucleotide sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity thereto.

[0444] Assays to determine the relative expression levels of genes or the function of proteins encoded by those genes in modified cells (e.g., T-iγδT cells) can be performed using a variety of methods. For example, the relative expression level of the FAS gene in modified γδT cells (e.g., T-iγδT cells) compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or to γδT cells before modification (e.g., T-iγδT cells) can be measured using quantitative polymerase chain reaction (qPCR), digital PCR (dPCR), droplet digital PCR (ddPCR), or nucleotide sequencing (e.g., next-generation sequencing). ddPCR can be performed using appropriate nucleic acid quantification kits or instruments, such as the Qubit BR dsDNA assay (Thermo Fisher Scientific), Qubit HS dsDNA assay (Thermo Fisher Scientific), Nanodrop spectrophotometer (Thermo Fischer Scientific), Stunner spectrophotometer (Unchained Labs), and Lunatic spectrophotometer (Unchained Labs). Nucleotide sequencing can be performed using NextSeq (Ilumina), GridION (Oxford Nanopore Technologies), Revio System (Pacific Biosciences), etc.

[0445] E.β-2-microglobulin (B2M) genome disruption This disclosure provides modified γδT cells, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) comprising one, two, three, four, five, six, or seven genomic disruptions and / or the incorporation of one, two, three, or four exogenous polynucleotides, one of which is a genomic disruption of the endogenous β2-microglobulin (B2M) gene. In some embodiments, the modified cells are modified T-iγδT cells comprising a genomic disruption of the endogenous B2M gene. In some embodiments, the modified cells are modified (e.g., with genomic disruption in one or more of SOCS1, CISH, BIM, FAS, CIITA, and TRAC, and / or incorporation of one or more of B2M-HLA-E and signaling factors) or non-modified γδT-iPSCs, modified or non-modified T-iHSCs, modified or non-modified T-iCLPs, or γδT cells differentiated from modified or non-modified T-iγδT cells. In some embodiments, the genomic disruption is in at least two or all three of SOCS1, CISH, and BIM, and further in CIITA and / or B2M, and optionally further in FAS. In some embodiments, the modified cells are derived from primary γδT cells.

[0446] In some embodiments, the modified cells include genomic disruption within the endogenous B2M gene that suppresses or eliminates B2M gene expression compared to an unmodified control. In some embodiments, the genomic disruption within the B2M gene is a loss-of-function nucleic acid mutation (e.g., a knockout). In some embodiments, the loss-of-function nucleic acid mutation within the B2M gene is an insertion, deletion, substitution, or any combination thereof. In some embodiments, the genomic disruption within the B2M gene is an endonuclease-mediated insertion or deletion (indel). In some embodiments, the loss-of-function nucleic acid mutation within the B2M gene is an endonuclease-mediated indel, such as a targeted knockout that can be obtained by a CRISPR-Cas system designed to target B2M (e.g., Cas9, Cas12, or MAD7).

[0447] In some embodiments, the genomic disruption of the endogenous B2M gene is located within exon 2. In some embodiments, the genomic disruption is a loss-of-function nucleic acid mutation within exon 2. In some embodiments, the genomic disruption is located within a target sequence in B2M that includes the nucleotide sequence of SEQ ID NO: 56, or a nucleotide sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity thereto.

[0448] Assays to determine the relative expression levels of genes or the function of proteins encoded by those genes in modified cells (e.g., T-iγδT cells) can be performed using a variety of methods. For example, the relative expression level of the B2M gene in modified γδT cells (e.g., T-iγδT cells) compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or to original γδT cells (e.g., T-iγδT cells) can be measured using quantitative polymerase chain reaction (qPCR), digital PCR (dPCR), droplet digital PCR (ddPCR), or nucleotide sequencing (e.g., next-generation sequencing). ddPCR can be performed using appropriate nucleic acid quantification kits or instruments, such as the Qubit BR dsDNA assay (Thermo Fisher Scientific), Qubit HS dsDNA assay (Thermo Fisher Scientific), Nanodrop spectrophotometer (Thermo Fischer Scientific), Stunner spectrophotometer (Unchained Labs), and Lunatic spectrophotometer (Unchained Labs). Nucleotide sequencing can be performed using NextSeq (Ilumina), GridION (Oxford Nanopore Technologies), Revio System (Pacific Biosciences), etc.

[0449] Genome disruption of F class II transactivator (CIITA) This disclosure provides modified γδT cells, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) comprising one, two, three, four, five, six, or seven genomic disruptions and / or the incorporation of one, two, three, or four exogenous polynucleotides, one of which is a genomic disruption of the endogenous class II transactivator (CIITA) gene. In some embodiments, the modified cells are modified T-iγδT cells comprising a genomic disruption of the endogenous CIITA gene. In some embodiments, the modified cells are modified (e.g., with genomic disruption in one or more of SOCIS1, CISH, BIM, FAS, B2M, and TRAC, and / or incorporation of one or more of B2M-HLA-E and signaling factors) or non-modified γδT-iPSCs, modified or non-modified T-iHSCs, modified or non-modified T-iCLPs, or γδT cells differentiated from modified or non-modified T-iγδT cells. In some embodiments, the genomic disruption is in at least two or all three of SOCS1, CISH, and BIM, and further in CIITA and / or B2M, and optionally further in FAS. In some embodiments, the modified cells are derived from primary γδT cells.

[0450] In some embodiments, the modified cells include genomic disruption within the endogenous CIITA gene that suppresses or eliminates CIITA gene expression compared to the unmodified control. In some embodiments, the genomic disruption within the CIITA gene is a loss-of-function nucleic acid mutation (e.g., knockout). In some embodiments, the loss-of-function nucleic acid mutation within the CIITA gene is an insertion, deletion, substitution, or any combination thereof. In some embodiments, the genomic disruption within the CIITA gene is an endonuclease-mediated insertion / deletion (indel). In some embodiments, the loss-of-function nucleic acid mutation within the CIITA gene is an endonuclease-mediated indel, such as a targeted knockout that can be obtained by a CRISPR-Cas system designed to target CIITA (e.g., Cas9, Cas12, or MAD7).

[0451] In some embodiments, the genomic disruption of the endogenous CIITA gene is located within exon 3. In some embodiments, the genomic disruption is a loss-of-function nucleic acid mutation within exon 3. In some embodiments, the genomic disruption is located within a target sequence in CIITA that includes the nucleotide sequence of SEQ ID NO: 59, or a nucleotide sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity thereto.

[0452] Assays to determine the relative expression levels of a gene or the function of the protein encoded by that gene in modified cells (e.g., T-iγδT cells) can be performed using a variety of methods. For example, the relative expression level of the CIITA gene in modified γδT cells (e.g., T-iγδT cells) compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or to γδT cells before modification (e.g., T-iγδT cells) can be measured using quantitative polymerase chain reaction (qPCR), digital PCR (dPCR), droplet digital PCR (ddPCR), or nucleotide sequencing (e.g., next-generation sequencing). ddPCR can be performed using appropriate nucleic acid quantification kits or instruments, such as the Qubit BR dsDNA assay (Thermo Fisher Scientific), Qubit HS dsDNA assay (Thermo Fisher Scientific), Nanodrop spectrophotometer (Thermo Fischer Scientific), Stunner spectrophotometer (Unchained Labs), and Lunatic spectrophotometer (Unchained Labs). Nucleotide sequencing can be performed using NextSeq (Ilumina), GridION (Oxford Nanopore Technologies), Revio System (Pacific Biosciences), etc.

[0453] G.TRAC genome disruption This disclosure provides modified γδT cells, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) comprising one, two, three, four, five, six, or seven genomic disruptions and / or the incorporation of one, two, or three exogenous polynucleotides, one of which is a genomic disruption of the TRAC gene. In some embodiments, the modified cells are modified T-iγδT cells comprising a genomic disruption within the endogenous TRAC gene. In some embodiments, the modified cells are modified γδT-iPSCs (e.g., with genomic disruption in one or more of SOCIS1, CISH, BIM, FAS, B2M, and CIITA, and / or incorporation of one or more of B2M-HLA-E and signaling factors) or non-modified γδT-iPSCs, modified or non-modified T-iHSCs, modified or non-modified T-iCLPs, or γδT cells differentiated from modified or non-modified T-iγδT cells. In some embodiments, the modified cells are derived from primary γδT cells.

[0454] In some embodiments, the modified cells include genomic disruption within the endogenous TRAC gene that suppresses or eliminates TRAC gene expression compared to the unmodified control. In some embodiments, the genomic disruption within the TRAC gene is a loss-of-function nucleic acid mutation (e.g., knockout). In some embodiments, the loss-of-function nucleic acid mutation within the TRAC gene is an insertion, deletion, substitution, or any combination thereof. In some embodiments, the genomic disruption within the TRAC gene is an endonuclease-mediated insertion / deletion (indel). In some embodiments, the loss-of-function nucleic acid mutation within the TRAC gene is an endonuclease-mediated indel, such as a targeted knockout that can be obtained by a CRISPR-Cas system designed to target TRAC (e.g., Cas9, Cas12, or MAD7).

[0455] Assays to determine the relative expression levels of genes or the function of proteins encoded by those genes in modified cells (e.g., T-iγδT cells) can be performed using a variety of methods. For example, the relative expression levels of SOCIS1, CISH, BIM, FAS, B2M, CIITA, or TRAC genes in modified γδT cells (e.g., T-iγδT cells) compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or to original γδT cells (e.g., T-iγδT cells) can be measured using quantitative polymerase chain reaction (qPCR), digital PCR (dPCR), droplet digital PCR (ddPCR), or nucleotide sequencing (e.g., next-generation sequencing). ddPCR can be performed using appropriate nucleic acid quantification kits or instruments, such as the Qubit BR dsDNA assay (Thermo Fisher Scientific), Qubit HS dsDNA assay (Thermo Fisher Scientific), Nanodrop spectrophotometer (Thermo Fischer Scientific), Stunner spectrophotometer (Unchained Labs), and Lunatic spectrophotometer (Unchained Labs). Nucleotide sequencing can be performed using NextSeq (Ilumina), GridION (Oxford Nanopore Technologies), Revio System (Pacific Biosciences), etc.

[0456] H.B2M-HLA-E integration This disclosure provides modified γδT cells, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) comprising one, two, three, four, five, six, or seven genomic disruptions and / or the incorporation of one, two, three, or four exogenous polynucleotides, one of which is the incorporation of B2M-HLA-E. In some embodiments, the modified cells are modified T-iγδT cells containing an exogenous polynucleotide encoding the B2M-HLA-E gene. In some embodiments, the modified cells are γδT cells differentiated from modified iPSCs (e.g., alone or with genomic disruption in one or more of SOCS1, CISH, BIM, FAS, B2M, CIITA, TRAC, and / or incorporation of one or more signaling factors). In some embodiments, the modified cells are derived from primary γδT cells.

[0457] In some embodiments, modified cells contain an exogenous polynucleotide encoding B2M-HLA-E, resulting in B2M-HLA-E expression compared to an unmodified control. In some embodiments, the exogenous polynucleotide encoding B2M-HLA-E is incorporated into modified cells by knock-in. In some embodiments, the knock-in is a targeted knock-in obtainable by a CRISPR-Cas system (e.g., Cas9, Cas12, or MAD7) targeting a genomic locus of interest, and is combined with a donor template containing an exogenous polynucleotide that is incorporated into the genomic locus of interest by homologous recombination repair. In some embodiments, the exogenous polynucleotide encoding B2M-HLA-E is inserted into the B2M locus. In some embodiments, the exogenous polynucleotide encoding B2M-HLA-E is incorporated into modified cells by a lentiviral vector.

[0458] In some embodiments, B2M-HLA-E is a fusion protein comprising at least a portion of B2M fused with at least a portion of HLA-E. In some embodiments, B2M-HLA-E comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, B2M-HLA-E is essentially or consists of the amino acid sequence of SEQ ID NO: 85.

[0459] In some embodiments, a CAG promoter was used to induce expression from an exogenous polynucleotide encoding B2M-HLA-E.

[0460] I. Signal conversion factors This disclosure provides modified γδT cells, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) comprising one, two, three, four, five, six, or seven genomic disruptions and / or the incorporation of one, two, three, or four exogenous polynucleotides, one of which is the incorporation of a signal transmutation factor (e.g., IFNγ signal transmutation factor). In some embodiments, the modified cells are modified T-iγδT cells comprising one or more exogenous polynucleotides encoding a signal transmutation factor (e.g., IFNγ signal transmutation factor). In some embodiments, the modified cells are γδT cells differentiated from modified iPSCs (e.g., alone or with genomic disruption in one or more of SOCS1, CISH, BIM, FAS, B2M, CIITA, TRAC, and / or incorporation of B2M-HLA-E). In some embodiments, the modified cells are derived from primary γδT cells.

[0461] In some embodiments, the signal-converting factor is a chimeric structure of two or more different cytokine receptors, where the extracellular structure of the signal-converting factor is derived from one cytokine receptor A, and the intracellular structure of the signal-converting factor is derived from another cytokine receptor B, and the binding of cytokine A to the extracellular structure of cytokine receptor A transmits, passes through, or converts intracellular signaling via the intracellular structure of cytokine receptor B, thereby activating or improving intracellular signaling and activator of transcription (STAT) signaling that is not activated, little activated, or poorly activated by the innate cytokine receptor A when binding to cytokine A. In some embodiments, the extracellular structure of the signal-converting factor is derived from the same or different cytokine receptors, or includes one or more extracellular domains derived from one or more cytokine receptors. In some embodiments, the intracellular structure of the signal-converting factor is derived from the same or different cytokine receptors, or includes one or more intracellular domains derived from one or more cytokine receptors.

[0462] In some embodiments, the extracellular structure of the signal-converting factor is derived from an interferon-gamma receptor. In some embodiments, the intracellular structure of the signal-converting factor is derived from an interleukin-2 receptor. In some embodiments, the extracellular and intracellular structures of the signal-converting factor are linked via a transmembrane structure, which includes a transmembrane domain derived from the same or different cytokine receptor from which the extracellular and intracellular structures of the signal-converting factor originate. In some embodiments, the transmembrane structure includes a transmembrane domain derived from the same cytokine receptor from which the extracellular or intracellular structure of the signal-converting factor originates. In some embodiments, the signal-converting factor includes a membrane proximal region between the ECD and the intracellular domain, which is derived from the same or different cytokine receptor from which the extracellular and intracellular structures of the signal-converting factor originate. In some embodiments, the membrane proximal region is derived from the same cytokine receptor from which the extracellular domain of the signal-converting factor originates, or / or the same cytokine receptor from which the intracellular domain of the signal-converting factor originates. In some embodiments, the signal-converting factor further includes a signal peptide that enables trafficking of the signal-converting factor to the cell membrane. In some embodiments, the signal peptide is removed from the signal-converting factor after localization to the membrane.

[0463] IFNγ signal-converting factor In some embodiments, the extracellular structure of the IFNγ signal-converting factor includes both the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the extracellular domain (ECD) of interferon-gamma receptor 2 (IFNGR2). In some embodiments, the intracellular structure of the IFNγ signal-converting factor includes the intracellular domain of interleukin-2 receptor subunit gamma (IL2RG) and the intracellular domain of interleukin-2 receptor subunit beta (IL2RB), where one ECD of IFNGR1 and one of IFNGR2 are fused with one ICD of IL2RG and one of IL2RB to form a first IFNγ fusion protein subunit, and the other ECD is linked or fused with the other ICD to form a second IFNγ fusion protein subunit. In some embodiments, the IFNγ signal-converting factor further includes the transmembrane domains of IFNGR1 and IFNGR2, or the IFNγ signal-converting factor further includes the transmembrane domains of IL2RG and IL2RB. In some embodiments, the IFNγ signal-converting factor further comprises the proximal membrane regions of IFNGR1 and IFNGR2, or the IFNγ signal-converting factor further comprises the proximal membrane regions of IL2RG and IL2RB. In some embodiments, one of the extracellular domains of IFNGR1 or IFNGR2 is fused sequentially with the transmembrane domain and the intracellular domain of IL2RG to form a first IFNγ fusion protein subunit, and the other of the extracellular domains of IFNGR2 or IFNGR1 is fused sequentially with the transmembrane domain and the intracellular domain of IL2RB to form a second IFNγ fusion protein subunit. In some embodiments, one of the extracellular domains of IFNGR1 or IFNGR2 is fused sequentially with the proximal membrane region, the transmembrane domain and the intracellular domain of IL2RG to form a first IFNγ fusion protein subunit, and the other of the extracellular domains of IFNGR2 or IFNGR1 is fused sequentially with the proximal membrane region, the transmembrane domain and the intracellular domain of IL2RB to form a second IFNγ fusion protein subunit. In some embodiments, the first IFNγ fusion protein subunit and the second IFNγ fusion protein subunit of the IFNγ signal-converting factor further comprise a signal peptide.

[0464] In some embodiments, the signal peptide of the first IFNγ fusion protein subunit comprises the amino acids of SEQ ID NO: 79 or SEQ ID NO: 80, and the signal peptide of the second IFNγ fusion protein subunit comprises the amino acid sequence of SEQ ID NO: 79 or SEQ ID NO: 80.

[0465] In some embodiments, the amino acid sequence of the signal peptide of the first IFNγ fusion protein subunit is SEQ ID NO: 79, and the amino acid sequence of the signal peptide of the second IFNγ fusion protein subunit is SEQ ID NO: 80. In some embodiments, the amino acid sequence of the extracellular domain of the first IFNγ fusion protein subunit is SEQ ID NO: 63, and the amino acid sequence of the extracellular domain of the second IFNγ fusion protein subunit is SEQ ID NO: 64. In some embodiments, the amino acid sequence of the intracellular domain of the first IFNγ fusion protein subunit is SEQ ID NO: 69, and the amino acid sequence of the intracellular domain of the second IFNγ fusion protein subunit is SEQ ID NO: 70. In some embodiments, the amino acid sequence of the transmembrane domain of the first IFNγ fusion protein subunit is SEQ ID NO: 67, and the amino acid sequence of the transmembrane domain of the second IFNγ fusion protein subunit is SEQ ID NO: 68. The amino acid sequence of the proximal membrane region of the first IFNγ fusion protein subunit is SEQ ID NO: 66, and the amino acid sequence of the proximal membrane region of the second IFNγ fusion protein subunit is SEQ ID NO: 66.

[0466] In some embodiments, the present disclosure provides an IFNγ signaling converter comprising a first IFNγ fusion protein subunit and a second IFNγ fusion protein subunit, wherein the first IFNγ fusion protein subunit comprises the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the intracellular domain of interleukin-2 receptor subunit gamma (IL2RG), and the second IFNγ fusion protein subunit comprises the ECD of interferon-gamma receptor 2 (IFNGR2) and the intracellular domain of interleukin-2 receptor subunit beta (IL2RB). In some embodiments, the first IFNγ fusion protein subunit of the IFNγ signaling converter comprises the amino acid sequence of SEQ ID NO: 71, and the second IFNγ fusion protein subunit of the IFNγ signaling converter comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the first IFNγ fusion protein subunit of the IFNγ signal-converting factor is essentially composed of or derived from the amino acid sequence of SEQ ID NO: 71, and the second IFNγ fusion protein subunit of the IFNγ signal-converting factor is essentially composed of or derived from the amino acid sequence of SEQ ID NO: 72. In some embodiments, the first exogenous polynucleotide encodes the amino acid sequence containing SEQ ID NO: 81 (the first IFNγ fusion protein subunit having the signal peptide), and the second exogenous polynucleotide encodes the amino acid sequence containing SEQ ID NO: 82 (the second IFNγ fusion protein subunit having the signal peptide).

[0467] In some embodiments, the modified cells contain one or more exogenous polynucleotides that result in the expression of IFNγ signaling factor compared to the unmodified control. In some embodiments, one or more exogenous polynucleotides encoding IFNγ signaling factor are incorporated into the modified cells by knock-in. In some embodiments, the knock-in is a targeted knock-in obtainable by a CRISPR-Cas system (e.g., Cas9, Cas12, or MAD7) targeting a genomic locus of interest and is combined with a donor template containing an exogenous polynucleotide that is incorporated into the genomic locus of interest by homologous recombination repair. In some embodiments, one or more exogenous polynucleotides encoding IFNγ signaling factor are incorporated into the modified cells by a lentiviral vector.

[0468] In some embodiments, a first exogenous polynucleotide encoding the first IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the SOCS1 locus. In some embodiments, a second exogenous polynucleotide encoding the second IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the ROSA26 locus.

[0469] In some embodiments, a first exogenous polynucleotide encoding the first IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the ROSA26 locus. In some embodiments, a second exogenous polynucleotide encoding the second IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the SOCS1 locus.

[0470] In some embodiments, a CAG promoter was used to induce expression from a first exogenous polynucleotide encoding a first IFNγ fusion protein subunit and a second exogenous polynucleotide encoding a second IFNγ fusion protein subunit.

[0471] J. Single, double, triple, quadruple, quadruple, and hexagram genome disruption This disclosure provides single, double, triple, quadruple, quintuple, or hexagene-modified γδT cells, for example, iPSC-derived γδT cells (and their precursor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)))). This disclosure provides single, double, triple, quadruple, quintuple, or hexavalent gene-modified γδT cells, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))). In some embodiments, the modified cells are T-iγδT cells containing genomic disruption of the endogenous SOCS1 and CISH genes. In some embodiments, the modified cells are T-iγδT cells containing genomic disruption of the endogenous SOCS1, CISH, and BIM genes. In some embodiments, the modified cells are T-iγδT cells containing genomic disruptions of the endogenous SOCS1, CISH, BIM, and FAS genes. In some embodiments, the modified cells are T-iγδT cells containing genomic disruptions of the endogenous SOCS1, CISH, BIM, B2M, and CIITA genes. In some embodiments, the modified cells are T-iγδT cells containing genomic disruptions of the endogenous SOCS1, CISH, BIM, FAS, B2M, and CIITA genes. In some embodiments, the modified cells are differentiated from γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells, and the modification may occur at any of these cell developmental stages and be carried over to the mature state of γδT, or the modification may occur at the mature state of γδT, i.e., the T-iγδT stage.

[0472] In some embodiments, the modified cells include genomic disruption within the endogenous SOCS1 gene that suppresses or eliminates the expression of SOCS1 and CISH genes.

[0473] This disclosure provides modified iPSC-derived γδT cells (and their progenitor cells (e.g., γδT-iPSC, γδT-iHSC, γδT-iCLP)) including genomic disruption within the endogenous SOCS1 gene that suppresses or eliminates SOCS1 expression, genomic disruption within the endogenous CISH gene that suppresses or eliminates CISH expression, and optionally genomic disruption within the endogenous BIM gene that suppresses or eliminates BIM expression.

[0474] This disclosure provides modified iPSC-derived γδT cells (and their progenitor cells (e.g., γδT-iPSC, γδT-iHSC, γδT-iCLP)) including genomic disruption within the endogenous SOCS1 gene that suppresses or eliminates SOCS1 expression, genomic disruption within the endogenous CISH gene that suppresses or eliminates CISH expression, genomic disruption within the endogenous BIM gene that optionally suppresses or eliminates BIM expression, and genomic disruption within the endogenous FAS gene that optionally suppresses or eliminates FAS expression.

[0475] This disclosure provides modified iPSC-derived γδT cells (and their progenitor cells (e.g., γδT-iPSC, γδT-iHSC, γδT-iCLP)) including genomic disruption within the endogenous SOCS1 gene to suppress or eliminate SOCS1 expression, genomic disruption within the endogenous CISH gene to suppress or eliminate CISH expression, genomic disruption within the endogenous BIM gene to optionally suppress or eliminate BIM expression, genomic disruption within the endogenous B2M gene to optionally suppress or eliminate B2M expression, and genomic disruption within the endogenous CIITA gene to optionally suppress or eliminate CIITA expression.

[0476] This disclosure provides modified iPSC-derived γδT cells (and their progenitor cells (e.g., γδT-iPSC, γδT-iHSC, γδT-iCLP)) including genomic disruption within the endogenous SOCS1 gene to suppress or eliminate SOCS1 expression, genomic disruption within the endogenous CISH gene to suppress or eliminate CISH expression, genomic disruption within the endogenous BIM gene to optionally suppress or eliminate BIM expression, genomic disruption within the endogenous FAS gene to optionally suppress or eliminate FAS expression, genomic disruption within the endogenous B2M gene to optionally suppress or eliminate B2M expression, and genomic disruption within the endogenous CIITA gene to optionally suppress or eliminate CIITA expression.

[0477] In some embodiments, BIM EL and BIM L The expression level and / or function of splice variants are reduced in modified cells compared to unmodified controls. In some embodiments, BIM S The expression level and / or function of the splice variant are preserved in the modified cells.

[0478] In some embodiments, genome disruption is performed by administering a guide RNA to cells, which may be a single guide RNA (sgRNA), and an endonuclease, or nucleic acid encoding an endonuclease, is also administered to the cells. Further information regarding guide RNA and endonucleases can be found in other sections of this disclosure, for example, Section V: Gene Editing Systems.

[0479] This also includes populations of cells containing single, double, triple, quadruple, quintuple, or hexagram genome disruptions as described herein.

[0480] In some embodiments, the cells are stored for a certain period before use. In some embodiments, this period is several weeks or several months. In some embodiments, this period is several months or several years.

[0481] In some embodiments, the cells are cryopreserved in liquid nitrogen at -196°C.

[0482] III. Modified cells containing CARs (e.g., anti-CD19 CARs) Modified cells as described herein, for example, the modified cells described in Section II, further comprise exogenous polynucleotides encoding chimeric antigen receptors (CARs), for example, CARs that specifically bind to human CD19 (anti-CD19 CARs).

[0483] This disclosure provides modified γδT cells, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) comprising one, two, three, four, five, six, or seven genomic disruptions and / or the incorporation of one, two, three, or four exogenous polynucleotides, one of which is the incorporation of an anti-CD19 CAR. In some embodiments, the modified cells are modified T-iγδT cells containing an exogenous polynucleotide encoding an anti-CD19 CAR. In some embodiments, the modified cells are γδT cells differentiated from modified iPSCs (e.g., with genomic disruption in one or more of SOCS1, CISH, BIM, FAS, B2M, CIITA, TRAC, and / or incorporation of one or more of B2M-HLA-E and signaling factors). In some embodiments, the modified cells are derived from primary γδT cells.

[0484] In some embodiments, modified cells contain an exogenous polynucleotide encoding an anti-CD19 CAR, resulting in anti-CD19 CAR expression compared to an unmodified control. In some embodiments, the exogenous polynucleotide encoding the anti-CD19 CAR is incorporated into modified cells by knock-in. In some embodiments, the knock-in is a targeted knock-in obtainable by a CRISPR-Cas system (e.g., Cas9, Cas12, or MAD7) targeting a genomic locus of interest, and is combined with a donor template containing an exogenous polynucleotide that is incorporated into the genomic locus of interest by homologous recombination repair. In some embodiments, the exogenous polynucleotide encoding the anti-CD19 CAR is inserted into the AAVS1 locus, which is a safer insertion region. In some embodiments, the exogenous polynucleotide encoding the anti-CD19 CAR is incorporated into modified cells by a lentiviral vector.

[0485] In some embodiments, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the anti-human CLL1 CAR comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the anti-CD19 CAR is essentially composed of or consists of the amino acid sequence of SEQ ID NO: 83 or SEQ ID NO: 84.

[0486] In some embodiments, the anti-CD19 CAR further comprises a signal peptide (e.g., including the amino acid sequence of SEQ ID NO: 73) that enables the trafficking of the CAR to the cell membrane. In some embodiments, the signal peptide is removed from the signal converter after localization to the membrane.

[0487] In some embodiments, a CAG promoter was used to induce expression from an exogenous polynucleotide encoding an anti-CD19 CAR.

[0488] IV. Functional enhancement of proliferation and killing activity In some embodiments, the disclosure provides modified γδT cells having functional enhancements to proliferation and / or tumor-killing activity, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))). More specifically, the functional enhancement is achieved by activation or improvement of signaling and activator-of-transcription (STAT) signaling in the cells. Methods are also disclosed for improving the proliferation and / or tumor-killing activity of modified γδT cells, such as iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) by activating or improving activating cellular signaling and activator-of-transcription (STAT) signaling with active compounds.

[0489] In some embodiments, a method for functionally enhancing proliferation and / or tumor-killing activity is achieved by introducing one or more exogenous polynucleotides encoding signal-converting factors (IFNγ signal-converting factors) into iPSCs or γδT cells.

[0490] Further information regarding signal conversion factors, such as the IFNγ signal conversion factor, can be found in other sections of this disclosure, for example, "Section II(I): Signal Conversion Factors."

[0491] In some embodiments, the amino acid sequence of the ECD of the first IFNγ fusion protein subunit is SEQ ID NO: 63, and the amino acid sequence of the ECD of the second IFNγ fusion protein subunit is SEQ ID NO: 64. In some embodiments, the amino acid sequence of the ECD of the first IFNγ fusion protein subunit is SEQ ID NO: 69, and the amino acid sequence of the ECD of the second IFNγ fusion protein subunit is SEQ ID NO: 70. In some embodiments, the amino acid sequence of the ECD of the first IFNγ fusion protein subunit is SEQ ID NO: 67, and the amino acid sequence of the ECD of the second fusion protein subunit is SEQ ID NO: 68. The amino acid sequence of the membrane-proximal region of the first IFNγ fusion protein subunit is SEQ ID NO: 65, and the amino acid sequence of the membrane-proximal region of the second IFNγ fusion protein subunit is SEQ ID NO: 66.

[0492] In some embodiments, the present disclosure relates to modified γδT cells containing an IFNγ signaling converter, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))), wherein the IFNγ signaling converter comprises a first IFNγ fusion protein subunit and a second IFNγ fusion protein subunit, the first IFNγ fusion protein subunit comprising the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the interleukin-2 receptor subunit gamma Also disclosed are modified γδT cells comprising an intracellular domain of (IL2RG), wherein the second IFNγ fusion protein subunit comprises the ECD of interferon-gamma receptor 2 (IFNGR2) and the intracellular domain of interleukin-2 receptor subunit beta (IL2RB), optionally further comprising an exogenous polynucleotide encoding a chimeric antigen receptor (CAR), optionally having the exogenous polynucleotide encoding the CAR inserted at the AAVS1 locus, optionally encoding a CAR that specifically binds to human CD19, optionally comprising SEQ ID NO: 83 or SEQ ID NO: 84. In some embodiments, the first IFNγ fusion protein subunit of the IFNγ signaling converter comprises the amino acid sequence of SEQ ID NO: 71, and the second IFNγ fusion protein subunit of the IFNγ signaling converter comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the first IFNγ fusion protein subunit of the IFNγ signal-converting factor is essentially composed of or derived from the amino acid sequence of SEQ ID NO: 71, and the second IFNγ fusion protein subunit of the IFNγ signal-converting factor is essentially composed of or derived from the amino acid sequence of SEQ ID NO: 72. In some embodiments, the first exogenous polynucleotide encodes the amino acid sequence containing SEQ ID NO: 81 (the first IFNγ fusion protein subunit having the signal peptide), and the second exogenous polynucleotide encodes the amino acid sequence containing SEQ ID NO: 82 (the second IFNγ fusion protein subunit having the signal peptide).

[0493] In some embodiments, the amino acid sequence of the first IFNγ fusion protein subunit is SEQ ID NO: 71, and the amino acid sequence of the transmembrane domain of the second IFNγ fusion protein subunit is SEQ ID NO: 72.

[0494] Modified γδT cells having functional enhancements to proliferation and / or tumor-killing activity as disclosed herein, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) may be pre- or further subjected to gene editing to suppress cytokine signaling as disclosed in other sections of this disclosure.

[0495] V. Gene editing systems This specification discloses gene editing systems optimized to disrupt the genomes of cells (e.g., γδT cells, iγδT cells, iPSCs, iHSCs, iCLP cells, or immature iT cells) within the genes of SOCS1;CISH;BIM;SOCS1 and CISH;SOCS1, CISH, and BIM;SOCS1, CISH, and FAS;SOCS1, CISH, BIM, and FAS;B2M, CIITA, CISH, SOCS1, and BIM;B2M, CIITA, CISH, SOCS1, BIM, and FAS.

[0496] A. Regular arrangement clusters of short palindromic sequences (CRISPR) In some embodiments, the gene editing system used to modify γδT cells, for example, iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)) is a regularly arranged cluster (CRISPR) system of short palindromic sequences, comprising one or more guide RNAs and an endonuclease or a nucleic acid encoding a nuclease. The CRISPR gene editing system comprises, for example, a guide RNA including a single guide RNA (sgRNA) or crRNA, and a nuclease including, for example, an endonuclease or a nucleic acid encoding a nuclease (e.g., a nucleic acid encoding an endonuclease).

[0497] In some embodiments, the guide RNA comprises a guide sequence that binds to a target sequence within SOCS1, CISH, BIM, B2M, CIITA, and / or FAS in the cells used herein (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells). In some embodiments, genomic disruption within the SOCS1, CISH, BIM, B2M, CIITA, or FAS genes is performed by administering a nuclease (e.g., an endonuclease), multiple nucleases (e.g., multiple endonucleases), or nucleic acids encoding multiple endonucleases, and a guide RNA or multiple guide RNAs to the cells used herein (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells).

[0498] In some embodiments, the administration of nucleases and guide RNAs to the cells used herein (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) is performed sequentially, with the first administration being a guide RNA targeting a first gene (e.g., SOCS1), followed by a guide RNA targeting a second gene (e.g., CISH). In some embodiments, a guide RNA targeting a third gene (e.g., BIM) may be administered. In some embodiments, a guide RNA targeting a fourth gene (e.g., FAS) may be administered. In some embodiments, a guide RNA targeting a fifth gene (e.g., B2M) may be administered. In some embodiments, a guide RNA targeting a sixth gene (e.g., CIITA) may be administered. The sequential administration of gene-targeting guide RNAs can be performed in any order.

[0499] This specification discloses sgRNA or crRNA nucleotide sequences that bind to a target sequence within the SOCS1 gene and include one of sequence numbers 5, 29, 11, 37, and nucleotide sequences having at least 70% identity with them (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). This specification discloses sgRNA or crRNA nucleotide sequences that bind to a target sequence within a CISH gene and include one of sequence numbers 3, 27, 4, 28, 12, 38, and nucleotide sequences having at least 70% identity with them (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). This specification discloses sgRNA or crRNA nucleotide sequences that bind to a target sequence within the BIM gene and include one of sequence numbers 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, 36, or any nucleotide sequence having at least 70% identity with these (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). This specification discloses sgRNA nucleotide sequences that bind to a target sequence in the FAS gene and include one of sequence numbers 60, 61, or any nucleotide sequence having at least 70% identity with them (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).This specification discloses sgRNA nucleotide sequences that bind to a target sequence in the B2M gene and include one of sequence numbers 54, 55, or any nucleotide sequence having at least 70% identity with them (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). This specification discloses sgRNA nucleotide sequences that bind to a target sequence in the CIITA gene and include one of sequence numbers 57, 58, or any nucleotide sequence having at least 70% identity with them (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0500] This specification discloses a gene editing system comprising one or more guide RNAs (e.g., sgRNA or crRNA) and an endonuclease or nucleic acid encoding an endonuclease, wherein one or more guide RNAs comprise a guide sequence or sgRNA / crRNA sequence comprising the following sequences: (1) Any one of sequence numbers 5, 29, 11, and 37, (2) Any one of sequence numbers 3, 27, 4, 28, 12, and 38, (3) Any one of sequence numbers 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, and 36, (4) Either one of sequence numbers 60 and 61, (5) Either one of sequence numbers 54 and 55, (6) Either one of sequence numbers 57 and 58, (7) Any one of sequence numbers 5, 29, 11, and 37 and any one of sequence numbers 3, 27, 4, 28, 12, and 38, (8) Any one of sequence numbers 5, 29, 11, and 37, any one of sequence numbers 3, 27, 4, 28, 12, and 38, and any one of sequence numbers 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, and 36, (9) Any one of sequence numbers 5, 29, 11, and 37; any one of sequence numbers 3, 27, 4, 28, 12, and 38; any one of sequence numbers 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, and 36; and any one of sequence numbers 60 and 61. (10) Any one of sequence numbers 5, 29, 11, and 37; any one of sequence numbers 3, 27, 4, 28, 12, and 38; any one of sequence numbers 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, and 36; any one of sequence numbers 54 and 55; and any one of sequence numbers 57 and 58, or (11) Any one of sequence numbers 5, 29, 11, and 37; any one of sequence numbers 3, 27, 4, 28, 12, and 38; any one of sequence numbers 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, and 36; any one of sequence numbers 54 and 55; any one of sequence numbers 57 and 58; and any one of sequence numbers 60 and 61.

[0501] This specification discloses a gene editing system comprising one or more guide RNAs (e.g., sgRNA or crRNA) and an endonuclease or nucleic acid encoding an endonuclease, wherein one or more guide RNAs comprise a guide sequence or sgRNA / crRNA sequence comprising the following sequences: (1) One or more sequences in which one or more of sequence numbers 5, 29, 11, 37 differ from sequence numbers 5, 29, 11, and 37 by 5, 4, 3, 2, or 1 or fewer nucleotides, (2) One or more sequences in which one or more of sequence numbers 3, 27, 4, 28, 12, 38 differ from any of sequence numbers 3, 27, 4, 28, 12, and 38 by 5, 4, 3, 2, or 1 or fewer nucleotides, (3) Sequence IDs 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, and 36, and one or more sequences in which 5, 4, 3, 2, or 1 or fewer nucleotides differ from any one of Sequence IDs 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, and 36, (4) One or more sequences that differ from any one of sequence numbers 60, 61, and sequence numbers 60 and 61 by 5, 4, 3, 2, or 1 or fewer nucleotides, (5) One or more sequences in which one or more of sequence numbers 54, 55, and sequence numbers 54 and 55 differ from 5, 4, 3, 2, or 1 or fewer nucleotides, (6) One or more sequences that differ from any one of sequence numbers 57, 58, and sequence numbers 57 and 58 by 5, 4, 3, 2, or 1 or fewer nucleotides, (7) One or more sequences that differ from any one of sequence numbers 5, 29, 11, 37 and sequence numbers 5, 29, 11, and 37 by 5, 4, 3, 2, or 1 or fewer nucleotides, and one or more sequences that differ from any one of sequence numbers 3, 27, 4, 28, 12, 38 and sequence numbers 3, 27, 4, 28, 12, and 38 by 5, 4, 3, 2, or 1 or fewer nucleotides, (8) One or more sequences that differ from any one of sequence numbers 5, 29, 11, 37 and sequence numbers 5, 29, 11, and 37 by 5, 4, 3, 2, or 1 or fewer nucleotides; one or more sequences that differ from any one of sequence numbers 3, 27, 4, 28, 12, 38 and sequence numbers 3, 27, 4, 28, 12, and 38 by 5, 4, 3, 2, or 1 or fewer nucleotides; and one or more sequences that differ from any one of sequence numbers 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, 36 and sequence numbers 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, and 36 by 5, 4, 3, 2, or 1 or fewer nucleotides. (9) One or more sequences that differ from any one of sequence numbers 5, 29, 11, 37 and sequence numbers 5, 29, 11, and 37 by 5, 4, 3, 2, or 1 or fewer nucleotides; one or more sequences that differ from any one of sequence numbers 3, 27, 4, 28, 12, 38 and sequence numbers 3, 27, 4, 28, 12, and 38 by 5, 4, 3, 2, or 1 or fewer nucleotides; sequence numbers 2, 26, 6 , 32, 7, 33, 8, 34, 9, 35, 10, 36 and one or more sequences of sequence numbers 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, and 36 that differ from any one of 5, 4, 3, 2, or 1 or fewer nucleotides, and one or more sequences of sequence numbers 60, 61 and 60 and 61 that differ from any one of 5, 4, 3, 2, or 1 or fewer nucleotides, (10) One or more sequences that differ from any one of sequence numbers 5, 29, 11, 37 and sequence numbers 5, 29, 11, and 37 by 5, 4, 3, 2, or 1 or fewer nucleotides; one or more sequences that differ from any one of sequence numbers 3, 27, 4, 28, 12, 38 and sequence numbers 3, 27, 4, 28, 12, and 38 by 5, 4, 3, 2, or 1 or fewer nucleotides; sequence numbers 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, 36 and sequence numbers 2, 26, 6 , one or more sequences that differ from any one of 32, 7, 33, 8, 34, 9, 35, 10, and 36 by 5, 4, 3, 2, or 1 or fewer nucleotides, one or more sequences that differ from any one of SEQ ID NOs. 54, 55, and SEQ ID NOs. 54 and 55 by 5, 4, 3, 2, or 1 or fewer nucleotides, and one or more sequences that differ from any one of SEQ ID NOs. 57, 58, and SEQ ID NOs. 57 and 58 by 5, 4, 3, 2, or 1 or fewer nucleotides, (11) One or more sequences that differ from any one of sequence numbers 5, 29, 11, 37 and sequence numbers 5, 29, 11, and 37 by 5, 4, 3, 2, or 1 or fewer nucleotides; one or more sequences that differ from any one of sequence numbers 3, 27, 4, 28, 12, 38 and sequence numbers 3, 27, 4, 28, 12, and 38 by 5, 4, 3, 2, or 1 or fewer nucleotides; one or more sequences that differ from any one of sequence numbers 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, and 36 One or more sequences that differ from 5, 4, 3, 2, or 1 or fewer nucleotides; one or more sequences that differ from 5, 4, 3, 2, or 1 or fewer nucleotides from any one of sequence numbers 54, 55, and sequence numbers 54 and 55; one or more sequences that differ from 5, 4, 3, 2, or 1 or fewer nucleotides from any one of sequence numbers 57, 58, and sequence numbers 57 and 58; and one or more sequences that differ from 5, 4, 3, 2, or 1 or fewer nucleotides from any one of sequence numbers 60, 61, and sequence numbers 60 and 61.

[0502] This specification discloses a gene editing system comprising one or more sgRNAs and an endonuclease or a nucleic acid encoding an endonuclease, wherein one or more sgRNAs include a guide sequence that binds to a target sequence in a target gene. In some embodiments, the target sequence is (1) One or more of sequence numbers 17, 23, 15, 16, and 24, (2) One or more of sequence numbers 14, 18, 19, 20, 21, and 22, (3) Sequence ID 62, (4) Sequence ID 56, (5) Sequence ID 59, (6) One or more of sequence numbers 17, 23, 15, 16, and 24, and one or more of sequence numbers 14, 18, 19, 20, 21, and 22, (7) One or more of sequence numbers 17, 23, 15, 16, and 24, one or more of sequence numbers 14, 18, 19, 20, 21, and 22, and sequence number 62, (8) One or more of sequence numbers 17, 23, 15, 16, and 24, one or more of sequence numbers 14, 18, 19, 20, 21, and 22, sequence number 56, and sequence number 59, or (9) Includes one or more of sequence numbers 17, 23, 15, 16, and 24, one or more of sequence numbers 14, 18, 19, 20, 21, and 22, sequence number 62, sequence number 56, and sequence number 59. In some embodiments, the target sequence includes: (1) One or more nucleotide sequences that have at least 70% (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with any one or more of sequence numbers 17, 23, 15, 16, and 24, (2) One or more nucleotide sequences that have at least 70% (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with any one or more of sequence numbers 14, 18, 19, 20, 21, and 22, (3) One or more nucleotide sequences that are identical to sequence number 62 by at least 70% (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), (4) One or more nucleotide sequences that are identical to sequence number 56 by at least 70% (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of sequence number 56, (5) One or more nucleotide sequences that are identical to sequence number 59 by at least 70% (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), (6) One or more nucleotide sequences having at least 70% identity (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) with SEQ ID NOs. 17, 23, 15, 16, and 24, and one or more nucleotide sequences having at least 70% identity (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with SEQ ID NOs. 14, 18, 19, 20, 21, and 22, (7) One or more nucleotide sequences having at least 70% identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) with SEQ ID NOs. 14, 18, 19, 20, 21, 22, and SEQ ID NOs. 14, 18, 19, 20, 21, 22 One or more nucleotide sequences having identity of %, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, as well as one or more nucleotide sequences having identity of at least 70% (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) with SEQ ID NO: 62, (8) One or more nucleotide sequences having at least 70% identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) with SEQ ID NOs. 14, 18, 19, 20, 21, 22, and One or more nucleotide sequences having uniformity, one or more nucleotide sequences having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with SEQ ID NO: 56, and one or more nucleotide sequences having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with SEQ ID NO: 59, (9) One or more nucleotide sequences having at least 70% identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) with SEQ ID NOs. 14, 18, 19, 20, 21, 22, and SEQ ID NOs. 14, 18, 19, 20, 21, 22 have at least 70% identity (e.g., at least One or more nucleotide sequences having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, one or more nucleotide sequences having at least 70% identity (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with SEQ ID NO: 56, One or more nucleotide sequences that are at least 70% (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 59, and one or more nucleotide sequences that are at least 70% (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 62.

[0503] Examples of endonucleases suitable for use in the disclosed gene editing systems include, for example, Cas endonucleases and other endonucleases well known in the art. In some embodiments, the Cas endonucleases are Cas9, Cas12i2, Cas12a, Cas12b, Cas12c, Cas13a(C2c2), Cas13b, or MAD7.

[0504] In some embodiments, the endonuclease used in the CRISPR gene editing system is MAD7 endonuclease, and the target sequence of the guide RNA in the CRISPR gene editing system includes SEQ ID NO: 22, and / or the guide RNA includes a sequence that differs from SEQ ID NO: 22 by 5, 4, 3, 2, or one or fewer nucleotides. In some embodiments, the endonuclease used in the CRISPR gene editing system is MAD7 endonuclease, and the target sequence of the guide RNA in the CRISPR gene editing system includes SEQ ID NO: 23, and / or the guide RNA includes a sequence that differs from SEQ ID NO: 11 by 5, 4, 3, 2, or one or fewer nucleotides. In some embodiments, the endonuclease used in the CRISPR gene editing system is MAD7 endonuclease, and the target sequence of the guide RNA in the CRISPR gene editing system includes SEQ ID NO: 24, and / or the guide RNA includes a sequence that differs from SEQ ID NO: 12 by 5, 4, 3, 2, or one or fewer nucleotides.

[0505] In some embodiments, the endonuclease used in the CRISPR gene editing system is a Cas9 endonuclease, and the target sequence of the guide RNA for the CRISPR gene editing system includes one of sequence numbers 14, 18, 19, 20, and 21, and / or the guide RNA includes a sequence that differs from one of sequence numbers 2, 6, 7, 8, and 9 by 5, 4, 3, 2, or one or fewer nucleotides. In some embodiments, the endonuclease used in the CRISPR gene editing system is a Cas9 endonuclease, and the target sequence of the guide RNA for the CRISPR gene editing system includes sequence number 17, and / or the guide RNA includes a sequence that differs from sequence number 5 by 5, 4, 3, 2, or one or fewer nucleotides. In some embodiments, the endonuclease used in the CRISPR gene editing system is Cas9 endonuclease, and the target sequence of the guide RNA in the CRISPR gene editing system includes either SEQ ID NO: 15 or 16, and / or the guide RNA includes a sequence that differs from SEQ ID NO: 3 or 4 by 5, 4, 3, 2, or one or fewer nucleotides. In some embodiments, the endonuclease used in the CRISPR gene editing system is Cas9 endonuclease, and the target sequence of the guide RNA in the CRISPR gene editing system includes SEQ ID NO: 62, and / or the guide RNA includes a sequence that differs from SEQ ID NO: 61 by 5, 4, 3, 2, or one or fewer nucleotides. In some embodiments, the endonuclease used in the CRISPR gene editing system is Cas9 endonuclease, and the target sequence of the guide RNA in the CRISPR gene editing system includes SEQ ID NO: 56, and / or the guide RNA includes a sequence that differs from SEQ ID NO: 55 by 5, 4, 3, 2, or one or fewer nucleotides. In some embodiments, the endonuclease used in the CRISPR gene editing system is a Cas9 endonuclease, the target sequence of the guide RNA of the CRISPR gene editing system includes SEQ ID NO: 59, and / or the guide RNA includes a sequence that differs from SEQ ID NO: 58 by 5, 4, 3, 2, or 1 or fewer nucleotides.

[0506] In some embodiments, the guide RNA includes SEQ ID NO: 5 (SOCS1), SEQ ID NO: 29 (SOCS1), SEQ ID NO: 11 (SOCS1), SEQ ID NO: 37 (SOCS1), SEQ ID NO: 27 (CISH), SEQ ID NO: 28 (CISH), SEQ ID NO: 12 (CISH), SEQ ID NO: 38 (CISH), SEQ ID NO: 2 (BIM), SEQ ID NO: 26 (BIM), SEQ ID NO: 6 (BIM), SEQ ID NO: 32 (BIM), SEQ ID NO: 7 (BIM), SEQ ID NO: 33 (BIM), SEQ ID NO: 8 (BIM), SEQ ID NO: 34 (BIM), SEQ ID NO: 9 (BIM), SEQ ID NO: 35 (BIM), SEQ ID NO: 10 (BIM), SEQ ID NO: 36 (BIM), SEQ ID NO: 61 (FAS), SEQ ID NO: 60 (FAS), SEQ ID NO: 55 (B2M), SEQ ID NO: 54 (B2M), SEQ ID NO: 58 (CIITA), or SEQ ID NO: 57 (CIITA).

[0507] In some embodiments, the guide RNA comprises a CRISPR RNA (crRNA) sequence, the crRNA sequence containing 2'-O-methyl modifications at each of the first three positions of the 5' end of the crRNA, and three 2'-O-methyl modifications at the 3' end, but not at the last base of the crRNA (for example, in the case of a 56-nucleotide crRNA, bases 53, 54, and 55 of the crRNA are modified, but base 56 is not; in the case of a 60-nucleotide crRNA, bases 57, 58, and 59 are modified, but base 60 is not). Furthermore, the crRNA sequence contains phosphorothioate bonds between each of the first three bases of the 5' end and phosphorothioate bonds between each of the last three bases of the 3' end. In some embodiments, the last base is modified to facilitate use with MAD7. In some embodiments, the guide RNA binds to MAD7. In some embodiments, the methyl modifications at the 5' end and the phosphorothioate bonds at the 3' end are for increasing the stability of the crRNA.

[0508] In some embodiments, the crRNA is approximately 56 nucleotides long. In some embodiments, the crRNA sequence is 56 nucleotides long and includes 2'-O-methyl modifications at positions 53, 54, and 55 of the 3' end of the crRNA sequence. In some embodiments, the crRNA sequence is 56 nucleotides long and includes 2'-O-methyl modifications at positions 53, 54, and 55 of the 3' end of the crRNA sequence and 2'-O-methyl modifications at positions 1, 2, and 3 of the 5' end. In some embodiments, the crRNA sequence is 56 nucleotides long and includes 2'-O-methyl modifications at positions 53, 54, and 55 of the 3' end of the crRNA sequence and 2'-O-methyl modifications at positions 1, 2, and 3 of the 5' end, phosphorothioate bonds between each base at the first three positions of the 5' end of the crRNA sequence and phosphorothioate bonds between each base at the last three positions of the 3' end. In some embodiments, the crRNA includes SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, which have the modification patterns described above. In some embodiments, the last nucleotide at the 3' end of the crRNA is modified with a nucleotide that fixes the binding of the crRNA to MAD7. In some embodiments, the last nucleotide at the 3' end of the crRNA is modified with a pseudoknot that fixes the binding of the crRNA to MAD7.

[0509] B. Programmable addition via site-specific targeting elements (PASTE) In some embodiments, the gene editing system used to modify the cells of this specification (e.g., iγδT cells, iPSCs, iHSCs, iCLP cells, or immature iT cells) is programmable addition (PASTE) via site-directed targeting elements (e.g., Arnall MTN, Ioannidi EI, Schmitt-Ulms C, Krajeski RN, Lim J, Villiger L, Zhou W, Jiang K, Garushyants SK, Roberts N, Zhang L, Vakulskas CA, Walker JA 2nd, Kadina AP, Zepeda AE, Holden K, Ma H, Xie J, Gao G, Foquet L, Bial G, Donnelly SK, Miyata Y, Radiloff DR, Henderson JM, Ujita A, Abudayyeh OO, Gootenberg JS. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat (See Biotechnol. 2023 Apr;41(4):500-512). The PASTE gene editing system comprises an attachment site-containing guide RNA (atgRNA) incorporating a guide RNA, for example, a prime editing guide RNA (pegRNA) sequence that designates a target site and codes for the desired genomic disruption, and an attachment site for serine integrase. PASTE further comprises a reverse transcriptase and CRISPR-Cas9 nickase fused with serine integrase. In some embodiments, the pegRNA targets SOCS1. In some embodiments, the pegRNA targets CISH. In some embodiments, the pegRNA targets BIM. In some embodiments, the pegRNA targets B2M. In some embodiments, the pegRNA targets CIITA. In some embodiments, the pegRNA targets FAS.In some embodiments, the PASTE gene is transfected into γδT cells used to produce modified T-iγδT cells, such as iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)).

[0510] C. Prime Editing In some embodiments, the gene editing system used to modify cells (e.g., iγδT cells, iPSCs, iHSCs, iCLP cells, or immature iT cells) as used herein is prime editing (see Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019 Dec;576(7785):149-157). The prime editing gene editing system includes, for example, a guide RNA containing a pegRNA that specifies a target site and encodes the desired genomic disruption, and a CRISPR-Cas9 endonuclease fused to a reverse transcriptase. In some embodiments, the pegRNA targets SOCS1. In some embodiments, the pegRNA targets CISH. In some embodiments, the pegRNA targets BIM. In some embodiments, the pegRNA targets B2M. In some embodiments, the pegRNA targets CIITA. In some embodiments, the pegRNA targets FAS. In some embodiments, the primed gene editing system is transfected into γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)) used to produce modified T-iγδT cells.

[0511] D. Base editing In some embodiments, the gene editing system used to modify cells (e.g., iγδT cells, iPSCs, iHSCs, iCLP cells, or immature iT cells) as described herein is base editing (see Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage. Nature. 2017 Nov 23;551(7681):464-471). Base editing comprises a base editor, including, for example, a cytosine base editor or an adenine base editor, and a guide sequence used to target the base editor to a specific nucleotide sequence. The base editor comprises Cas9 nickase and a nucleoside deaminase (e.g., cytosine deaminase or adenine deaminase). Cas9 nickase cleaves one strand of DNA in the cell's genome at a specific location determined by a guide sequence, and nucleoside deaminase removes an amino group from a specific type of nucleoside (e.g., cytosine or adenine). In some embodiments, the guide sequence targets SOCS1. In some embodiments, the guide sequence targets CISH. In some embodiments, the guide sequence targets BIM. In some embodiments, the sequence targets B2M. In some embodiments, the guide sequence targets CIITA. In some embodiments, the guide sequence targets FAS. In some embodiments, the base-editing gene-editing system is transfected into γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)) used to create modified T-iγδT cells.

[0512] E. Transcription activator-like effector nucleases (TALENs) In some embodiments, the gene editing system used to modify cells (e.g., iγδT cells, iPSCs, iHSCs, iCLP cells, or immature iT cells) as described herein is a transcription activator-like effector nuclease (TALEN) (see Christian M, Cermark T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010;186:757-761; and Miller JC, Tan S, Qiao G, Barlow KA, et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 2011;29:143-148). Furthermore, see Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucl Acids Res. 2011;39:9283-9293). A TALEN gene editing system comprises a TALEN, which is a restriction endonuclease (e.g., Fokl) fused to a transcription activator-like effector (TALE) engineered to bind to a desired DNA sequence and promote DNA cleavage at a specific site. In some embodiments, two different TALENs each target different regions of SOCS1. In some embodiments, two different TALENs each target distinct regions of CISH. In some embodiments, two different TALENs each target different regions of BIM. In some embodiments, two different TALENs each target different regions of B2M. In some embodiments, two different TALENs each target different regions of CIITA.In some embodiments, two different TALENs target different regions of the FAS. In some embodiments, the TALENs are transfected into γδT cells used to create modified T-iγδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)).

[0513] F. zinc finger nuclease (ZFN) In some embodiments, the gene editing system used to modify cells as described herein (e.g., iγδT cells, iPSCs, iHSCs, iCLP cells, or immature iT cells) is a zinc finger nuclease (ZFN) (see, e.g., Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: Zinc finger fusions to FokI cleavage domain. Proc Natl Acad Sci USA. 1996;93:1156-1160). In some embodiments, the ZFN comprises an endonuclease (e.g., FokI) fused to a zinc finger, with each zinc finger domain of the zinc finger targeting a sequence. In some embodiments, two different ZFNs each target different regions of SOCS1. In some embodiments, two different ZFNs each target different regions of CISH. In some embodiments, two different ZFNs each target different regions of BIM. In some embodiments, two different ZFNs each target different regions of B2M. In some embodiments, two different ZFNs each target different regions of CIITA. In some embodiments, two different ZFNs each target different regions of FAS. In some embodiments, the ZFNs are transfected into γδT cells used to produce modified T-iγδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)).

[0514] G. endonuclease This specification describes restriction endonucleases for use in the methods described herein and for use as components of gene editing systems. For example, homing endonucleases and meganucleases are provided. In some embodiments, meganucleases, restriction endonucleases, and / or homing endonucleases are operated to confer genomic disruption or loss-of-function nucleic acid mutations to BIM, SOCS, and CISH; SOCS1 and CISH, and optionally one or more of BIM, B2M, CIITA, and / or FAS in cells of this specification (e.g., iγδT cells, iPSCs, iHSCs, iCLP cells, or immature iT cells). In some embodiments, one meganuclease targets SOCS1 and a second meganuclease targets CISH. In some embodiments, a third meganuclease targets BIM. In some embodiments, a fourth meganuclease targets B2M. In some embodiments, a fifth meganuclease targets CIITA. In some embodiments, a sixth meganuclease targets FAS. In some embodiments, one restriction endonuclease targets SOCS1 and a second restriction endonuclease targets CISH. In some embodiments, a third restriction endonuclease targets BIM. In some embodiments, a fourth restriction endonuclease targets B2M. In some embodiments, a fifth restriction endonuclease targets CIITA. In some embodiments, a sixth restriction endonuclease targets FAS. In some embodiments, one homing endonuclease targets SOCS1 and a second restriction endonuclease targets CISH. In some embodiments, a third homing endonuclease targets BIM. In some embodiments, a fourth homing endonuclease targets B2M. In some embodiments, a fifth homing endonuclease targets CIITA. In some embodiments, a sixth homing endonuclease targets the FAS.In some embodiments, restriction endonucleases, meganucleases, and / or homing endonucleases are transfected into γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)) used to produce modified T-iγδT cells.

[0515] H. Transfection This disclosure provides methods for modifying cells used herein (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) by transfection, including, for example, electroporation (e.g., Nucleofection® (Lonza Cologne GmbH, Cologne Germany)), physical transfection (e.g., microinjection, particle bombardment, or phototransfection), lipid-mediated transfection, diethylaminoethyl (DEAE)-dextran transfection, calcium phosphate precipitation, cationic polymer transfection, or viral transfection.

[0516] Cells can be transfected (e.g., nucleofected) with a gene editing system to induce genome disruption or loss-of-function nucleic acid mutations. In some embodiments, the gene editing system comprises, for example, an endonuclease containing Cas9 or MAD7 and an sgRNA targeting SOCS1, CISH, BIM, B2M, CIITA, or FAS. In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are subjected to sequential transfection with the gene editing system. For example, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and an sgRNA targeting SOCS1, and then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting CISH and, optionally, a second endonuclease or the first endonuclease. For example, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and a CISH-targeting sgRNA, and then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a SOCS1-targeting sgRNA and, optionally, a second endonuclease or the first endonuclease.

[0517] In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and an sgRNA targeting SOCS1; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting CISH and optionally with a second endonuclease or the first endonuclease; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting BIM and optionally with a third endonuclease, a second endonuclease, and / or the first endonuclease. In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and a CISH-targeting sgRNA; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a SOCS1-targeting sgRNA and optionally a second endonuclease or the first endonuclease; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a BIM-targeting sgRNA and optionally a third endonuclease, a second endonuclease, and / or the first endonuclease.In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and an sgRNA targeting SOCS1; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting BIM and optionally a second endonuclease or the first endonuclease; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting CISH and optionally a third endonuclease, a second endonuclease, and / or the first endonuclease. In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and a CISH-targeting sgRNA; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a BIM-targeting sgRNA and optionally a second endonuclease or the first endonuclease; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a SOCS1-targeting sgRNA and optionally a third endonuclease, a second endonuclease, and / or the first endonuclease.In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and a BIM-targeting sgRNA; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a SOCS1-targeting sgRNA and optionally a second endonuclease or the first endonuclease; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a CISH-targeting sgRNA and optionally a third endonuclease, a second endonuclease, and / or the first endonuclease. In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and a BIM-targeting sgRNA; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a CISH-targeting sgRNA and optionally a second endonuclease or the first endonuclease; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a SOCS1-targeting sgRNA and optionally a third endonuclease, a second endonuclease, and / or the first endonuclease.

[0518] In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and an sgRNA targeting SOCS1; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting CISH and optionally with a second endonuclease or the first endonuclease; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting FAS and optionally with a third endonuclease, a second endonuclease, and / or the first endonuclease. In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and a CISH-targeting sgRNA; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a SOCS1-targeting sgRNA and optionally a second endonuclease or the first endonuclease; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a FAS-targeting sgRNA and optionally a third endonuclease, a second endonuclease, and / or the first endonuclease.In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and an sgRNA targeting SOCS1; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting FAS and optionally a second endonuclease or the first endonuclease; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting CISH and optionally a third endonuclease, a second endonuclease, and / or the first endonuclease. In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and a CISH-targeting sgRNA; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a FAS-targeting sgRNA and optionally a second endonuclease or the first endonuclease; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a SOCS1-targeting sgRNA and optionally a third endonuclease, a second endonuclease, and / or the first endonuclease.In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and an sgRNA targeting FAS; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting SOCS1 and optionally a second endonuclease or the first endonuclease; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting CISH and optionally a third endonuclease, a second endonuclease, and / or the first endonuclease. In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and an sgRNA targeting FAS; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting CISH and optionally with a second endonuclease or the first endonuclease; then cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting SOCS1 and optionally with a third endonuclease, a second endonuclease, and / or the first endonuclease.

[0519] In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and an sgRNA targeting SOCS1, and further cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting CISH and optionally a second endonuclease or the first endonuclease, and further cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with an sgRNA targeting BIM and optionally a third endonuclease, the second endonuclease, and It transfects the cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) with sgRNA targeting B2M, and optionally with a fourth, third, second, or first endonuclease, and further transfects the cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) with sgRNA targeting CIITA, and optionally with a fifth, fourth, third, second, and / or first endonuclease. In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with SOCS1-targeting sgRNA, CISH-targeting sgRNA, BIM-targeting sgRNA, B2M-targeting sgRNA, and CIITA-targeting sgRNA in any order. In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease, a second endonuclease, a third endonuclease, a fourth endonuclease, a fifth endonuclease, or any combination thereof in any order.

[0520] In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease and SOCS1-targeting sgRNA, and further cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with CISH-targeting sgRNA and optionally a second endonuclease or the first endonuclease. Transfect cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) with sgRNA targeting BIM, and optionally a third endonuclease, a second endonuclease, or a first endonuclease, and further transfect cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) with B2M. The target sgRNA is transfected with the fourth, third, second, or first endonuclease, and then the cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with the sgRNA targeting CIITA and, if applicable, the fifth, fourth, third, or second endonuclease. Transfect with crease and / or the first endonuclease, and further transfect cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) with sgRNA targeting FAS, and optionally with the sixth, fifth, fourth, third, second endonuclease, and / or the first endonuclease.In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with SOCS1-targeting sgRNA, CISH-targeting sgRNA, BIM-targeting sgRNA, B2M-targeting sgRNA, CIITA-targeting sgRNA, B2M-targeting sgRNA, and FAS-targeting sgRNA in any order. In some embodiments, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) are transfected with a first endonuclease, a second endonuclease, a third endonuclease, a fourth endonuclease, a fifth endonuclease, a sixth endonuclease, or any combination thereof in any order.

[0521] VI. Method for producing modified cells In some embodiments, methods are provided for inhibiting the expression of one or more of the SOCS1 gene, CISH gene, and BIM gene in γδT cells, for example, iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))).

[0522] In some embodiments, methods are provided for inhibiting the expression of the SOCS1 gene and the CISH gene in γδT cells, for example, iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))). In some embodiments, methods are provided for inhibiting the expression of the SOCS1 gene, the CISH gene, and the BIM gene in γδT cells, for example, iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))). In some embodiments, methods are provided for inhibiting the expression of the SOCS1 gene, CISH gene, BIM gene, and FAS gene in γδT cells, for example, iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))). In some embodiments, methods are provided for inhibiting the expression of the SOCS1 gene, CISH gene, BIM gene, B2M gene, and CIITA gene in γδT cells, for example, iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))). In some embodiments, methods are provided for inhibiting the expression of the SOCS1 gene, CISH gene, BIM gene, B2M gene, CIITA gene, and FAS gene in γδT cells, for example, iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))). In some embodiments, the method suppresses or eliminates the expression of SOCS1 and CISH. In some embodiments, the method suppresses or eliminates the expression of SOCS1, CISH, and BIM.In some embodiments, the method suppresses or eliminates the expression of SOCS1, CISH, BIM, and FAS. In some embodiments, the method suppresses or eliminates the expression of SOCS1, CISH, BIM, B2M, and FAS. In some embodiments, the method suppresses or eliminates the expression of SOCS1, CISH, BIM, B2M, CIITA, and FAS. In some embodiments, BIM. EL and BIM L The expression level of splice variants is reduced in modified cells compared to unmodified controls (e.g., unmodified T-iγδT cells). In some embodiments, BIM S The expression level of splice variants is maintained in modified cells.

[0523] This specification describes methods for inhibiting the expression of SOCS1 and / or CISH genes in γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)) used to produce modified T-iγδT cells. The methods involve using a gene editing system (e.g., a gene editing system described in Section V) to modify γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)). For example, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLP cells, or immature T-iγδT cells) can be brought into contact with a guide RNA and an endonuclease or nucleic acid encoding an endonuclease, and such contact results in genomic disruption within the endogenous SOCS1 gene, which suppresses or eliminates SOCS1 expression, and / or genomic disruption within the endogenous CISH gene, which suppresses or eliminates CISH expression. In some embodiments, genomic disruption within SOCS1 and / or CISH is performed by administering the guide RNA to the cells of this specification (e.g., iγδT cells, iPSCs, iHSCs, iCLP cells, or immature iT cells). In some embodiments, the guide RNA is sgRNA. In some embodiments, the guide RNA comprises one or more of SEQ ID NOs: 5, 29, 11, 37 and / or one or more of SEQ ID NOs: 3, 27, 4, 28, 12, or 38. In some embodiments, the endonuclease administered to the cells is a Cas endonuclease. In some embodiments, the endonuclease administered to the cells is a MAD7 endonuclease. The MAD7 endonuclease is an engineered class 2 type VA CRISPR-Cas(Cas12a / Cpf1) system.

[0524] This specification describes methods for inhibiting the expression of the BIM gene in γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)) used to produce modified T-iγδT cells. The methods involve using a gene editing system (e.g., a gene editing system described in Section V) to modify γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)). For example, cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) can be brought into contact with guide RNA and an endonuclease or nucleic acid encoding an endonuclease, and such contact results in genomic disruption within the endogenous BIM gene that suppresses or eliminates BIM expression, and the genomic disruption is located in exon 2C of the endogenous BIM gene, and BIM EL and BIM L The expression level and / or function of splice variants are reduced in modified cells compared to unmodified controls, and BIM S The expression level and / or function of the splice variant are preserved in the modified cells. In some embodiments, genomic disruption within the BIM is performed by administering a guide RNA to the cells of this specification (e.g., iγδT cells, iPSCs, iHSCs, iCLP cells, or immature iT cells). In some embodiments, the guide RNA is an sgRNA. In some embodiments, the guide RNA comprises one or more of SEQ ID NOs: 2, 26, 8, 34, 9, 35, 10, and 36. In some embodiments, the endonuclease administered to the cells is a Cas endonuclease. In some embodiments, the endonuclease administered to the cells is a MAD7 endonuclease. The MAD7 endonuclease is an engineered class 2 type VA CRISPR-Cas (Cas12a / Cpf1) system.

[0525] This specification describes methods for inhibiting the expression of SOCS1, CISH, and BIM genes by inducing gene disruption within the SOCS1, CISH, and BIM genes in γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)). The methods include using a gene editing system (e.g., a gene editing system described in Section V) to modify γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)). For example, cells of this specification (e.g., iγδT cells, iPSCs, iHSCs, iCLPs, or immature iT cells) can be brought into contact with guide RNA and an endonuclease or nucleic acid encoding an endonuclease, and such contact results in genomic disruption within the endogenous SOCS1 gene, which suppresses or eliminates SOCS1 expression; genomic disruption within the endogenous CISH gene, which suppresses or eliminates CISH expression; and genomic disruption within the endogenous BIM gene, which suppresses or eliminates BIM expression. In some embodiments, BIM EL and BIM L The expression level and / or function of splice variants are reduced in modified γδT cells compared to unmodified γδT cells, or compared to pre-modification γδT cells (e.g., T-iγδT). In some embodiments, BIM S The expression level and / or function of the splice variant are preserved in modified γδT cells.

[0526] This specification describes methods for inhibiting the expression of SOCS1, CISH, BIM, and FAS genes by inducing gene disruption within the SOCS1, CISH, BIM, and FAS genes in γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)). The methods include using a gene editing system (e.g., a gene editing system described in Section V) to modify γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)). For example, cells of this specification (e.g., iγδT cells, iPSCs, iHSCs, iCLPs, or immature iγδT cells) can be brought into contact with guide RNA and an endonuclease or nucleic acid encoding an endonuclease, and such contact results in genomic disruption within the endogenous SOCS1 gene, which suppresses or eliminates SOCS1 expression; genomic disruption within the endogenous CISH gene, which suppresses or eliminates CISH expression; genomic disruption within the endogenous BIM gene, which suppresses or eliminates BIM expression; and genomic disruption within the endogenous FAS gene, which suppresses or eliminates FAS expression. In some embodiments, BIM EL and BIM L The expression level and / or function of splice variants are reduced in modified γδT cells compared to unmodified γδT cells, or compared to pre-modification γδT cells (e.g., T-iγδT). In some embodiments, BIM S The expression level and / or function of the splice variant are preserved in modified γδT cells.

[0527] This specification describes methods for inhibiting the expression of SOCS1, CISH, BIM, B2M, and CIITA genes by inducing gene disruption within the SOCS1, CISH, BIM, B2M, and CIITA genes in γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)). The methods involve using a gene editing system to modify γδT cells, e.g., iPSC-derived γδT cells, and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells)). For example, the cells of this specification (e.g., iγδT cells, iPSCs, iHSCs, iCLPs, or immature iT cells) can be brought into contact with a guide RNA and an endonuclease or nucleic acid encoding an endonuclease, and such contact results in genomic disruption within the endogenous SOCS1 gene, which suppresses or eliminates the expression of SOCS1; genomic disruption within the endogenous CISH gene, which suppresses or eliminates the expression of CISH; genomic disruption within the endogenous BIM gene, which suppresses or eliminates the expression of BIM; genomic disruption within the endogenous B2M gene, which suppresses or eliminates the expression of B2M; and genomic disruption within the endogenous CIITA gene, which suppresses or eliminates the expression of CIITA. In some embodiments, BIM EL and BIM L The expression level and / or function of splice variants are reduced in modified γδT cells compared to unmodified γδT cells, or compared to pre-modification γδT cells (e.g., T-iγδT). In some embodiments, BIM S The expression level and / or function of the splice variant are preserved in modified γδT cells.

[0528] Described herein is a method of inhibiting the expression of SOCS1, CISH, BIM, B2M, CIITA, and FAS genes by causing genetic disruption within the SOCS1, CISH, BIM, B2M, CIITA, and FAS genes of γδ T cells, e.g., iPSC-derived γδ T cells, and progenitor cells thereof (e.g., iPSCs (e.g., γδ T-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδ T-iHSCs, γδ T-iCLPs, and immature T-iγδ T cells)). The method comprises using a gene editing system to modify γδ T cells, e.g., iPSC-derived γδ T cells, and progenitor cells thereof (e.g., iPSCs (e.g., γδ T-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδ T-iHSCs, γδ T-iCLPs, and immature T-iγδ T cells)). For example, a cell herein (e.g., an iγδ T cell, iPSC, iHSC, iCLP, or immature iT cell) can be contacted with a guide RNA and an endonuclease or a nucleic acid encoding an endonuclease, wherein said contacting results in genomic disruption within the endogenous SOCS1 gene that suppresses or eliminates SOCS1 expression, genomic disruption within the endogenous CISH gene that suppresses or eliminates CISH expression, genomic disruption within the endogenous BIM gene that suppresses or eliminates BIM expression, genomic disruption within the endogenous B2M gene that suppresses or eliminates B2M expression, genomic disruption within the endogenous CIITA gene that suppresses or eliminates CIITA expression, and genomic disruption within the endogenous FAS gene that suppresses or eliminates FAS expression. In some embodiments, BIM EL and BIM L splice variant expression level and / or function is reduced in modified γδ T cells compared to unmodified γδ T cells, or compared to γδ T cells before modification (e.g., T-iγδ T cells). In some embodiments, BIM S splice variant expression level and / or function is retained in the modified γδ T cells.

[0529] In some embodiments, genomic disruption and / or loss-of-function mutations of the SOCS1 and BIM genes, SOCS1, CISH, and BIM genes, SOCS1, CISH, BIM, and FAS genes, SOCS1, CISH, BIM, B2M, and CIITA genes, or SOCS1, CISH, BIM, B2M, CIITA, and FAS genes are performed by administering guide RNA to cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells). In some embodiments, the guide RNA is sgRNA. In some embodiments, guide RNA targeting the SOCS1 gene includes one or more of SEQ ID NOs: 5, 29, 11, and 37; guide RNA targeting the CISH gene includes one or more of SEQ ID NOs: 3, 27, 4, 28, 12, or 38; guide RNA targeting the BIM gene includes one or more of SEQ ID NOs: 2, 26, 6, 32, 7, 33, 8, 34, 9, 35, 10, and 36; guide RNA targeting the FAS gene includes one or more of SEQ ID NOs: 60 and 61; guide RNA targeting the B2M gene includes one or more of SEQ ID NOs: 54 and 55; and guide RNA targeting the CIITA gene includes one or more of SEQ ID NOs: 57 and 58. In some embodiments, the nuclease is administered to cells (e.g., T-iγδT cells, γδT-iPSCs, T-iHSCs, T-iCLPs, or immature T-iγδT cells) together with the guide RNA. In some embodiments, the nuclease is an endonuclease. In some embodiments, the endonuclease administered to the cells is a Cas endonuclease. In some embodiments, the endonuclease administered to the cells is a Cas9 endonuclease or a MAD7 endonuclease.

[0530] In some embodiments, genomic disruption of the endogenous SOCS1 gene suppresses or eliminates SOCS1 gene expression in modified γδT cells (e.g., T-iγδT cells) compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or pre-modification γδT cells (e.g., T-iγδT cells). In some embodiments, the genomic disruption within the SOCS1 gene is located in exon 2 of the SOCS1 gene. In some embodiments, the genomic disruption within the SOCS1 gene is a loss-of-function nucleic acid mutation. In some embodiments, the genomic disruption within the SOCS1 gene is a loss-of-function nucleic acid mutation in exon 2 of the SOCS1 gene. In some embodiments, the genomic disruption or loss-of-function nucleic acid mutation within the SOCS1 gene is an insertion, deletion, substitution, or any combination thereof. In some embodiments, the genome disruption occurs within a target sequence in SOCS1 that includes one of the nucleotide sequences of SEQ ID NO: 17, SEQ ID NO: 23, or a nucleotide sequence having at least 70% identity with them (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0531] In some embodiments, genomic disruption of the endogenous CISH gene suppresses or eliminates the expression of the CISH gene in modified γδT cells (e.g., T-iγδT cells) compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or pre-modification γδT cells (e.g., T-iγδT cells). In some embodiments, the genomic disruption within the CISH gene is located in exon 3, exon 4, or a combination thereof. In some embodiments, the genomic disruption within the CISH gene is a loss-of-function nucleic acid mutation. In some embodiments, the genomic disruption within the CISH gene is a loss-of-function nucleic acid mutation located in exon 3, exon 4, or a combination thereof. In some embodiments, the genomic disruption or loss-of-function nucleic acid mutation within the CISH gene is an insertion, deletion, substitution, or any combination thereof. In some embodiments, the genome disruption occurs within a target sequence in CISH containing any one of the nucleotide sequences of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 24, or a nucleotide sequence having at least 70% identity with them (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0532] In some embodiments, genomic disruption of the endogenous BIM gene in modified γδT cells results in a splice variant of BIM. EL The expression level and / or function of is reduced compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or pre-modification γδT cells (e.g., T-iγδT cells) derived from γδT-iPSCs. In some embodiments, genomic disruption of the endogenous BIM gene reduces the expression level and / or function of BIM, which is a splice variant of BIM. EL The expression level and / or function of is reduced compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or pre-modification γδT cells (e.g., T-iγδT cells) derived from γδT-iPSCs. In some embodiments, genomic disruption of the endogenous BIM gene in modified γδT cells results in a splice variant of BIM, BIM EL and BIML The expression level and / or function of is reduced compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or pre-modification γδT cells (e.g., T-iγδT cells) derived from γδT-iPSCs. In some embodiments, genomic disruption of the endogenous BIM gene in modified γδT cells results in a splice variant of BIM, BIM s The expression level and / or function of the gene are preserved compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or pre-modification γδT cells (e.g., T-iγδT cells) derived from γδT-iPSCs. In some embodiments, the genomic disruption of the BIM gene is located in exon 2C of the endogenous BIM gene. In some embodiments, the genomic disruption of the BIM gene is a loss-of-function nucleic acid mutation within exon 2C of the endogenous BIM gene. In some embodiments, the loss-of-function nucleic acid mutation is an insertion, deletion, or substitution. In some embodiments, the genome disruption occurs within a target sequence in the BIM that includes any one nucleotide sequence from SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, or a nucleotide sequence having at least 70% identity with these (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0533] In some embodiments, genomic disruption of the endogenous FAS gene suppresses or eliminates FAS gene expression in modified γδT cells (e.g., T-iγδT cells) compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or pre-modification γδT cells (e.g., T-iγδT cells). In some embodiments, the genomic disruption within the FAS gene is located in exon 1 of the FAS gene. In some embodiments, the genomic disruption within the FAS gene is a loss-of-function nucleic acid mutation. In some embodiments, the genomic disruption within the FAS gene is a loss-of-function nucleic acid mutation in exon 1 of the FAS gene. In some embodiments, the genomic disruption or loss-of-function nucleic acid mutation within the FAS gene is an insertion, deletion, substitution, or any combination thereof. In some embodiments, the genome disruption occurs within a target sequence in the FAS that contains the nucleotide sequence of SEQ ID NO: 62, or a nucleotide sequence having at least 70% identity thereto (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0534] In some embodiments, genomic disruption of the endogenous B2M gene suppresses or eliminates B2M gene expression in modified γδT cells (e.g., T-iγδT cells) compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or pre-modification γδT cells (e.g., T-iγδT cells). In some embodiments, the genomic disruption within the B2M gene is located in exon 2 of the B2M gene. In some embodiments, the genomic disruption within the B2M gene is a loss-of-function nucleic acid mutation. In some embodiments, the genomic disruption within the B2M gene is a loss-of-function nucleic acid mutation in exon 2 of the B2M gene. In some embodiments, the genomic disruption or loss-of-function nucleic acid mutation within the B2M gene is an insertion, deletion, substitution, or any combination thereof. In some embodiments, the genome disruption occurs within a target sequence in B2M that includes the nucleotide sequence of SEQ ID NO: 56, or a nucleotide sequence having at least 70% identity thereto (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0535] In some embodiments, genomic disruption of the endogenous CIITA gene suppresses or eliminates CIITA gene expression in modified γδT cells (e.g., T-iγδT cells) compared to unmodified γδT cells (e.g., unmodified T-iγδT cells) or pre-modification γδT cells (e.g., T-iγδT cells). In some embodiments, the genomic disruption within the CIITA gene is located in exon 3 of the CIITA gene. In some embodiments, the genomic disruption within the CIITA gene is a loss-of-function nucleic acid mutation. In some embodiments, the genomic disruption within the CIITA gene is a loss-of-function nucleic acid mutation in exon 3 of the CIITA gene. In some embodiments, the genomic disruption or loss-of-function nucleic acid mutation within the CIITA gene is an insertion, deletion, substitution, or any combination thereof. In some embodiments, the genome disruption occurs within a target sequence in CIITA that includes the nucleotide sequence of SEQ ID NO: 59, or a nucleotide sequence having at least 70% identity thereto (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).

[0536] In some embodiments, genomic disruption or loss-of-function nucleic acid mutations within the SOCS1, CISH, BIM, B2M, CIITA, and / or FAS genes are insertions, deletions, substitutions, or any combination thereof.

[0537] In some embodiments, the method involves contacting cells with a guide RNA and a nuclease or nucleic acid encoding the nuclease, wherein the contact results in genomic disruption within the endogenous SOCS1 gene, which suppresses or eliminates SOCS1 expression, and genomic disruption within the endogenous CISH gene, which suppresses or eliminates CISH expression. If BIM is also to be disrupted, a guide RNA that inhibits the expression of the endogenous BIM gene is used. If B2M is also to be disrupted, a guide RNA that inhibits the expression of the endogenous B2M gene is used. If CIITA is also to be disrupted, a guide RNA that inhibits the expression of the endogenous CIITA gene is used. If FAS is also to be disrupted, a guide RNA that inhibits the expression of the endogenous FAS gene is used.

[0538] Further information regarding guide RNA and endonucleases can be found in other sections of this disclosure, for example, Section V: Gene Editing Systems.

[0539] Further information regarding the cells used in the inhibitory methods can be found in other sections of this disclosure, for example, Section II: Modified Cells and Section III: Modified Cells Including CARs, e.g., Anti-CD19 CARs.

[0540] In some embodiments, a method is provided for introducing one or more exogenous polynucleotides encoding IFNγ signaling factors into γδT cells, for example, iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))).

[0541] In some embodiments, a method is provided for introducing one or more exogenous polynucleotides encoding anti-CD19 CAR, B2M-HLA-E, and IFNγ signaling converter into γδT cells, for example, iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))).

[0542] In some embodiments, the introduction of one or more exogenous polynucleotides into γδT cells, e.g., iPSC-derived γδT cells (and their progenitor cells (e.g., iPSCs (e.g., γδT-iPSCs), or intermediate cells differentiated from iPSCs (e.g., γδT-iHSCs, γδT-iCLPs, and immature T-iγδT cells))) is performed by knock-in. In some embodiments, the knock-in is a targeted knock-in obtainable by a CRISPR-Cas system (e.g., Cas9, Cas12, or MAD7) targeting a genomic locus of interest, and is combined with a donor template containing an exogenous polynucleotide that is incorporated into the genomic locus of interest by homologous recombination repair.

[0543] In some embodiments, this disclosure provides modified cells prepared by methods of inhibiting expression and / or introducing exogenous polynucleotides as described herein.

[0544] This disclosure provides a method for producing modified γδT cells, comprising (i) producing modified iPSC cells as described herein (e.g., sections II and III), and (ii) differentiating the modified iPSC cells into modified γδT cells.

[0545] VII. Pharmaceutical Compositions Pharmaceutical compositions comprising either modified cells or populations of cells (e.g., modified γδT) are also included. In some embodiments, the pharmaceutical composition comprises modified cells or populations of cells (e.g., modified γδT) and a pharmaceutically acceptable carrier.

[0546] VIII. Treatment method This specification describes methods for treating diseases and disorders using modified cells, cell populations, or pharmaceutical compositions described herein.

[0547] In some embodiments, the Disclosure provides a method for killing CD19-positive B cells having abnormal B-cell function, comprising administering a therapeutically effective amount of the modified γδT cells or the pharmaceutical composition described herein to a target subject of interest. In some embodiments, the B cells are CD19-positive B cells from a patient with diffuse large B-cell lymphoma (DLBCL) or systemic lupus erythematosus (SLE).

[0548] In some embodiments, the present disclosure provides a method for treating DLBCL or SLE, comprising administering a therapeutically effective amount of modified γδT cells or a pharmaceutical composition described herein to a subject of interest.

[0549] In some embodiments, this disclosure also provides modified γδT cells or pharmaceutical compositions described herein for therapeutic use.

[0550] In some embodiments, the Disclosure also provides modified γδT or pharmaceutical compositions described herein for use in the treatment of DLBCL or SLE.

[0551] In some embodiments, this disclosure provides the use of modified cells (e.g., modified γδT cells, modified iPSC cells as described herein) or pharmaceutical compositions as described herein in the manufacture of agents for the treatment of DLBCL or SLE.

[0552] The abbreviation "e.g., (for example)" derives from the Latin phrase exempli gratia, and is used in the present specification to indicate non-limiting examples. Accordingly, the abbreviation "e.g., (for example)" is synonymous with the term "for example". The abbreviation "i.e., (that is)" derives from the Latin phrase id est, and is used in the present specification to indicate a non-limiting rephrasing or clarification. Accordingly, the abbreviation "i.e., (that is)" is synonymous with the term "that is".

[0553] Section headings, materials, methods, and examples are illustrative only and not intended to be limiting.

[0554] Examples Example 1. CISH - / - SOCS1 - / - BIM - / - Triple Knockout (TKO) TiPSC- Preparation of γδ T cells Example 1.1 Methods Nucleofection To generate target gene knockouts in T-iγδT cells obtained from Vδ2 γδT-derived iPSCs (iPSC-derived γδT, wherein the iPSCs are generated from primary Vδ2 γδT cells), 5 × 10 6T-iγδT cells were sequentially nucleofected in each Nucleocuvette container using 200 pmol of Alt-R® Sp HiFi Cas9 nuclease V3 (Integrated DNA Technologies (Coralville, IA), catalog number 1081060) and 1,000 pmol of synthetic single guide RNA (sgRNA) with Lonza P3 primary cell 4D-Nucleofector® X kit L (Lonza (Basel, Switzerland), catalog number V4XP-3024) and Lonza 4D Nucleofector® core and X unit (Lonza, catalog number AAF-1003X). To generate double knockout (DKO) or triple knockout (TKO) T-iγδT cells, the T-iγδT cells were sequentially nucleofected to ensure the knockout efficiency of each target gene. Nucleofected T-iγδ T cells were administered 1 × 10¹⁶ doses every two days before the next round of nucleofection in medium supplemented with recombinant human IL-7 (10 ng / mL; PeproTech (Cranbury, NJ), catalog number: 200-07) and recombinant human IL-15 (10 ng / mL, PeproTech, catalog number: 200-15) (STEMdiffAPEL2 medium (STEMCELL, catalog number: 05275) supplemented with penicillin-streptomycin (100 U / mL, Gibco, catalog number: 15140-122) and AA2P (50 ng / mL, Cayman), in a medium containing 1 × 10¹⁶ doses every two days prior to the next round of nucleofection. 6 The cells were subcultured at a cell density of cells / mL.

[0555] Example 1.2 Nucleofection of T cells by T-iγδT Cas9 nuclease and sgRNA To generate single, double, or triple target gene knockout T-iγδT cells, 5 × 10 6Each T-iγδT cell was sequentially nucleofected in each Nucleocuvette container using Lonza P3 primary cell 4D-Nucleofector® X kit L (Lonza, catalog number V4XP-3024) and Lonza 4D Nucleofector® core and X unit (Lonza, catalog number AAF-1003X) with 200 pmol of Alt-R® Sp.HiFi Cas9 nuclease V3 (Integrated DNA Technologies, catalog no. 1081060) and 1,000 pmol of synthetic single guide RNA (sgRNA) as listed in Table 1. Each target gene was knocked out with one synthetic sgRNA, except for CISH knocked out using two sgRNAs (CISH #3 and #4) simultaneously. To ensure the knockout efficiency of each target gene, T-iγδT cells were sequentially nucleofected. Since the cytotoxic effect and viability of T-iγδT cells are affected by nucleofection, all groups were subjected to a total of three rounds of nucleofection. In the single knockout group, T-iγδT cells were nucleofected with target gene sgRNA(s) in the first round of nucleofection, and then scrambled sgRNA(s) were nucleofected in the second and third rounds of nucleofection.For TKO / DKO, T-iγδT cells were sequentially nucleofected in the order shown in parentheses in Figure 1 and below: In TKO (BIM+SOCS1+CISH), T-iγδT cells were nucleofected with sgRNA targeting BIM, then sgRNA targeting SOCS1, and finally sgRNA targeting CISH. In TKO (SOCS1+BIM+CISH), T-iγδT cells were nucleofected with sgRNA targeting SOCS1, then BIM. In DKO (SOCS1+CISH), T-iγδT cells are nucleofected with SOCS1-targeted sgRNA, followed by CISH-targeted sgRNA. In TKO (SOCS1+CISH+BIM), T-iγδT cells are nucleofected with SOCS1-targeted sgRNA, followed by CISH-targeted s...

Claims

1. A modified iPSC, γδT cell, or intermediate cell differentiated from an iPSC, comprising: genomic disruption within the endogenous SOCS1 gene that suppresses or eliminates the expression of functional cytokine signaling repressor 1 (SOCS1) protein; and genomic disruption within the endogenous CISH gene that suppresses or eliminates the expression of functional cytokine-inducible SH2-containing protein (CISH) protein.

2. The modified cell according to claim 1, wherein the genome disruption is located in exon 2 of the SOCS1 gene, and optionally the genome disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

3. The modified cell according to claim 1 or 2, wherein the genome disruption is located in exon 3 or exon 4 of the CISH gene, and optionally the genome disruption is a loss-of-function nucleic acid mutation, and optionally the mutation is an insertion, deletion, or substitution.

4. The modified cell according to any one of claims 1 to 3, wherein the genome disruption is located within a target sequence in SOCS1 including SEQ ID NO: 17 or SEQ ID NO: 23, and / or the genome disruption is located within a target sequence in CISH including SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:

24.

5. The modified cell according to any one of claims 1 to 4, wherein the genome disruption in SOCS1 and CISH is carried out by administering a guide RNA to the cell, and optionally the guide RNA is a single guide RNA (sgRNA) or crRNA, and an endonuclease or nucleic acid encoding the endonuclease is also administered to the cell.

6. The modified cell according to claim 5, wherein the guide RNA includes one or more guide sequences from sequence numbers 5, 11, 3, 4, and 12, or one or more sgRNA or crRNA sequences from sequence numbers 29, 37, 27, 28, and 38.

7. A modified cell according to any one of claims 1 to 6, further comprising genomic disruption within an endogenous Bcl-2-interacting cell death mediator (BIM) gene.

8. The modified cell according to claim 7, wherein the genome disruption within the BIM gene is located within exon 2C of the BIM gene.

9. BIM ＥＬ and BIM Ｌ The expression level and / or function of the splice variant were reduced in the modified cells compared to the unmodified control, and BIM Ｓ The modified cell according to claim 7 or 8, wherein the expression level and / or function of the splice variant are maintained in the modified cell.

10. The modified cell according to any one of claims 7 to 9, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 2C of the endogenous BIM gene, and the mutation may be an insertion, deletion, or substitution.

11. The modified cell according to any one of claims 7 to 10, wherein the genome disruption is located within a target sequence in the BIM containing any one of sequence numbers 14, 18, 19, 20, 21, and 22.

12. The modified cell according to any one of claims 7 to 11, wherein the genome disruption within the BIM is carried out by administering a guide RNA to the cell, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cell.

13. The modified cell according to claim 12, wherein the guide RNA includes one of the guide sequences from sequence numbers 2, 6, 7, 8, 9, and 10, or one or more sgRNA or crRNA sequences from sequence numbers 26, 32, 33, 34, 35, and 36.

14. A modified cell according to any one of claims 1 to 13, further comprising genomic disruption within an endogenous FAS gene that suppresses or eliminates the expression of a functional FAS cell surface cell death receptor (FAS) protein.

15. The modified cell according to claim 14, wherein the genome disruption within the FAS gene is located within exon 1 of the FAS gene.

16. The modified cell according to claim 14 or 15, wherein the genome disruption is a loss-of-function nucleic acid mutation in exon 1 of the endogenous FAS gene, and the mutation may be an insertion, deletion, or substitution.

17. The modified cell according to any one of claims 14 to 16, wherein the genome disruption is located within a target sequence in the FAS containing sequence number 62.

18. The modified cell according to any one of claims 14 to 17, wherein the genome disruption within the FAS is carried out by administering a guide RNA to the cell, wherein the guide RNA is a single guide RNA (sgRNA) or crRNA, and optionally an endonuclease or a nucleic acid encoding the endonuclease is also administered to the cell.

19. The modified cell according to claim 18, wherein the guide RNA includes the guide sequence of SEQ ID NO: 61 or the sgRNA of SEQ ID NO:

60.

20. A modified cell according to any one of claims 1 to 19, further comprising one or more exogenous polynucleotides encoding an IFNγ signaling factor, wherein the IFNγ signaling factor comprises a first IFNγ fusion protein subunit and a second IFNγ fusion protein subunit, the first IFNγ fusion protein subunit comprising the extracellular domain (ECD) of interferon-gamma receptor 1 (IFNGR1) and the intracellular domain of interleukin-2 receptor subunit gamma (IL2RG), and the second IFNγ fusion protein subunit comprising the ECD of interferon-gamma receptor 2 (IFNGR2) and the intracellular domain of interleukin-2 receptor subunit beta (IL2RB).

21. The modified cell according to claim 20, wherein a first exogenous polynucleotide encoding the first IFNγ fusion protein subunit of the IFNγ signaling factor is inserted at the SOCS1 locus.

22. The modified cell according to any one of claims 20 to 21, wherein a second exogenous polynucleotide encoding the second IFNγ fusion protein subunit of the IFNγ signal-converting factor is inserted at the ROSA26 locus.

23. The modified cell according to any one of claims 20 to 22, wherein the first IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 71, the second IFNγ fusion protein subunit of the IFNγ signal-converting factor comprises the amino acid sequence of SEQ ID NO: 72, and optionally the first exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO: 81, and the second exogenous polynucleotide encodes an amino acid sequence comprising SEQ ID NO:

82.

24. A modified cell according to any one of claims 1 to 23, further comprising an exogenous polynucleotide encoding a chimeric antigen receptor (CAR).

25. The modified cell according to claim 24, wherein the exogenous polynucleotide encoding the CAR is inserted into the AAVS1 locus.

26. The modified cell according to claim 24 or 25, wherein the exogenous polynucleotide encodes a CAR that specifically binds to human CD19.

27. The modified cell according to claim 26, wherein the CAR that specifically binds to human CD19 contains the amino acid of SEQ ID NO: 83 or SEQ ID NO:

84.

28. The modified cell according to any one of claims 1 to 27, wherein the iPSC is derived from primary γδT cells (γδT-iPSCs).

29. The modified cell according to any one of claims 1 to 28, wherein the γδT cells are iPSC-derived γδT (iγδT) cells, and optionally the γδT cells are T-iPSC-derived γδT cells (T-iγδT).

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