WO2025097031A1

WO2025097031A1 - Compositions and methods for cell therapy

Info

Publication number: WO2025097031A1
Application number: PCT/US2024/054231
Authority: WO
Inventors: Brian C. Thomas; Cristina Noel BUTTERFIELD; Cindy CASTELLE; Steven FLAMMER; Artur KARASYOV; Kyungyoon KWON; Kazim Kerim Moncal; Michael Spelman; Meghan STORLIE; Emily SUTER; Megan VAN OVERBEEK; Tyler Waite; Wenshi Wang
Original assignee: Metagenomi Inc
Current assignee: Metagenomi Inc
Priority date: 2023-11-02
Filing date: 2024-11-01
Publication date: 2025-05-08
Anticipated expiration: 2026-05-02

Abstract

Described herein are methods, compositions, and systems for the treatment of cancer and other conditions.

Description

COMPOSITIONS AND METHODS FOR CELL THERAPY

CROSS-REFERENCE

[0001] This application claims the benefit and priority to U.S. Provisional Patent Application No. 63/595,713 filed November 2, 2023. which is incorporated by reference in its entirety herein.

SUMMARY

[0002] Described herein, in certain embodiments, are methods for engineering a yd T cell, comprising contacting the y5 T cell using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M- locus. [0003] Described herein, in certain embodiments, are methods for engineering a yd T cell, comprising contacting the yd T cell using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus.

[0004] In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the engineered system further comprises one or more donor nucleic acids. In some embodiments, the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the donor nucleic acid encodes a first cytokine. In some embodiments, the donor nucleic acid further encodes a second CAR. In some embodiments, the donor nucleic acid further encodes a second cytokine. In some embodiments, the first CAR and the second CAR comprises an extracellular antigen binding domain, and wherein the extracellular antigen binding domain binds to a tumor-associated antigen selected from the group consisting of MUC16, FOLRL BCMA, CLDN6, SLC34A2, and TAG72. In some embodiments, the extracellular antigen binding domain of the first CAR binds to MUC16 or FOLRL In some embodiments, the extracellular antigen binding domain of the second CAR binds to MUC 16 or FOLR1. In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv). In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-12. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 10. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 11. In some embodiments, the first or the second CAR comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 28-31. In some embodiments, the first cytokine is IL-12 or IL-15. In some embodiments, the second cytokine is IL-12 or IL-15. In some embodiments, the donor nucleic acid comprises a first homology arm and a second homology arm; and wherein the first homology arm comprises a sequence located on the 5 ’ side of the target nucleic acid sequence and the second homolog)’ arm comprises a sequence located on the 3’ side of the target nucleic acid sequence. In some embodiments, the first homology arm comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 46, 48, 50, and 52. In some embodiments, the second homology arm comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 47. 49. 51, and 53.

[0005] Described herein, in certain embodiments, are pharmaceutical compositions comprising the engineered y5 T cell produced by the method described herein.

[0006] Described herein, in certain embodiments, are gene editing systems comprising: a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127. 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR).

[0007] Described herein, in certain embodiments, are gene editing systems comprising: a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17. TRAC, and B2M locus; and cone or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the donor nucleic acid encodes a first cytokine. In some embodiments, the donor nucleic acid further encodes a second CAR. In some embodiments, the donor nucleic acid further encodes a second cytokine. In some embodiments, the first CAR and the second CAR comprises an extracellular antigen binding domain, and wherein the extracellular antigen binding domain binds to a tumor-associated antigen selected from the group consisting of MUC16, FOLR1, BCMA, CLDN6, SLC34A2, and TAG72. In some embodiments, the extracellular antigen binding domain of the first CAR binds to MUC16 or FOLR1. In some embodiments, the extracellular antigen binding domain of the second CAR binds to MUC16 or FOLR1 . In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv). In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-12. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 10. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 11. In some embodiments, the first or the second CAR comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 28-31. In some embodiments, the first cytokine is IL-12 or IL-15. In some embodiments, the second cytokine is IL-12 or IL-15.

[0008] Described herein, in certain embodiments, are yo T cells comprising the gene editing system described herein.

[0009] Described herein, in certain embodiments, are methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered y§ T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus.

[0010] Described herein, in certain embodiments, are methods of killing a cancer cell comprising contacting the cancer cell with an engineered yo T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127. 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus.

[0011] Described herein, in certain embodiments, are methods of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered y5 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus.

[0012] Described herein, in certain embodiments, are methods of killing a cancer cell comprising contacting the cancer cell with an engineered y5 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176, wherein the base editor comprises an endonuclease domain that is deficient in nuclease activity: and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus.

[0013] In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the engineered system comprises one or more donor nucleic acids. In some embodiments, the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the donor nucleic acid encodes a first cytokine. In some embodiments, the donor nucleic acid further encodes a second CAR. In some embodiments, the donor nucleic acid further encodes a second cytokine. In some embodiments, the first CAR and the second CAR comprises an extracellular antigen binding domain, and wherein the extracellular antigen binding domain binds to a tumor-associated antigen selected from the group consisting of MUC16, FOLR1, BCMA, CLDN6, SLC34A2, and TAG72. In some embodiments, the extracellular antigen binding domain of the first CAR binds to MUC16 or FOLR1. In some embodiments, the extracellular antigen binding domain of the second CAR binds to MUC16 or FOLR1. In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv). In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-12. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 10. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 11. In some embodiments, the first CAR or the second CAR comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 28-31. In some embodiments, the first cytokine is IL-12 or IL-15. In some embodiments, the second cytokine is IL-12 or IL-15. In some embodiments, the cancer is characterized by tumor cells that exhibit cell surface expression of one or more tumor-associated antigens selected from the group consisting of MUC16, FOLR1, BCMA, CLDN6, SLC34A2, and TAG72. In some embodiments, the cancer is selected from the group consisting of ovarian, endometrial, lung, breast, brain, kidney, and colon cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer cell comprises tumor cells that exhibit cell surface expression of one or more tumor-associated antigens selected from the group consisting ofMUC16, FOLR1, BCMA, CLDN6, SLC34A2, and TAG72. In some embodiments, the cancer cell is selected from the group consisting of an ovarian, endometrial, lung, breast, brain, kidney, and colon cancer cell. In some embodiments, the cancer cell is an ovarian cancer cell. [0014] Described herein, in certain embodiments, are methods of modifying a TRAC and a PD-1 locus in a y5 T cell comprising contacting to the y8 T cell: a) a first engineered system comprising: i) a first endonuclease encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus; and b) a second engineered system comprising: i) a second endonuclease encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus.

[0015] Described herein, in certain embodiments, are methods of modifying a TRAC and an IL- 17 locus in a y6 T cell comprising contacting to the y3 T cell: a) a first engineered system comprising: i) a first endonuclease encoded by a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 127, 128, and 179; and ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus; and b) a second engineered system comprising: i) a second endonuclease encoded by a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 127, 128, and 179; and ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus.

[0016] Described herein, in certain embodiments, are methods of modifying a PD-1 and an IL-17 locus in a y8 T cell comprising contacting to the y5 T cell: a) a first engineered system comprising: i) a first endonuclease encoded by a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 127. 128, and 179; and ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL-17 locus; and b) a second engineered system comprising: i) a second endonuclease encoded by a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 127. 128, and 179; and ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL-17 locus.

[0017] Described herein, in certain embodiments, are methods of modifying a PD-1 and a TIM3 locus in a y5 T cell comprising contacting to the y5 T cell: a) a first engineered system comprising: i) a first endonuclease encoded by a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 127, 128, and 179; and ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: i) a second endonuclease encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus.

[0018] Described herein, in certain embodiments, are methods of modifying a PD-1 and a TIM3 locus in a y5 T cell comprising contacting to the y5 T cell: a) a first engineered system comprising: i) a first base editor encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176, wherein the first base editor comprises a first endonuclease domain that is deficient in nuclease activity; and ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease domain and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: i) a second base editor encoded by a sequence having at least 80% sequence identity to any one SEQ ID NOs: 175-176, wherein the second base editor comprises a second endonuclease domain that is deficient in nuclease activity; and ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease domain and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus.

[0019] In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 117-119. and 123. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 117-119, and 124-126. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 123-126. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 120-123. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 146-172. In some embodiments, the method further comprises introducing to the y5 T cell one or more donor nucleic acids. In some embodiments, the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the donor nucleic acid encodes a first cytokine. In some embodiments, the donor nucleic acid further encodes a second CAR. In some embodiments, the donor nucleic acid further encodes a second cytokine. In some embodiments, the first CAR and the second CAR comprises an extracellular antigen binding domain, and wherein the extracellular antigen binding domain binds to a tumor-associated antigen selected from the group consisting of MUC16, FOLR1, BCMA, CLDN6, SLC34A2. and TAG72. In some embodiments, the extracellular antigen binding domain of the first CAR binds to MUC16 or FOLR1. In some embodiments, the extracellular antigen binding domain of the second CAR binds to MUC16 or FOLR1. In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv). In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-12. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 10. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 11. In some embodiments, the first CAR or the second CAR comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 28-31. In some embodiments, the first cytokine is IL- 12 or IL-15. In some embodiments, the second cytokine is IL- 12 or IL-15. In some embodiments, the donor nucleic acid comprises a first homology arm and a second homology arm; and wherein the first homology arm comprises a sequence located on the 5 ’ side of the target nucleic acid sequence and the second homology arm comprises a sequence located on the 3’ side of the target nucleic acid sequence. In some embodiments, the first homology arm comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 46, 48, 50, and 52. In some embodiments, the second homology' arm comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 47. 49. 51, and 53.

[0020] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure.

Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0022] FIGs. 1A and IB depict graphs show ing the isolation of gamma delta T-cells from healthy donor PBMCs. FIG. 1A illustrates that negative selection of gamma delta T-cells from peripheral blood mononuclear cells (PBMCs) from a representative healthy donor leukopak results in high purity of target population with <11% contaminating cells. FIG. IB illustrates that the isolation process yields gamma delta population that can be distinguished into VD1 and VD2 subsets.

[0023] FIGs. 2A and 2B depict barplots showing a summary of y5 T cell purity post-isolation and subset prevalence by healthy donor. FIG. 2A shows barplots illustrating effective, high purity isolation of y5 T-cells from low starting prevalence (<5%) in PBMCs (n=12). FIG. 2B shows barplots illustrating that VD1 and VD2 prevalence varies by donor (donors are indicated on the x axis), with generally higher prevalence of VD2 cells and a small subset of VD1-VD2- y8 T-cells.

[0024] FIGs. 3A and 3B depict barplots showing that primary y5 T cell subtypes exhibit different memory phenotypes. FIG. 3A shows that across 10 healthy donors after the 10-day (D: DI 0) expansion process, VD1 y5 T-cells maintain a range of naive (TN), central memory (TCM), effector memory⁷ (TEM) and effector memory⁷ expressing CD45RA+ (TEMRA) T-cells. FIG. 3B shows that across 10 healthy donors after the 10-day (D10) expansion process, VD2 y6 T-cells predominantly differentiated into a TEM population.

[0025] FIGs. 4A and 4B depict graphs showing that y5T cells produce effector cytokines in a dose-dependent manner. y5T cells from a representative donor stimulated with phorbol myristate acetate (PMA) and ionomycin (indicated as PMAI) for 24 hours produced IFNy (FIG. 4A) and TNFa (FIG. 4B) in a dose-dependent manner. PMA/I concentration (indicated as [PMAI]) fold is shown on the x axis, where lx PMA/1 = 81 nM PMA and 1.34 pM ionomycin.

[0026] FIGs. 5A and 5B depict graphs showing that unmodified y3T cells demonstrate innate killing proficiency against liquid and solid tumor. FIG. 5A: K562 cells w ere labeled with CellTrace™ Violet (CTV) dye and co-cultured with expanded y5 T cells. % dead target cells are indicated on the y axis and Effector: Target ratio is indicated on the x axis. After 24 hours, the viability of CTV+ target cells was assessed by flow cytometry to measure % dead target cells. All donor-derived y5 T cells (donors are indicated by the key) exhibited dose-dependent cytotoxic activity⁷ against K562 cells (n=6). FIG. 5B: TOV-112D-GFP-Luciferase cells were cocultured with expanded y6 T cells. Donors are indicated by the key. After 24 hours, D-luciferin w as added to wells, incubated, and luminescence was detected by microplate reader. % cytotoxicity (y axis) w as calculated by subtracting sample relative luminescence units (RLU) from target only RLU, divided by target only RLU. Effector: Target ratio is indicated on the x axis.

[0027] FIG. 6 depicts an illustration of the cell engineering workflow for y5 T cells. y6 Tells are isolated from healthy donors on DO and subsequently activated for 3 days. Cells are removed from activation on D3. Genome editing reagents are delivered on D4, either via electroporation (mRNA or protein RNP) or via LNP (mRNA). Edited cells are then cultured for 3+ days. Various assays are run on D7 or later to assess engineered cells at the genomic, proteomic, phenotypic, and therapeutic level.

[0028] FIGs. 7A and 7B depict graphs showing checkpoint inhibitor expression in expanded yd T cells. Expression of checkpoint inhibitors PD-1 and TIM3 was measured by flow' cytometry in VD1+ and VD2+ yd T cell subsets over a 10-day expansion period. FIG. 7A shows that VD1+ yd T cells express high levels of PD-1 in response to activation stimuli (n=12 donors). FIG. 7B show s that VD2+ yd T cells express increasing levels of TIM3 over the 10-day expansion period (n=l l donors).

[0029] FIGs. 8A and 8B depict barplots showing editing of checkpoint inhibitor genes in yd T cells. Donor-derived yd T cells were edited on D4 post-isolation using MG nucleases and guides. Editing efficiency is measured through quantification of insertion-deletion (indel) frequency in gDNA extracted from edited y5 T cells on D7 post-isolation (% Indels). FIG. 8A shows editing efficiency (% Indels) of PDCD1 gene (encoding PD-1) across donors (x axis) utilizing Guide #H3. FIG. 8B shows editing efficiency (% Indels) of HAVCR2 (encoding TIM3) across donors utilizing Guide #A1 and Guide #A4.

[0030] FIGs. 9A and 9B depict barplots showing protein knockdown of checkpoint inhibitors in yd T cells. Surface protein expression of edited yd T cells was measured via flow cy tometry' to assess protein knockdown. FIG. 9A shows PD-1 surface expression for yd T cells edited using PDCD1 Guide #H3. Cells were repeatedly stimulated with immobilized immu510 antibody for 24 hours weekly starting at Dl l (repeat stimulation #1: Dl l, #2: DI 8, #3: D25). After each stimulation, cells w ere divided into a stimulated and unstimulated condition for subsequent stimulations. Data shown for all cell conditions at D26 post-isolation for a single donor. FIG. 9B shows TIM3 surface expression for yd T cells edited using HAVCR2 Guide #A1 across multiple donors. Data shown for all cell conditions at DI 1 post-isolation.

[0031] FIG. 10 depicts barplots showing editing of IL17A for gene KO in yd T cells. Donor- derived yd T cells were edited on D4 post-isolation using MG nucleases and guides. Editing efficiency’ (% indel) is measured through quantification of insertion-deletion (indel) frequency in gDNA extracted from edited yd T cells on D7 post-isolation. Editing efficiency (% indel) of IL17A gene (encoding IL-17A) across donors utilizing Guide #1, #4, #7, #12, and #13.

[0032] FIGs. 11A-11D depict barplots show ing LNP delivery’ of editing reagents to yd T cells. Lipid nanoparticles (LNP) were formulated using copackaging MG enzyme mRNA with guide RNAs. LNP were delivered to yd T cells on D4 post-isolation. Editing efficiency (% indel) is measured through quantification of insertion-deletion (indel) frequency' in gDNA extracted from edited yd T cells on D7 post-isolation. FIG. 11A show's editing efficiency (% indel) of TRAC and PDCD1 genes using Guide #6 and Guide #H3, respectively. Editing efficiency (% indel) at each target site was dose dependent. Data shown for a single donor. FIG. 11B shows the comparison of editing efficiency (% indel) of TRAC and PDCD1 genes using two different reagent delivery modalities (LNP and electroporation) across multiple donors. FIG. 11C shows the estimated yield (estimated relative engineered cell count) of edited y5 T cells using two different reagent delivery’ modalities (LNP and electroporation). Data shown for a single donor. FIG. 11D shows simultaneous editing (% indel) of two genes using two different reagent delivery modalities (LNP and electroporation). LNP were dosed at two different concentrations. Data shown for a single donor.

[0033] FIGs. 12A and 12B depict an illustration of CAR cassette design and production. FIG. 12A shows a schematic diagram representing the general architecture of the MG-0100 CAR expression cassette(s). Sites 1 -5 within the schematic represent modular domains that can be changed to alter characteristics of the CAR: 1 = homology’ regions determining integration site; 2 = promoter sequence that can be swapped to modulate expression levels of the CAR; 3 = scFv binding moiety that determines the antigen target of the CAR; 4 = costimulatory domain that can be swapped to fine-tune the intracellular signaling activity’ upon CAR binding; 5 = tagBFP reporter that can be swapped for any alternative fluorescent reporter, or protein tag for detection/purification. FIG. 12B shows a schematic representation of various CAR and TAA target molecules related to MG-0100. Created with BioRender.

[0034] FIG. 13 depicts a schematic representation of molecules involved in CAR/TCR signaling cascade. Bars within cytoplasmic tails of proteins shown represent ITAM-, ITIM-, and ITSM- domains. FIG. adapted from Sievers, Nico M et al. “CARs: Beyond T Cells and T Cell-Derived Signaling Domains.’' International Journal of Molecular Sciences 21 (2020).

[0035] FIG. 14 depicts a schematic representation of ddPCR assays used to characterize sitespecific integration efficiency. Target amplicon 1 and 3 represent the junctions of the genomic locus with the integrated transgene, and amplicon 2 represents a transgene-specific region to allow for measurement of transgene copies relative to potential ectopic copies from the donor AAV. Created with BioRender.

[0036] FIG. 15 depicts graphs showing flow cytometry characterization of CAR/transgene payload expression. y5 T cells are edited to co-express a CAR receptor and tagBFP fluorescent cassette reporter. Flow cytometry’ data collected on the Attune NxT is gated for live y5 T cells and BFP reporter expression is quantified (gating strategy from left to right).

[0037] FIG. 16 depicts a schematic representation of bicistronic transgene architecture. Example (generic) design of bicistronic cassette for insertion at MG-nuclease edited site in y8 T cells. Bicistronic or multi cistronic design enables multiple genes to be expressed from single cassette. Promoter 1 and Promoter 2 independently drive expression of Payload 1 and Payload 2, respectively.

[0038] FIG. 17 depicts a bar plot showing the insertion of various CAR designs (CAR1, CAR2, and CAR3) at specific sites for multiple donors. The %CAR+ is indicated on the y axis.

[0039] FIG. 18 depicts boxplots showing the insertion of CAR into different sites (%CAR+). Flanking homology sequences present in the CAR delivery vector allow for site-specific integration of the expression cassette into a genetic locus of our choosing, as relevant to MG- 0100 biology.

[0040] FIGs. 19A-19D depict bar plots showing dual site integration of CAR cassettes (%CAR+). Dual delivery of AAV-CARs with homology regions targeting the pair of edited sites (TRAC+PD-1. TRAC+IL-17, or PD-l+IL-17) demonstrates the feasibility of multiplexed engineering in this cell type. FIG. 19A shows results for AAV only transduction control samples. FIG. 19B shows results for Single site KO, with single site-specific AAV CAR delivery. FIG. 19C shows results for dual site KO, with single site-specific AAV CAR delivery. FIG. 19D shows results for dual site KO, with dual site-specific AAV CAR delivery. Data represented as mean ± SD of replicate samples (n=3).

[0041] FIGs. 20A and 20B depict bar plots showing dual site integration of CAR cassettes. Dual editing of donor-derived y5 T cells (n=l) using MG3-6 mRNA with TRAC and/or PD-l-specific sgRNA, or in combination with MG29-1 RNP (targeting IL- 17), resulted in variable levels of combined indel frequencies (%indels site 1 x %indels site 2). Dual delivery of AAV-CARs with homology regions targeting the pair of edited sites (TRAC+PD-1, TRAC+IL-17, or PD-l+IL-17) demonstrates the feasi bi 1 i ty of multiplexed engineering in this cell type. FIG. 20A shows results for dual site KO (% Dual KO). FIG. 20B shows results for dual site KI (% Dual KI). Data represented as mean ± SD of replicate samples (n=3).

[0042] FIG. 21 depicts bar plots showing non-viral DNA delivery of CAR cassette in yd T cells: Data from healthy-donor derived (n=2) y5 T cells modified using MG3-6 enzyme with TRAC- specific sgRNA, and co-delivery of msDNA-CAR at varying doses. %Viability and %CAR+ measurements were averaged across replicates (n=3) and used to visualize the percent of Viable- CAR+ y§ T cells post-engineering for all conditions in each donor.

[0043] FIG. 22 depicts a schematic representation of the analytical pipeline used for KO candidate nomination. A comprehensive tree diagram depicting the analytical tools used for the pipeline in the order at which they occur. Most analysis downstream including visualization, pseudotime analysis, and differential gene expression were accomplished.

[0044] FIG. 23 depicts results of tumor-associated antigen expression characterization of EOC cell lines. To better understand surface receptor expression of potential therapeutic targets, biomarkers were screened using qPCR, Flow-Cytometry, and ICC. Readouts were calculated via the 2-AACt method for qPCR and binary determination of signal via fluorescence for FC and ICC

[0045] FIG. 24 depicts bar plots showing the cell count or viability⁷ of donor y5 T cells that w ere electroporated with an exemplary⁷ cytosine base editor (CBE) or nuclease mRNA. Isolated and purified donor y5 T cells (n=3 donors) electroporated with 3-6/8 CBE mRNA exhibited limited toxicity⁷ in terms of overall cell count and viability⁷ measurements 72 hours post-electroporation, relative to control CBE and MG3-6 nuclease. Nuclease mRNA was also delivered to cells w⁷ith sgRNA for lead MG3-6 TRAC site, with minimal impact on readouts.

[0046] FIGs. 25A-25C depict data related to 3-6/8 CBE mRNA delivered to primary donor y6 T cells (n=2) with an array of candidate sgRNAs targeting HAVCR2 (TIM3) or PDCD1 (PD-1). FIG. 25A depicts tw⁷o bar plots showing the protein knock-out efficiency⁷ of HAVCR2 (TIM3) and PDCD1 (PD-1)., respectively. FIG. 25B depicts two bar plots assessing the impact on cell viability⁷ of TIM3 and PD1 protein knock-out efficiency for guides (HAVCRg2 or PDCDlgl) when paired with CBE-139, CBE-152, or CBE control mRNA. FIG. 25C depicts two bar plots showing the percent of protein knock-out across all TIM3 guides and all PD1 guides using CBE- 139.

[0047] FIGs. 26A-26E depict data related to using isolated and purified donor y8 Ts (n=l donor), w ith guides for TIM3 (g2) and PD-1 (gl) identified in a screen and used with CBE-139 mRNA, either individually or together, to assess feasibility of multiplexing base edits. Exemplary MG3-6 nuclease and guides for both sites w ere included for comparison. FIG. 26A depicts a bar graph showing the Viability⁷ measurements , FIG. 26B depicts a bar graph showing cell counts, FIG. 26C depicts a bar graph showing surface expression of PD1, and FIG. 26D depicts a bar graph showing surface expression of TIM3. All were assessed 3 days post-editing via flow cytometry readout. FIG. 26E depicts the results of a CRISPresso analysis used to determine the rate of C>T conversion at each base within the spacer sequence; data shown represents HAVCRg2 spacer.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0048] The Sequence Listing filed herewith provides exemplary⁷ poly nucleotide and polypeptide sequences for use in methods, compositions, and systems according to the disclosure. Below are exemplary descriptions of sequences therein.

[0049] SEQ ID NOs: 1-5 show the nucleotide sequences of promoters.

[0050] SEQ ID NOs: 6-7 show⁷ the nucleotide sequences of signal peptides. [0051] SEQ ID NOs: 8 and 21-24 show the nucleotide sequences of reporters. [0052] SEQ ID NOs: 9-12 show the nucleotide sequences of scFVs. [0053] SEQ ID NOs: 13-15 show the nucleotide sequences of linkers.

[0054] SEQ ID NO: 16 shows the nucleotide sequence of a TM domain.

[0055] SEQ ID NOs: 17 and 87-101 show the nucleotide sequences of costimulatory domains.

[0056] SEQ ID NOs: 18 and 102-111 show the nucleotide sequences of ITAM domains.

[0057] SEQ ID NOs: 19-20 show the nucleotide sequences of self-cleaving peptides.

[0058] SEQ ID NOs: 25-27 show the nucleotide sequences of cytokines.

[0059] SEQ ID NOs: 28-31 show the nucleotide sequences of CARs.

[0060] SEQ ID NOs: 32-34 show the nucleotide sequences of expression cassettes.

[0061] SEQ ID NOs: 35-45 show the nucleotide sequences of spacers.

[0062] SEQ ID NOs: 46-53 show the nucleotide sequences of homology domains.

[0063] SEQ ID NOs: 54-64 show the nucleotide sequences of NGS amplicons.

[0064] SEQ ID NOs: 65-86 show the nucleotide sequences of primers.

[0065] SEQ ID NOs: 112-115 show the nucleotide sequences of fused CARs.

[0066] SEQ ID NOs: 116-126 and 146-172 show the nucleotide sequences of guide RNAs.

[0067] SEQ ID NO: 127 shows the nucleotide sequence encoding the MG3-6 mRNA.

[0068] SEQ ID NO: 128 shows the nucleotide sequence encoding the MG21-1 mRNA.

[0069] SEQ ID NO: 179 shows the nucleotide sequence encoding the MG29-1 mRNA.

[0070] SEQ ID NOs: 130-145 and 180-205 show amino acid sequences of nuclear localization signals (NLSs).

[0071] SEQ ID NOs: 173-174 show nucleotide sequences of primers.

[0072] SEQ ID NO: 175 shows the nucleotide sequence of cytosine base editor (CBE) 139- 52vl4_MG3-6-3-8.

[0073] SEQ ID NO: 176 shows the nucleotide sequence of cy tosine base editor (CBE) 152- 6vl3 MG3-6 3-8.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0074] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[0075] The practice of some methods disclosed herein employ, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology⁷, genomics, and recombinant DNA. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel. et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies. A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0076] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”.

[0077] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0078] The term “nucleotide,” as used herein, refers to a base-sugar-phosphate combination. Contemplated nucleotides include naturally occurring nucleotides and synthetic nucleotides. Nucleotides are monomeric units of a nucleic acid sequence (e.g, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine tnphosphate (CTP). guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP. and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein encompasses dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of ddNTPs include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled or detectably labeled, such as using moieties comprising optically detectable moieties (e.g, fluorophores) or quantum dots. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels of nucleotides include but are not limited fluorescein, 5- carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy- X-rhodamine (ROX), 4-(4'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue. Oregon Green, Texas Red, Cyanine and 5-(2'-aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, |TAMRA|dUTP. [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP. [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dRl 10]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City . Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, IL; Fluorescein- 15 -dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6- dUTP, IR770-9-dATP, Fluorescein- 12-ddUTP, Fluorescein- 12-UTP, and Fluorescein-15-2'- dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY- TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP. Cascade Blue-7-dUTP, fluorescein- 12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP. Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. The term nucleotide encompasses chemically modified nucleotides. An exemplary chemically-modified nucleotide is biotin-dNTP. Nonlimiting examples of biotinylated dNTPs include, biotin-dATP (e.g. bio-N6-ddATP, biotin-14- dATP), biotin-dCTP ( .g., biotin- 11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g, biotin-11- dUTP, biotin-16-dUTP, biotm-20-dUTP).

[0079] The terms “polynucleotide,” “oligonucleotide.” and “nucleic acid” are used interchangeably to refer to a polymenc form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multistranded form. Contemplated polynucleotides include a gene or fragment thereof. Exemplary' polynucleotides include, but are not limited to, DNA, RNA. coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozy mes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. In a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA. A polynucleotide can be exogenous or endogenous to a cell and/or exist in a cell-free environment. The term polynucleotide encompasses modified polynucleotides (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure are imparted before or after assembly of the polymer. Non-limiting examples of modifications include: 5-bromouraciL peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0080] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer is interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary or tertiary structure (e g., domains). The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. The terms “amino acid” and “amino acids,” as used herein, refer to natural and non-natural amino acids, including, but not limited to, modified amino acids. Modified amino acids include amino acids that have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. The term “amino acid” includes both D-amino acids and L-amino acids.

[0081] The terms “engineered,” “synthetic,” and “artificial” are used interchangeably herein to refer to an object that has been modified by human intervention. For example, the terms refer to a polynucleotide or polypeptide that is non-naturally occurring. An engineered peptide has, but does not require, low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity ) to a naturally occurring human protein. For example, VPR and VP64 domains are synthetic transactivation domains. Non-limiting examples include the following: a nucleic acid modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid synthesized in vitro with a sequence that does not exist in nature; a protein modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein acquiring a new function or property. An “engineered” system comprises at least one engineered component.

[0082] As used herein, “operably linked”, “operable linkage”, “operatively linked”, or grammatical equivalents thereof refer to an arrangement of genetic elements, e.g., a promoter, an enhancer, a poly adenylation sequence, etc., wherein an operation (e.g., movement or activation) of a first genetic element has some effect on the second genetic element. The effect on the second genetic element can be, but need not be, of the same type as operation of the first genetic element. For example, two genetic elements are operably linked if movement of the first element causes an activation of the second element. For instance, a regulatory element, which may comprise promoter and/or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.

[0083] As used herein, the term “complex” refers to a joining of at least two components. The two components may each retain the properties/activities they had prior to forming the complex or gain properties as a result of forming the complex. The joining includes, but is not limited to, covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, Van der Waals interactions, and hydrophobic bond), use of a linker, fusion, or any other suitable method. Contemplated components of the complex include polynucleotides, polypeptides, or combinations thereof. For example, a complex comprises an endonuclease and a guide polynucleotide.

[0084] As used herein, a “guide nucleic acid” or “guide polynucleotide” refers to a nucleic acid that may hybridize to a target nucleic acid and thereby directs an associated nuclease to the target nucleic acid. A guide nucleic acid is, but is not limited to. RNA (guide RNA or gRNA), DNA. or a mixture of RNA and DNA. A guide nucleic acid can include a crRNA or a tracrRNA or a combination of both. The term guide nucleic acid encompasses an engineered guide nucleic acid and a programmable guide nucleic acid to specifically bind to the target nucleic acid. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. The strand of a double-stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid is the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand, and therefore is not complementary’ to the guide nucleic acid is called noncompl ementary strand. A guide nucleic acid having a polynucleotide chain is a “single guide nucleic acid.” A guide nucleic acid having two polynucleotide chains is a “double guide nucleic acid.” If not otherw ise specified, the term “guide nucleic acid” is inclusive, referring to both single guide nucleic acids and double guide nucleic acids. A guide nucleic acid may comprise a segment referred to as a “nucleic acidtargeting segment” or a “nucleic acid-targeting sequence,” or a “spacer.” A nucleic acid-targeting segment can include a sub-segment referred to as a “protein binding segment” or “protein binding sequence” or “Cas protein binding segment.” [0085] The term “donor nucleic acid’' refers to a polynucleotide that includes an exogenous polynucleotide sequence (e.g.. a nucleic acid sequence for a therapeutic gene) and one or more polynucleotide sequences for mediating recombination such as by via non-homologous end joining (NHEJ) or homology⁷ directed repair (HDR).

[0086] The term “locus’" refers to a location on a chromosome or DNA molecule corresponding to a gene or a physical or phenotypic feature.

[0087] The term “binds” or any grammatical variation thereof (e.g., binding, etc.) when made in reference to the binding of two molecules (e.g., extracellular antigen binding domain of a CAR and a tumor-associated antigen, etc.) refer to an interaction of the two molecules that is dependent upon the presence of a particular structure on one or both of the molecules.

[0088] The term “tumor antigen” or “tumor associated antigen” as used herein refers to a substance, produced by tumor cells, which is recognized by the immune system and which elicits an antigen specific immune response in a host (e.g., which is presented by MHC complexes). In some embodiments, a tumor antigen is on the surface of a tumor cell. In some embodiments, the tumor antigen is presented by an MHC/HLA.

[0089] As used herein, the terms “treatment,” “treating,” and the like, in some embodiments, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of affecting a partial or complete cure for a disease and/or symptoms of the disease. “Treatment,” as used herein, may include treatment of a disease or disorder e.g., cancer) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g.. including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Treating may refer to any indicia of success in the treatment or amelioration or prevention of a cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms; or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present invention to prevent, delay, alleviate, arrest or inhibit development of the symptoms or conditions associated with diseases (e.g, cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject. [0090] The terms “recipient,” “individual.” “subject,” “host,” and “patient,” are used interchangeably herein and in some embodiments, refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory', zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc. In some embodiments, the mammal is human. None of these terms require the supervision of medical personnel.

[0091] As used herein, the term "administering", “administration”, or “administer” means delivering a composition or pharmaceutical composition as described herein to a target cell or a subject. The compositions or pharmaceutical compositions described herein are designed for delivery to subjects in need thereof by any suitable route or a combination of different routes. [0092] The term “sequence identity” or “percent identity” in the context of two or more nucleic acids or polypeptide sequences, generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, e.g., BLASTP using parameters of a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and the PAM30 scoring matrix setting gap costs at 9 to open gaps and 1 to extend gaps for sequences of less than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https://blast.ncbi.nlm.nih.gov); CLUSTALW with the Smith-Waterman homology search algorithm parameters with a match of 2, a mismatch of -1, and a gap of -1; MUSCLE with default parameters; MAFFT with parameters of a retree of 2 and max iterations of 1000; Novafold with default parameters; HMMER hmmalign with default parameters.

[0093] The term “optimally aligned” in the context of two or more nucleic acids or polypeptide sequences, generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that have been aligned to maximal correspondence of amino acids residues or nucleotides, for example, as determined by the alignment producing a highest or “optimized” percent identity score.

[0094] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see, for e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993)). The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

Overview

[0095] Adoptive cell therapy (ACT) is a form of cancer treatment based on delivering tumorspecific immune cells to a patient to attack and eliminate the patient's cancer. ACT involves the use of T-cells that are isolated from a patient's body and expanded ex vivo to re-infuse back into the patient. These T-cells are designed to target specific antigens expressed on cell surfaces of cancer cells. The tumor specificity is obtained by genetically inserting a chimeric antigen receptor (CAR) into the isolated T cell to enhance the recognition of tumor cell surface antigens. Commonly, CARs comprise a single chain fragment variable (scFv) of an antibody specific for a tumor associated antigen (TAA) that is fused to the signaling domain of a TCR.

[0096] Gamma delta T-cells (y8 T) are a population of cytotoxic T-cells that comprise 1-5% of human peripheral blood mononuclear cells (PBMCs). The majority⁷ of y5 T cells belong to V51 (<50%) or V62 (50-95%) subsets, which are predominantly compartmentalized in tissue or peripheral blood, respectively. These subsets have distinct phenotypes and functional properties. An advantage of y6 T cell-based immunotherapies is HLA-independent direct recognition of any innate response to malignant cells via their T-cell receptor (TCR) and natural killer cell receptors (NKR). This innate response involves the production of effector cytokines such as interferon gamma (IFNy) and tumor necrosis factor alpha (TNFa). and secretion of cytotoxic granules including granzymes and perforin. The bridge that y5 T cells provide between innate and adaptive immunity⁷ against malignant cells imparts a significant and unique advantage as a cell therapy platform.

[0097] In addition to their innate potential as a vehicle for cell therapy, y6 T cells can be engineered to further enhance their ability to penetrate dense tumors, persist within a patient, maintain potency within an immunosuppressive tumor microenvironment, and improve on-target on-tumor specificity⁷. Manipulation of y5 T genes utilizing CRISPR-based editing systems is one strategy for precisely augmenting these therapeutic functions. The implementation of Type II and Type V nucleases may facilitate knockout and knock-in of genes in donor-derived y§ T cells.

CRISPR/Cas Enzymes

[0098] The discovery of new Cas enzy mes with unique functionality and structure offers the potential to further gene editing technologies, improving speed, specificity, functionality, and ease of use. Relative to the predicted prevalence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems in microbes and the sheer diversity of microbial species, relatively few functionally characterized CRISPR/Cas enzy mes exist in the literature. This is partly because a huge number of microbial species may not be readily cultivated in laboratory conditions. Metagenomic sequencing from natural environmental niches containing large numbers of microbial species may offer the potential to drastically increase the number of new CRISPR/Cas systems characterized and speed the discovery' of new oligonucleotide editing functionalities. A recent example of the fruitfulness of such an approach is demonstrated by the 2016 discovery of CasX/CasY CRISPR systems from metagenomic analysis of natural microbial communities.

[0099] CRISPR/Cas systems are RNA-directed nuclease complexes that function as an adaptive immune system in microbes. In their natural context, CRISPR/Cas systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally are made up of two parts: (i) an array of short repetitive sequences (30-40 bp) separated by short spacer sequences, which encode the RNA-based targeting element; and (ii) ORFs encoding the Cas nuclease. Efficient nuclease targeting of a particular target nucleic acid sequence generally requires both (i) complementary hybridization between the first 6-8 nucleic acids of the target nucleic acid and a crRNA guide: and (ii) presence of a protospacer-adjacent motif (PAM) sequence within a certain vicinity of the target nucleic acid sequence depending on the specific Cas nuclease (the PAM usually being a sequence not commonly represented within the host genome). Depending on the exact function and organization of the system, CRISPR-Cas systems are commonly organized into 2 classes, 5 types and 16 subtypes based on shared functional characteristics and evolutionary similarity.

[0100] Class 1 CRISPR-Cas systems have large, multi-subunit effector complexes, and include Types I, III, and IV Cas nucleases.

[0101] Type I CRISPR-Cas systems are considered of moderate complexity in terms of components. In Type I CRISPR-Cas systems, the array of RNA-targeting elements is transcribed as a long precursor crRNA (pre-crRNA) that is processed at repeat elements to liberate short, mature crRNAs that direct the nuclease complex to nucleic acid targets when they are followed by a suitable short consensus sequence called a protospacer-adjacent motif (PAM). This processing occurs via an endoribonuclease subunit (Cas6) of a large endonuclease complex called Cascade, which also includes a nuclease (Cas3) protein component of the crRNA-directed nuclease complex. Cas I nucleases function primarily as DNA nucleases.

[0102] Type III CRISPR systems are characterized by the presence of a central nuclease, known as CaslO, alongside a repeat-associated mysterious protein (RAMP) that includes Csm or Cmr protein subunits. Like in Type I systems, the mature crRNA is processed from a pre-crRNA using a Cas6-like enzyme. Unlike Type I and II systems, type III systems appear to target and cleave DNA-RNA duplexes (such as DNA strands being used as templates for an RNA polymerase).

[0103] Type IV CRISPR-Cas systems possess an effector complex that consists of a highly reduced large subunit nuclease (csfl ), two genes for RAMP proteins of the Cas5 (csfl) and Cas7 (csf2) groups, and, in some cases, a gene for a predicted small subunit; such systems are commonly found on endogenous plasmids.

[0104] Class 2 CRISPR-Cas systems generally have single-polypeptide multidomain nuclease effectors, and comprise Types II, V and VI.

[0105] Type II CRISPR-Cas systems are considered the simplest in terms of components. In Type II CRISPR-Cas systems, the processing of the CRISPR array into mature crRNAs does not require the presence of a special endonuclease subunit, but rather a small trans-encoded crRNA (tracrRNA) with a region complementary to the array repeat sequence; the tracrRNA interacts with both its corresponding effector nuclease (e.g. Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate a mature effector enzyme loaded with both tracrRNA and crRNA. Cas II nucleases are identified as DNA nucleases. Type 2 effectors generally exhibit a structure comprising a RuvC-like endonuclease domain that adopts the RNase H fold with an unrelated HNH nuclease domain inserted within the folds of the RuvC-like nuclease domain. The RuvC-like domain is responsible for the cleavage of the target (e.g., crRNA complementary ) DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand.

[0106] Type V CRISPR-Cas systems are characterized by a nuclease effector (e.g. Cas 12) structure similar to that of Type II effectors, comprising a RuvC-like domain. Similar to Type II, most (but not all) Type V CRISPR systems use a tracrRNA to process pre-crRNAs into mature crRNAs. However, unlike Type II systems which requires RNAse III to cleave the pre-crRNA into multiple crRNAs. Type V systems are capable of using the effector nuclease itself to cleave pre-crRNAs. Like Type-II CRISPR-Cas systems, Type V CRISPR-Cas systems are again identified as DNA nucleases. Unlike Type II CRISPR-Cas systems, some Type V enz mes (e.g., Casl2a) appear to have a robust single-stranded nonspecific deoxyribonuclease activity that is activated by the first crRNA directed cleavage of a double-stranded target sequence.

[0107] Type VI CRISPR-Cas systems have RNA-guided RNA endonucleases. Instead of RuvC- like domains, the single polypeptide effector of Type VI systems (e.g. Casl3) include two HEPN ribonuclease domains. Differing from both Type II and V systems, Type VI systems also appear to not require a tracrRNA for processing of pre-crRNA into crRNA. Similar to Type V systems, however, some Type VI systems (e.g., C2C2) appear to possess robust single-stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA directed cleavage of a target RNA.

Gene Editing Systems

[0108] Described herein, in certain embodiments, are engineered y8 T cells modified by contacting the y8 T cells using an engineered system comprising: a) one or more endonucleases or one or more base editors; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the engineered y8 T cells of the disclosure are used for treating cancer.

[0109] Further described herein, in certain embodiments, are gene editing systems comprising: a) one or more endonucleases or one or more base editors; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR).

MG Endonucleases

[0110] Disclosed herein, in some embodiments, are gene editing systems and methods for treating cancer. In some embodiments, the systems and methods comprise one or more endonucleases. In some embodiments, the endonucleases are functional in prokaryotic or eukaryotic cells for in vitro, in vivo, or ex vivo applications. In some embodiments, the endonucleases are nucleic acid guided nucleases, chimeric nucleases, or fusion nucleases. In some embodiments, the gene editing system comprises one or more base editors. In some embodiments, the base editor comprises an endonuclease domain that is deficient in nuclease activity. In some embodiments, gene editing systems described herein comprise a means for cutting a target nucleic acid sequence.

[0111] In some embodiments, the endonuclease is MG29-1 (i.e., SEQ ID NO: 129 and 179). In some embodiments, the endonuclease is MG3-6 (i.e., SEQ ID NO: 127). In some embodiments, the endonuclease is MG21-1 (i.e., SEQ ID NO: 128). MG29-1 is a type V CRISPR nuclease, and MG3-6 and MG21-1 are type II CRISPR nucleases. In some embodiments, the PAM for MG29-1 is functionally defined in mammalian cells as KTTN (K = G or T, N = any base). In some embodiments, the PAM for MG3-6 is functionally defined in mammalian cells as AAANN (N = any base). In some embodiments, the PAM for MG21-1 is functionally defined in mammalian cells as NNRNR (R = A or G, N = any base).

[0112] In some embodiments, the endonuclease is encoded by a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 127. 128, and 179. In some embodiments, the endonuclease is encoded by a sequence having at least about 70% identity to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the endonuclease is encoded by a sequence having at least about 75% identity to any one of SEQ ID NOs: 127, 128. and 179. In some embodiments, the endonuclease is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the endonuclease is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the endonuclease is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the endonuclease is encoded by a sequence having at least about 95% identity' to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the endonuclease is encoded by a sequence having at least about 96% identity' to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the endonuclease is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the endonuclease is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the endonuclease is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the endonuclease is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 127, 128, and 179.

[0113] In some embodiments, the endonuclease comprises an amino acid sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 129. In some embodiments, the endonuclease comprises an amino acid sequence having at least about 70% identity to SEQ ID NO: 129. In some embodiments, the endonuclease comprises an amino acid sequence having at least about 75% identity to SEQ ID NO: 129. In some embodiments, the endonuclease comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 129. In some embodiments, the endonuclease comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 129. In some embodiments, the endonuclease comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 129. In some embodiments, the endonuclease comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 129. In some embodiments, the endonuclease comprises an amino acid sequence having at least about 96% identity⁷ to SEQ ID NO: 129. In some embodiments, the endonuclease comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 129. In some embodiments, the endonuclease comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 129. In some embodiments, the endonuclease comprises an amino acid sequence having at least about 99% identity⁷ to SEQ ID NO: 129. In some embodiments, the endonuclease comprises an amino acid sequence having 100% identity⁷ to SEQ ID NO: 129.

[0114] In some embodiments, the systems and methods described herein comprise a first endonuclease and a second endonuclease. In some embodiments, the first endonuclease and the second endonuclease is MG29-1 (i.e., SEQ ID NO: 129 and 179). In some embodiments, the first endonuclease and the second endonuclease is MG3-6 (i.e., SEQ ID NO: 127). In some embodiments, the first endonuclease and the second endonuclease is MG21-1 (i.e.. SEQ ID NO: 128). In some embodiments, the first endonuclease is MG29-1 (i.e., SEQ ID NO: 129 and 179) and the second endonuclease is MG3-6 (i.e., SEQ ID NO: 127). In some embodiments, the first endonuclease is MG29-1 (i.e., SEQ ID NO: 129 and 179) and the second endonuclease is MG21- I (i.e., SEQ ID NO: 128). In some embodiments, the first endonuclease is MG3-6 (i.e.. SEQ ID NO: 127) and the second endonuclease is MG21-1 (i.e., SEQ ID NO: 128).

[0115] In some embodiments, the first endonuclease and the second endonuclease is encoded by a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity⁷ to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the first endonuclease and the second endonuclease is encoded by a sequence having at least about 70% identity to any one of SEQ ID NOs: 127, 128. and 179. In some embodiments, the first endonuclease and the second endonuclease is encoded by a sequence having at least about 75% identity to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the first endonuclease and the second endonuclease is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 127, 128. and 179. In some embodiments, the first endonuclease and the second endonuclease is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the first endonuclease and the second endonuclease is encoded by a sequence having at least about 90% identity' to any one of SEQ ID NOs: 127, 128. and 179. In some embodiments, the first endonuclease and the second endonuclease is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the first endonuclease and the second endonuclease is encoded by a sequence having at least about 96% identity' to any one of SEQ ID NOs: 127, 128. and 179. In some embodiments, the first endonuclease and the second endonuclease is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the first endonuclease and the second endonuclease is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 127, 128. and 179. In some embodiments, the first endonuclease and the second endonuclease is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 127, 128, and 179. In some embodiments, the first endonuclease and the second endonuclease is encoded by a sequence having 100% identity' to any one of SEQ ID NOs: 127, 128, and 179.

[0116] In some embodiments, the base editor is encoded by a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 175 or SEQ ID NO:

176. In some embodiments, the base editor is encoded by a sequence having at least about 70% identity’ to SEQ ID NO: 175 or SEQ ID NO: 176. In some embodiments, the base editor is encoded by a sequence having at least about 75% identity’ to SEQ ID NO: 175 or SEQ ID NO: 176. In some embodiments, the base editor is encoded by a sequence having at least about 80% identity to SEQ ID NO: 175 or SEQ ID NO: 176. In some embodiments, the base editor is encoded by a sequence having at least about 85% identity to SEQ ID NO: 175 or SEQ ID NO: 176. In some embodiments, the base editor is encoded by a sequence having at least about 90% identity to SEQ ID NO: 175 or SEQ ID NO: 176. In some embodiments, the base editor is encoded by a sequence having at least about 95% identity to SEQ ID NO: 175 or SEQ ID NO: 176. In some embodiments, the base editor is encoded by a sequence having at least about 96% identity' to SEQ ID NO: 175 or SEQ ID NO: 176. In some embodiments, the base editor is encoded by a sequence having at least about 97% identity' to SEQ ID NO: 175 or SEQ ID NO: 176. In some embodiments, the base editor is encoded by a sequence having at least about 98% identity to SEQ ID NO: 175 or SEQ ID NO: 176. In some embodiments, the base editor is encoded by a sequence having at least about 99% identity to SEQ ID NO: 175 or SEQ ID NO: 176. In some embodiments, the base editor is encoded by a sequence having 100% identity to SEQ ID NO: 175 or SEQ ID NO: 176.

[0117] In some embodiments, the endonuclease or base editor comprises one or more fragments or domains of a nuclease, such as nucleic acid-guided nuclease. In some embodiments, the endonuclease or base editor comprises one or more fragments or domains of a nuclease from orthologs of organisms, genus, species, or other phylogenetic groups described herein. In some embodiments, the endonuclease or base editor comprises one or more fragments or domains from nuclease orthologs of different species.

[0118] In some embodiments, the endonuclease or base editor comprises one or more fragments or domains from at least two different nucleases. In some embodiments, the endonuclease or base editor comprises one or more fragments or domains from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleases. In some embodiments, the endonuclease or base editor comprises one or more fragments or domains from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleases from different species. In some embodiments, the endonuclease or base editor comprises 2 fragments or domains, each from a different nuclease. In some embodiments, the endonuclease or base editor comprises 3 fragments or domains, each from a different nuclease. In some embodiments, the endonuclease or base editor comprises 4 fragments or domains, each from a different nuclease. In some embodiments, the endonuclease or base editor comprises 5 fragments or domains, each from a different nuclease. In some embodiments, the endonuclease or base editor comprises 3 fragments or domains, wherein at least one fragment or domain is from a different nuclease. In some embodiments, the endonuclease or base editor comprises 4 fragments or domains, wherein at least one fragment or domain is from a different nuclease. In some embodiments, the endonuclease or base editor comprises 5 fragments or domains, wherein at least one fragment or domain is from a different nuclease.

[0119] In some embodiments, junctions between fragments or domains from different nucleases or species occur in stretches of unstructured regions. Unstructured regions in polynucleotides include, for example, regions that have no predicted secondary structure elements such as alpha helices or beta strands. Unstructured regions may include for example, regions which are exposed within a protein structure, loop regions, or regions that are not conserved within various protein orthologs as predicted by sequence or structural alignments.

[0120] In some embodiments, the endonuclease or base editor comprises one or more nuclear localization sequences (NLSs). In some embodiments, the NLS is at an N-terminus of the endonuclease or base editor. In some embodiments, the NLS is at a C-terminus of the endonuclease or base editor. In some embodiments, the NLS is at an N-terminus and a C- terminus of the endonuclease or base editor.

[0121] In some embodiments, the NLS comprises a sequence of any one of SEQ ID NOs: ISO- 145 and 180-205, or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%. at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having at least about 80% identity to SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having at least about 85% identity to SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having at least about 90% identity to SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having at least about 91% identity to SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having at least about 92% identity to SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having at least about 93% identity' to SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having at least about 94% identity to SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having at least about 95% identity to SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having at least about 96% identity to SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having at least about 97% identity to SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having at least about 98% identity to SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having at least about 99% identity to SEQ ID NOs: 130-145 and 180-205. In some embodiments, the NLS comprises a sequence having 100% identity' to SEQ ID NOs: 130-145 and 180-205.

Table 1: Example NLS sequences that can be used with endonuclease or base editors according to the disclosure

Guide Polynucleotides

[0122] The engineered systems and methods described herein, may comprise guide polynucleotides e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA for supplementing liver enzymes. In a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA. In some embodiments, the engineered systems and methods described herein comprise a means for directing the endonuclease or base editor to a particular location in the target nucleic acid.

[0123] In some embodiments, the guide polynucleotide is configured to form a complex with the endonuclease or base editor. In some embodiments, the guide polynucleotide binds to the endonuclease or base editor to form a complex. In some embodiments, the guide polynucleotide binds (e.g., non-covalently through electrostatic interactions or hydrogen bonds) to the endonuclease or base editor to form a complex. In some embodiments, the guide polynucleotide is fused to the endonuclease or base editor to form a complex.

[0124] In some embodiments, the guide polynucleotide comprises a spacer sequence. In some embodiments, the spacer sequence is configured to hybridize to a target nucleic acid sequence. In some embodiments, the endonuclease or base editor is configured to bind to a protospacer adjacent motif (PAM) sequence.

[0125] In some embodiments, the guide polynucleotide (e.g., gRNA) targets or hybridizes to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1. TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the guide polynucleotide (e.g., gRNA) targets a gene or a locus in a cell. In some embodiments, the locus is selected from the group consisting of a PD-1. TIM3, IL- 17, TRAC, and B2M locus. In some embodiments, the cell is a T cell. In some embodiments, the cell is a y5 T cell. [0126] In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. or at least about 99% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 116-126 and 146-172.

[0127] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary’ to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary⁷ to any one of SEQ ID NOs: 116- 126 and 146-172 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary⁷ to a sequence having at least about 85% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 116-126 and 146- 172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 97% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1 1 -126 and 146-172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 116-126 and 146-172.

[0128] In some embodiments, the systems and methods described herein comprise a first guide polynucleotide and a second guide polynucleotide. In some embodiments, the first engineered guide polynucleotide and the second guide polynucleotide target or hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus. In some embodiments, the first e guide polynucleotide and the second guide target a gene or a locus in a cell. In some embodiments, the locus is selected from the group consisting of a PD-1. TIM3, IL- 17, TRAC, and B2M locus. In some embodiments, the cell is a T cell. In some embodiments, the cell is a

T cell.

[0129] In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize to a target nucleic acid sequence within the TRAC or the IL-17 locus. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize to a target nucleic acid sequence within the PD-1 or the 11-17 locus. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus.

[0130] In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 116- 126 and 146-172.

[0131] In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize or target a sequence complementary to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize or target a sequence complementary to any one of SEQ ID NOs: 116-126 and 146-172 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize or target a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize or target a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize or target a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize or target a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize or target a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize or target a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize or target a sequence complementary' to a sequence having at least about 98% identity' to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize or target a sequence complementary' to a sequence having at least about 99% identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide hybridize or target a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 116-126 and 146-172.

[0132] In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 117-119, and 123. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identity' to any one of SEQ ID NOs: 117-119, and 124-126. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 123-126. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 120-123.

[0133] In some embodiments, the guide polynucleotides (e.g., guide RNAs) comprise various structural elements including but not limited to: a spacer sequence which binds to the protospacer sequence (target sequence), a crRNA, and an optional tracrRNA. In some embodiments, the genome editing system comprises a CRISPR guide RNA. In some embodiments, the guide RNA comprises a crRNA comprising a spacer sequence. In some embodiments, the guide RNA additionally comprises a tracrRNA or a modified tracrRNA.

[0134] In some embodiments, the guide polynucleotide comprises a sense sequence. In some embodiments, the guide polynucleotide comprises an anti-sense sequence. In some embodiments, the guide polynucleotide comprises nucleotide sequences other than the region complementary to or substantially complementary' to a region of a target sequence. For example, a crRNA is part or considered part of a guide polynucleotide, or is comprised in a guide polynucleotide, e.g., a crRNA:tracrRNA chimera.

[0135] In some embodiments, the guide polynucleotide comprises synthetic nucleotides or modified nucleotides. In some embodiments, the guide polynucleotide comprises one or more inter-nucleoside linkers modified from the natural phosphodi ester. In some embodiments, all of the inter-nucleoside linkers of the guide polynucleotide, or contiguous nucleotide sequence thereof, are modified. For example, in some embodiments, the inter nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage. In some embodiments, the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified inter- nucleoside linkers. In some embodiments, the guide polynucleotide comprises 1. 2, 3, 4, 5. 6, 7, 8, 9, 10, or more than 10 modified inter-nucleoside linkers (e.g.. phosphorothioate inter- nucleoside linkage).

[0136] In some embodiments, the guide polynucleotide comprises modifications to a ribose sugar or nucleobase. In some embodiments, the guide polynucleotide comprises one or more nucleosides comprising a modified sugar moiety', wherein the modified sugar moiety is a modification of the sugar moiety⁷ when compared to the ribose sugar moiety found in deoxyribose nucleic acid (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, replacement with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g, locked nucleic acids (LNA)), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g, UNA). In some embodiments, the sugar-modified nucleosides comprise bicyclohexose nucleic acids or tricyclic nucleic acids. In some embodiments, the modified nucleosides comprise nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example peptide nucleic acids (PNA) or morpholino nucleic acids. [0137] In some embodiments, the guide polynucleotide comprises one or more modified sugars. In some embodiments, the sugar modifications comprise modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2’ -OH group naturally found in DNA and RNA nucleosides. In some embodiments, substituents are introduced at the 2’, 3’, 4’, 5’ positions, or combinations thereof. In some embodiments, nucleosides with modified sugar moieties comprise 2‘ modified nucleosides, e.g., 2’ substituted nucleosides. A 2’ sugar modified nucleoside, in some embodiments, is a nucleoside that has a substituent other than H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradical, and comprises 2’ substituted nucleosides and LNA (2’-4’ biradical bridged) nucleosides. Examples of 2 ’-substituted modified nucleosides comprise, but are not limited to, 2’-O-alkyl-RNA, 2 -0- methyl-RNA, 2’-alkoxy-RNA. 2’-O-methoxy ethyl- RNA (MOE), 2’-amino-DNA, 2’-Fluoro- RNA, and 2’-F-ANA nucleoside. In some embodiments, the modification in the ribose group comprises a modification at the 2’ position of the ribose group. In some embodiments, the modification at the 2’ position of the ribose group is selected from the group consisting of 2’-O- methyl, 2’-fluoro, 2’-deoxy, and 2’ -O-(2 -methoxy ethyl).

[0138] In some embodiments, the guide polynucleotide comprises one or more modified sugars. In some embodiments, the guide polynucleotide comprises only modified sugars. In some embodiments, the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2’-O-methyl. In some embodiments, the modified sugar comprises a 2’ -fluoro. In some embodiments, the modified sugar comprises a 2’-O- methoxy ethyl group. In some embodiments, the guide polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified sugars (e.g., comprising a 2’-O-methyl or 2’-fluoro).

[0139] In some embodiments, the guide polynucleotide comprises both inter-nucleoside linker modifications and nucleoside modifications. In some embodiments, the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified inter-nucleoside linkers and greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the guide polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified inter- nucleoside linkers (e.g., phosphorothioate inter-nucleoside linkage) and 1, 2, 3. 4, 5, 6, 7, 8, 9, 10. or more than 10 modified sugars (e.g., comprising a 2’-O-methyl or 2’-fluoro).

[0140] In some embodiments, the guide polynucleotide is 30-250 nucleotides in length. In some embodiments, the guide polynucleotide is more than 90 nucleotides in length. In some embodiments, the guide polynucleotide is less than 245 nucleotides in length. In some embodiments, the guide polynucleotide is 30, 40, 50, 60, 70, 80, 90, 100. 120, 140. 160, 180, 200, 220, 240, or more than 240 nucleotides in length. In some embodiments, the guide polynucleotide is about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 120, about 30 to about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200. about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 120, about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240, about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180. about 100 to about 200, about 100 to about 220. about 100 to about 240, about 160 to about 180, about 1 0 to about 200, about 160 to about 220, or about 160 to about 240 nucleotides in length.

MG Gene Editing Systems

[0141] Described herein, in certain embodiments, are engineered yb T cells, wherein the engineered yb T cells have been modified in one or more loci using an engineered system comprising: a) one or more endonucleases or one or more base editors; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease or the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1. T1M3, IL- 17, TRAC, and B2M locus.

[0142] The disclosure further provides, in certain embodiments, a gene editing system comprising: a) one or more endonucleases or one or more base editors; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease or the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR).

[0143] In some embodiments, the endonuclease or base editor induces a single-stranded break at or proximal to the target nucleic acid sequence. In some embodiments, the endonuclease or base editor induces a double-stranded break at or proximal to the target nucleic acid sequence. In some embodiments, the donor template is integrated into the target nucleic acid sequence at the doublestranded break. In some embodiments, the donor template is integrated into the target nucleic acid sequence at the double-stranded break via non-homologous end joining (NHEJ). In some embodiments, the donor template is integrated into the target nucleic acid sequence at the doublestranded break via homology-directed repair (HDR). [0144] In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 127. 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127. 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL- 17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1 , TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 127. 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). [0145] In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL- 17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 96% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD- 1 , TIM3, IL-17, TRAC, and B2M; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 97% sequence identity’ to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 98% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 99% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 100% sequence identity’ to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR).

[0146] In some embodiments, the engineered guide polynucleotide is a single guide nucleic acid. In some embodiments, the engineered guide polynucleotide is a dual guide nucleic acid. In some embodiments, the engineered guide polynucleotide is RNA. In some embodiments, the endonuclease is in a complex with the engineered guide polynucleotide. In some embodiments, the endonuclease is linked to the engineered guide polynucleotide.

[0147] In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 127. 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 70% sequence identity' to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex yvith the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity' to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 85% sequence identity⁷ to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex yvith the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127. 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 116-126 and 146- 172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL- 17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 1 16-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127. 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M wherein the engineered guide poly nucleotide comprises a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 116-126 and 146- 172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more endonucleases encoded by a sequence having at least 100% sequence identity⁷ to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1 , TIM3, IL- 17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR).

[0148] In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 70% sequence identity⁷ to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 85% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence wi thin a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 95% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 95% sequence identify to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 96% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 96% sequence identify to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 97% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 98% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 99% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17. TRAC, and B2M wherein the engineered guide polynucleotide comprises a sequence having at least 99% sequence identify to any one of SEQ ID NOs: 116-126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the gene editing system comprises a) one or more base editors encoded by a sequence having at least 100% sequence identify’ to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus, wherein the engineered guide polynucleotide comprises a sequence having at least 100% sequence identify to any one of SEQ ID NOs: 116- 126 and 146-172; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR).

[0149] In some embodiments, the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the donor nucleic acid further encodes a second CAR. In some embodiments, the first CAR and the second CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular antigen binding domain In certain cases, the first CAR and the second CAR comprises domains for additional co-stimulatory signaling.

[0150] In some embodiments, the first CAR and the second CAR comprises an extracellular antigen binding domain. In some embodiments, the extracellular antigen binding domain binds to a tumor-associated antigen. In some embodiments, the extracellular antigen binding domain binds to a tumor-associated antigen selected from the group consisting of MUC16, FOLR1, BCMA, CLDN6, SLC34A2. and TAG72. In some embodiments, the extracellular antigen binding domain of the first CAR binds to MUC16 or FOLR1. In some embodiments, the extracellular antigen binding domain of the second CAR binds to MUC 1 or FOLR1 . [0151] In some embodiments, the extracellular antigen binding domain comprises an antibody. In some embodiments, the extracellular antigen binding domain comprises an antibody fragment. In some embodiments the extracellular antigen binding domain comprises a T-cell receptor (TCR) variable alpha (Va) and variable beta (VB) domain, or a TCR variable delta (Vy) and variable gamma (V5) domain. In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv). In some embodiments, the scFv is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 9-12.

[0152] In some embodiments, the first CAR and the second CAR comprises a transmembrane (TM) domain. In some embodiments, the TM is selected from a group consisting of a CD2, CD3, CD4, CD16, CD64, CD28, CD8, and a 41 BBL TM domain. In some embodiments, the TM is a CD8 TM domain. In some embodiments, the TM domain is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16. In some embodiments, the first CAR and the second CAR comprises a hinge region. In some embodiments, the hinge region is selected from a hinge region of a CD4 or a CD8 hinge region.

[0153] In some embodiments, the first CAR and the second CAR comprises a signaling domain. In some embodiments, the signaling domain is a signaling domain of CD3 . In some embodiments, the signaling domain is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the signaling domain comprises one or more immunotyrosine activation motifs (ITAMs). In some embodiments, the ITAM domains are selected from the group consisting of an 1TAM domain of CD3 . CD3s, CD3y, CD35, an DAP12. In some embodiments, the ITAM domain is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 18 and 102-111. In some embodiments, the first CAR and the second CAR comprises no signaling domain.

[0154] In some embodiments, the first CAR and the second CAR comprises one or more costimulatory domains. In some embodiments, costimulatory domains are selected from a group consisting of a CD28, 41BB. CD2, CD4, CD27. CD30, 0X40, CD40, CD84. CD226, CD244, CD258, DR3, FCRL1, FCRL6, GITR, LAG3, SLAM, and TIM1 costimulatory domain. In some embodiments, the costimulatory domain is a CD28 costimulatory domain. In some embodiments, the costimulatory domain is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 17 and 87-101.

[0155] In some embodiments, the first CAR and the second CAR comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity⁷ to any one of SEQ ID NOs: 28-31. In some embodiments, the first CAR and the second CAR comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 28-31. In some embodiments, the first CAR and the second CAR comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 28-31. In some embodiments, the first CAR and the second CAR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 28-31. In some embodiments, the first CAR and the second CAR comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 28-31 . In some embodiments, the first CAR and the second CAR comprises a sequence having at least 96% sequence identity⁷ to any one of SEQ ID NOs: 28-31. In some embodiments, the first CAR and the second CAR comprises a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 28-31. In some embodiments, the first CAR and the second CAR comprises a sequence having at least 98% sequence identity⁷ to any one of SEQ ID NOs: 28-31. In some embodiments, the first CAR and the second CAR comprises a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 28-31. In some embodiments, the first CAR and the second CAR comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 28-31.

[0156] In some embodiments, the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the donor nucleic acid encodes a first cytokine. In some embodiments, the donor nucleic acid further encodes a second CAR. In some embodiments, the donor nucleic acid further encodes a second cytokine. In some embodiments, the first cytokine is IL-12 or IL-15. In some embodiments, the second cytokine is IL-12 or IL-15. In some embodiments, the first cytokine is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 25-27. In some embodiments, the second cytokine is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 25-27.

[0157] In some embodiments, the extracellular antigen binding domain of the first CAR binds to MUC16 and the first cytokine is IL- 12. In some embodiments, the extracellular antigen binding domain of the first CAR binds to MUC16 and the first cytokine is IL-15. In some embodiments, the extracellular antigen binding domain of the first CAR binds to FOLR1 and the first cytokine is IL-12. In some embodiments, the extracellular antigen binding domain of the first CAR binds to FOLR1 and the first cytokine is IL-15. In some embodiments, the first cytokine is IL- 12 and the second. In some embodiments, the first cytokine is IL-12 and the second cytokine is IL-15. [0158] In some embodiments, the extracellular antigen binding domain of the first CAR comprises a single chain variable fragment (scFv) that is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 9-12 and the first cytokine is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 25. In some embodiments, the extracellular antigen binding domain of the first CAR comprises a single chain variable fragment (scFv) that is encoded by a sequence having at least 70%, at least 80%, at least 85%. at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 10 and the first cytokine is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 25. In some embodiments, the extracellular antigen binding domain of the first CAR comprises a scFv that is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 11 and the first cytokine is encoded by a sequence having at least 80% sequence identity’ to SEQ ID NOs: 25. In some embodiments, the extracellular antigen binding domain of the first CAR comprises a scFv that is encoded by a sequence having at least 70%. at least 80%. at least 85%. at least 90%, at least 95%. at least 96%. at least 97%. at least 98%. at least 99%, or 100% sequence identity to SEQ ID NOs: 10 and the first cytokine is encoded by a sequence having at least 80% sequence identity' to SEQ ID NOs: 26. In some embodiments, the extracellular antigen binding domain of the first CAR comprises a scFv that is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 11 and the first cytokine is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 26. In some embodiments, the extracellular antigen binding domain of the first CAR comprises a scFv that is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 10 and the first cytokine is encoded by a sequence having at least 70% sequence identity’ to SEQ ID NOs: 27. In some embodiments, the extracellular antigen binding domain of the first CAR comprises a scFv that is encoded by a sequence having at least 70%. at least 80%. at least 85%, at least 90%, at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 11 and the first cytokine is encoded by a sequence having at least 80% sequence identity' to SEQ ID NOs: 27. [0159] In some embodiments, the first CAR comprises a sequence having at least 70% at least 80%. at least 85%. at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 28-31 and the first cytokine is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 25. In some embodiments, the first CAR comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity’ to SEQ ID NOs: 29 and the first cytokine is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 25. In some embodiments, the first CAR comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 30 and the In some embodiments, the first CAR comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 29 and the first cytokine is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 26. In some embodiments, the first CAR comprises a sequence having at least 70%, at least 80%, at least 85%. at least 90%. at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 30 and the first cytokine is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 26. In some embodiments, the first CAR comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 29 and the first cytokine is encoded by a sequence having at least 70% sequence identity to SEQ ID NOs: 27. In some embodiments, the first CAR comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 30 and the first cytokine is encoded by a sequence having at least 80% sequence identity' to SEQ ID NOs: 27.

[0160] In some embodiments, the donor nucleic acid comprises a first homology arm and a second homology' arm. In some embodiments, the first homology arm comprises a sequence located on the 5' side of the target nucleic acid sequence. In some embodiments, the second homology arm comprises a sequence located on the 3‘ side of the target nucleic acid sequence. In some embodiments, the first homology arm comprises a sequence located on the 5’ side of the target nucleic acid sequence and the second homology arm comprises a sequence located on the 3’ side of the target nucleic acid sequence. In some embodiments, the first homology arm comprises a sequence of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60. at least 70, at least 80, at least 90, at least 100, at least 1 10, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200, at least 250, at least 300, at least 400, at least 500, at least 750, or at least 1000 nucleotides. In some embodiments, the second homology arm comprises a sequence of at least 10, at least 20, at least 30. at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200, at least 250, at least 300, at least 400, at least 500, at least 750, or at least 1000 nucleotides. In some embodiments, the first homology arm comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 46, 48, 50, and 52. In some embodiments, the second homology arm comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 47, 49. 51, and 53.

[0161] In some embodiments, the donor nucleic acid comprises a polyadenylation signal. In some embodiments, the polyadenylation signal is at a C-terminus of a sequence encoding the first or the second CAR. In some embodiments, the polyadenylation signal is linked to a sequence encoding the first or the second CAR. In some embodiments, the polyadenylation signal is fused to a sequence encoding the first or the second CAR.

Cells

[0162] Described herein, in certain embodiments, is an engineered cell comprising the gene editing system described herein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a y5 T cell.

[0163] In some embodiments, the disclosure provides a T cell comprising a gene editing system comprising: a) one or more endonucleases encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more endonucleases encoded by a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more endonucleases encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more endonucleases encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more endonucleases encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127. 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more endonucleases encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more endonucleases encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more endonucleases encoded by a sequence having 100% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17. TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the cell is a yd T cell. In some embodiments, the engineered yd T cells of the disclosure are used for treating cancer.

[0164] In certain aspects, the disclosure provides an engineered yd T cell, wherein the engineered yd T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus. In some embodiments, the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the engineered yd T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the engineered yd T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus. In some embodiments, the engineered yd T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 95% sequence identity⁷ to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus. In some embodiments, the engineered y8 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the engineered y6 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the engineered y8 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus. In some embodiments, the engineered y6 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus. In some embodiments, the engineered y8 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having 100% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus.

[0165] In some embodiments, the disclosure provides a T cell comprising a gene editing system comprising: a) one or more base editors encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more base editors encoded by a sequence having at least 85% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD- 1 , TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more base editors encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more base editors encoded by a sequence having at least 96% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more base editors encoded by a sequence having at least 97% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more base editors encoded by a sequence having at least 98% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more base editors encoded by a sequence having at least 99% sequence identity⁷ to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-L TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the T cell comprises a gene editing system comprising: a) one or more base editors encoded by a sequence having 100% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the cell is a yd T cell. In some embodiments, the engineered yd T cells of the disclosure are used for treating cancer.

[0166] In certain aspects, the disclosure provides an engineered yd T cell, wherein the engineered yd T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 70% sequence identity⁷ to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the engineered yd T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the engineered yd T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 85% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus. In some embodiments, the engineered yd T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 90% sequence identity' to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus. In some embodiments, the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the engineered y8 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 96% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the engineered y8 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 97% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus. In some embodiments, the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 98% sequence identity⁷ to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus. In some embodiments, the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 99% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having 100% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. [0167] In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 116-126 and 146- 172.

[0168] In some embodiments, the engineered y5 T cell has been further modified to express a first (CAR). In some embodiments, the engineered y5 T cell has been further modified to express a first cytokine. In some embodiments, the engineered y5 T cell has been further modified to express a second CAR. In some embodiments, the engineered y8 T cell has been further modified to express a second cytokine. In some embodiments, the first CAR and the second CAR comprises an extracellular antigen binding domain, and the extracellular antigen binding domain binds to a tumor-associated antigen selected from the group consisting of MUC16. FOLR1, BCMA, CLDN6, SLC34A2, and TAG72. In some embodiments, the extracellular antigen binding domain of the first CAR binds to MUC16 or FOLR1. In some embodiments, the extracellular antigen binding domain of the second CAR binds to MUC16 or FOLR1. In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv). In some embodiments, the scFv is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity⁷ to any one of SEQ ID NOs: 9-12. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 10. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 11. In some embodiments, the first CAR or the second CAR comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 28-31. In some embodiments, the first cytokine is IL-12 or IL-15. In some embodiments, the second cytokine is IL- 12 or IL-15.

[0169] In some embodiments, the engineered system comprises one or more donor nucleic acids. In some embodiments, the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the donor nucleic acid encodes a first cytokine. In some embodiments, the donor nucleic acid further encodes a second CAR. In some embodiments, the donor nucleic acid further encodes a second cytokine. In some embodiments, the first CAR and the second CAR comprises an extracellular antigen binding domain, and the extracellular antigen binding domain binds to a tumor-associated antigen selected from the group consisting of MUC16, FOLR1, BCMA, CLDN6, SLC34A2. and TAG72. In some embodiments, the extracellular antigen binding domain of the first CAR binds to MUC16 or FOLR1 . In some embodiments, the extracellular antigen binding domain of the second CAR binds to MUC16 or FOLR1. In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv). In some embodiments, the scFv is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 9-12. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 10. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 11. In some embodiments, the first CAR or the second CAR comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity⁷ to any one of SEQ ID NOs: 28-31. In some embodiments, the first cytokine is IL-12 or IL-15. In some embodiments, the second cytokine is IL- 12 or IL- 15.

[0170] In some embodiments, the engineered yb T cell has one or more modifications within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the engineered yo T cell has a modification within a TRAC locus and a PD-1 locus. In some embodiments, the engineered yo T cell has a modification within a TRAC locus and an IL- 17 locus. In some embodiments, the engineered y5 T cell has a modification within a PD-1 locus and an IL- 17 locus. In some embodiments, the engineered yb T cell has a modification within a PD-1 locus and a TIM3 locus.

[0171] Further disclosed herein are methods of making an engineered yb T cell having a modification in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus. In some embodiments, the method of making an engineered yb T cell having a modification in one or more loci using an engineered system comprises a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered yo T cell having a modification in one or more loci using an engineered system comprises a) one or more endonucleases encoded by a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered y§ T cell having a modification in one or more loci using an engineered system comprises a) one or more endonucleases encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus. In some embodiments, the method of making an engineered y§ T cell having a modification in one or more loci using an engineered system comprises a) one or more endonucleases encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1 , TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered yo T cell having a modification in one or more loci using an engineered system comprises a) one or more endonucleases encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered y5 T cell having a modification in one or more loci using an engineered system comprises a) one or more endonucleases encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered yb T cell having a modification in one or more loci using an engineered system comprises a) one or more endonucleases encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-L TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered yb T cell having a modification in one or more loci using an engineered system comprises a) one or more endonucleases encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered y5 T cell having a modification in one or more loci using an engineered system comprises a) one or more endonucleases encoded by a sequence having 100% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL- 17, TRAC, and B2M locus.

[0172] Further disclosed herein are methods of making an engineered y5 T cell having a modification in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus. In some embodiments, the method of making an engineered y5 T cell having a modification in one or more loci using an engineered system comprises a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered y§ T cell having a modification in one or more loci using an engineered system comprises a) one or more base editors encoded by a sequence having at least 85% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered yo T cell having a modification in one or more loci using an engineered system comprises a) one or more base editors encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus. In some embodiments, the method of making an engineered y5 T cell having a modification in one or more loci using an engineered system comprises a) one or more base editors encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD- 1 , TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered y5 T cell having a modification in one or more loci using an engineered system comprises a) one or more base editors encoded by a sequence having at least 96% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence wi thin a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered y5 T cell having a modification in one or more loci using an engineered system comprises a) one or more base editors encoded by a sequence having at least 97% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus. In some embodiments, the method of making an engineered y8 T cell having a modification in one or more loci using an engineered system comprises a) one or more base editors encoded by a sequence having at least 98% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered y8 T cell having a modification in one or more loci using an engineered system comprises a) one or more base editors encoded by a sequence having at least 99% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of making an engineered y8 T cell having a modification in one or more loci using an engineered system comprises a) one or more base editors encoded by a sequence having 100% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus.

[0173] In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 116-126 and 146- 172.

[0174] In some embodiments, the engineered system comprises one or more donor nucleic acids. In some embodiments, the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR). In some embodiments, the donor nucleic acid encodes a first cytokine. In some embodiments, the donor nucleic acid further encodes a second CAR. In some embodiments, the donor nucleic acid further encodes a second cytokine. In some embodiments, the first CAR and the second CAR comprises an extracellular antigen binding domain, and the extracellular antigen binding domain binds to a tumor-associated antigen selected from the group consisting of MUC16, FOLR1, BCMA, CLDN6, SLC34A2, and TAG72. In some embodiments, the extracellular antigen binding domain of the first CAR binds to MUC16 or FOLR1. In some embodiments, the extracellular antigen binding domain of the second CAR binds to MUC 16 or FOLR1. In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv). In some embodiments, the scFv is encoded by a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 9-12. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 10. In some embodiments, the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 11. In some embodiments, the first CAR or the second CAR comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 28-31. In some embodiments, the first cytokine is IL-12 or IL-15. In some embodiments, the second cytokine is IL- 12 or IL-15.

[0175] In some embodiments, the donor nucleic acid comprises a first homology arm and a second homology arm; and the first homology arm comprises a sequence located on the 5’ side of the target nucleic acid sequence and the second homology arm comprises a sequence located on the 3’ side of the target nucleic acid sequence. In some embodiments, the first homology arm comprises a sequence having at least 70%, at least 80%, at least 85%. at least 90%. at least 95%. at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 46, 48, 50, and 52. In some embodiments, the second homology arm comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 47, 49, 51, and 53.

[0176] In some embodiments, the yb T cells (or gamma delta T cells) described herein is a 51, 52, 53, or 54 yb T cells, or a combination thereof. In some embodiments, the yb T cells are V51 or V52 y5 T cells. In some embodiments, the population of y5 T cells described herein are a combination of V51 and V52 y5 T cells. In some embodiments, the yb T cells are naive, effector memory, central memory, or terminally differentiated yb T-cells. In some embodiments, the y5 T cells are V54, V55, V57, and V58 y5 T cells. In some embodiments, the y5 T cells are Vy2, Vy3, Vy5, Vy8, Vy9, VylO, and Vyl 1 y5 T cells. In some embodiments, the y5 T cells are V51, V52, or V61 and V52 T cells.

[0177] In some embodiments, the engineered y5 T cell described herein are made from unmodified cells. In some embodiments, the engineered y5 T cell described herein are made from unmodified wild-type T cells. In some embodiments, a donor cell is used to make the engineered yd T cell described herein. In some embodiments, the donor cell is obtained from a subject. In some embodiments, the donor cell is obtained from an allogeneic donor. In some embodiments, the donor cell is obtained from an autologous donor. In some embodiments, the donor cell is mammalian donor cell. In some embodiments, the donor cell is a human cell. In some embodiments, the donor cell is a T cell. In some embodiments, the donor cell is a y5 T. In some embodiments, y5 T cells are obtained from an allogeneic or an autologous donor.

[0178] In some embodiments, the donor T cell is a primary T cell (e g., non-transformed and terminally differentiated T cells) obtained from one or more human donors. In some embodiments, the donor T cell are differentiated from precursor T cells obtained from one or more suitable donor or stem cells such as hematopoietic stem cells or inducible pluripotent stem cells (iPSC). In some embodiments, T cells from a T cell bank are used as the starting material for preparing the engineered y5 T disclosed herein.

[0179] In some embodiments, the yd T cells are partially or entirely purified or not purified. In some embodiments, yd T cells are expanded ex vivo. In some embodiments, the expansion is performed before or after, or before and after, the gene editing system is introduced into the yd T cell(s). In some embodiments, the engineered yd T cells described herein are stored, e g., cryopreserved, for use in adoptive cell therapy.

[0180] Provided herein, in certain embodiments, are pharmaceutical compositions comprising the engineered T cells of the disclosure. The pharmaceutical compositions described herein are designed for deliver}’ to subjects in need thereof by any suitable route or a combination of different routes. The cells can be formulated in a manner appropriate to the disease to be treated. Factors that determine formulation, include the particular disorder being treated, the clinical condition of the individual patient, the cause of the disorder, the site of deliver)’ of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0181] In some embodiments, therapeutic formulations comprising the cells disclosed herein are frozen, or prepared for administration with physiologically acceptable carriers, excipients or stabilizers (see e.g., Remington's Pharmaceutical Sciences 16th edition. Osol, A. Ed. (1980)), in the form of aqueous solutions. In some embodiments, the T cells of the disclosure are formulated for intravenous administration. In some embodiments, the composition comprises buffers such as neutral buffered saline or phosphate buffered saline. In some embodiment, the composition comprises physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline.

[0182] The engineered system described herein, or components thereof can be introduced into a cell in any suitable way, either stably or transiently. In some embodiments, the system described herein, or components thereof is transfected into a cell. In some embodiments, the cell is transduced or transfected with a nucleic acid construct that encodes the system described herein or components thereof. For example, a cell is transduced (e.g, with a virus encoding the engineered system described herein or components thereof), or transfected (e.g, with a plasmid encoding the engineered system described herein or components thereof) with a nucleic acid that encodes the engineered system described herein or components thereof, or the translated the engineered system described herein or components thereof. In some embodiments, the transduction is a stable or transient transduction. In some embodiments, cells expressing the engineered system described herein or components thereof or containing the engineered system described herein or components thereof are transduced or transfected with one or more gRNA molecules, for example, when the engineered system described herein or components thereof comprises a CRISPR nuclease. In some embodiments, a plasmid expressing the engineered system described herein or components thereof is introduced into cells through electroporation, transient (e.g, lipofection) and stable genome integration (e.g, piggyBac®) and viral transduction (for example lentivirus or AAV) or other methods known to those of skill in the art. In some embodiments, the gene editing system is introduced into the cell as one or more polypeptides. In some embodiments, delivery is achieved through the use of RNP complexes. Delivery methods to cells for polypeptides and/or RNPs are known in the art, for example by electroporation or by cell squeezing.

[0183] Exemplary methods of delivery of nucleic acids include lipofection, nucleofection, electroporation, stable genome integration (e.g., piggyBac®), microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g.. U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and SF Cell Line 4D-Nucleofector X Kit™ (Lonza)). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of WO 91/17424 and WO 91/16024. In some embodiments, the delivery is to cells (e.g.. in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). In some embodiments, the nucleic acid is comprised in a liposome or a nanoparticle that specifically targets a cell. Additional methods for the delivery' of nucleic acids to cells are known to those skilled in the art. See, for example, US 2003/0087817.

[0184] In some embodiments, the present disclosure provides a cell comprising a gene editing system described herein. In some embodiments, the cell expresses a gene editing system or parts thereof. In some embodiments, the cell is a human cell. In some embodiments, the cell is a human T cell. In some embodiments, the cell is a y8 T cell. In some embodiments, the cell is modified ex vivo. In some embodiments, the cell is modified in vivo.

[0185] Disclosed herein, in certain embodiments, are lipid nanoparticles comprising a gene editing system of the disclosure for delivery' into a cell. In certain embodiments, the engineered y8 T cell of the disclosure is made by introducing lipid nanoparticles comprising an engineered system into a cell. In some embodiments, the lipid nanoparticle comprises the engineered system or a nucleic acid encoding the engineered system. In some embodiments, the lipid nanoparticle comprises the one or more components of the engineered system. In some embodiments, the lipid nanoparticle comprises the endonuclease or a nucleic acid encoding the endonuclease. In some embodiments, the lipid nanoparticle comprises the engineered guide polynucleotide. In some embodiments, the lipid nanoparticle comprises the donor nucleic acid. In some embodiments, the lipid nanoparticle is tethered to the engineered system.

[0186] In some embodiments, lipid nanoparticles as described herein are 4-component lipid nanoparticles. Such nanoparticles can be configured for delivery of RNA or other nucleic acids (e.g., synthetic RNA, mRNA, or in vzfro-synthesized mRNA). Such nanoparticles can generally comprise: (a) a cationic lipid, (b) a neutral lipid (e.g., DSPC or DOPE), (c) a sterol (e.g., cholesterol or a cholesterol analog), or (d) a PEG-modified lipid (e.g., PEG-DMG).

[0187] In some embodiments, cationic lipid formulations include particles comprising either 3 or 4 or more components in addition to polynucleotide, primary construct, or RNA (e.g., mRNA). As an example, formulations with certain cationic lipids, include, but are not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (Cl 4 or greater alkyl chain length). As another example, formulations with certain lipidoids include, but are not limited to, C12-200 and may contain 50% cationic lipid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.

[0188] In some embodiments, the cationic lipid nanoparticle comprises a cationic lipid, a PEG- modified lipid, a sterol, and a non-cationic lipid. In some embodiments, the cationic lipid nanoparticle has a molar ratio of about 20-60% cationic lipid: about 5-25% non-cationic lipid: about 25-55% sterol; and about 0.5-15% PEG-modified lipid. In some embodiments, the cationic lipid nanoparticle comprises a molar ratio of about 50% cationic lipid, about 1.5% PEG-modified lipid, about 38.5% cholesterol, and about 10% non-cationic lipid. In some embodiments, the cationic lipid nanoparticle comprises a molar ratio of about 55% cationic lipid, about 2.5% PEG- modified lipid, about 32.5% cholesterol, and about 10% non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol. In some embodiments, the cationic lipid nanoparticle has a molar ratio of 50:38.5: 10: 1.5 of cationic lipid: cholesterol: PEG2000-DMG:DSPC or DMG:DOPE. In some embodiments, lipid nanoparticles as described herein comprise cholesterol, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,1 ‘-((2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethyl)azanediyl)bis(dodecan-2-ol) (C 12-200), and DMG-PEG-2000 at molar ratios of 47.5: 16:35:1.5.

Methods of Use

[0189] Provided herein are methods for treating cancer and other conditions using the gene editing systems and engineered y5 T cells described herein. Methods for treating cancer and other conditions comprise modifying one or more loci selected from the group consisting of a PD-1. TIM3, IL-17, TRAC, and B2M locus in a y8 T cell. In some embodiments, the y8 T cell are further modified to express a CAR that targets specific tumor cells. In some embodiments, the y5 T cells described herein are used to target undesired cells in an individual in need thereof. In some embodiments, the y5 T cells described herein are used to target tumor cells in an individual in need thereof, thereby treating cancer. In some embodiments, the engineered systems described herein are used to treat cancer in an individual in need thereof.

[0190] In some embodiments, the cancer comprises one or more malignant tumors. In some embodiments, the cancer is metastatic. Examples of cancers include, but are not limited to ovarian, endometrial, lung, breast, brain, kidney, and colon cancer.

[0191] In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y5 T cell, wherein the engineered y§ T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y8 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered 76 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17. TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered 76 T cell, wherein the engineered 78 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered 76 T cell, wherein the engineered 78 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered 78 T cell, wherein the engineered 78 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17. TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered 78 T cell, wherein the engineered y6 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y5 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y6 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, T1M3, IL- 17. TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y5 T cell, wherein the engineered y6 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having 100% sequence identity' to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 116-126 and 146- 172. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172.

[0192] In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y5 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y6 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1 , TIM3, IL- 17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y5 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 85% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y5 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y6 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL- 17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y5 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 96% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y6 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 97% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y5 T cell, wherein the engineered yo T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 98% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y8 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 99% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of treating cancer in a subject in need thereof, comprises administering to the subject a therapeutically effective amount of an engineered y6 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having 100% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL- 17, TRAC, and B2M locus. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 116-126 and 146-172.

[0193] In some embodiments, methods described herein are for killing or inhibiting a cancer cell. In some embodiments, the cancer cell is a cell undergoing early, intermediate or advanced stages of multi-step neoplastic progression. In some embodiments, cells in early, intermediate and advanced stages of neoplastic progression including “pre-neoplastic cells (i.e., “hyperplastic cells and dysplastic cells), and neoplastic cells in advanced stages of neoplastic progression of a dysplastic cell. In some embodiments, the engineered cells described herein exhibit in vitro and/or in vivo killing activity against a cancer cell. In some embodiments, the engineered cells described herein exhibit in vitro and/or in vivo killing activity against a cancer cell that exhibits cell surface expression of one or more tumor-associated antigens selected from the group consisting of MUC16, FOLR1 , BCMA, CLDN6, SLC34A2, and TAG72.

[0194] In some embodiments, the methods of killing a cancer cell comprises contacting the cancer cell with an engineered y6 T cell, wherein the y8 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered y5 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered y5 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a)one or more endonucleases encoded by a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered y8 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered 78 T cell, wherein the 76 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered 78 T cell, wherein the 78 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered 78 T cell, wherein the 78 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered 78 T cell, wherein the 78 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered 78 T cell, w herein the 78 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell wi th an engineered y5 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having 100% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus.

[0195] In some embodiments, the methods of killing a cancer cell comprises contacting the cancer cell with an engineered y6 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176: and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered y5 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered y5 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a)one or more base editors encoded by a sequence having at least 85% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered y5 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1 , TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered y5 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered y5 T cell, wherein the y6 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 96% sequence identity' to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered yo T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 97% sequence identity’ to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered yo T cell, wherein the y§ T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 98% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17. TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered y5 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 99% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus. In some embodiments, the method of killing a cancer cell comprises contacting the cancer cell with an engineered y5 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having 100% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus.

[0196] Described herein are also methods of modifying a TRAC and a PD-1 locus in a y5 T cell comprising contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 70% sequence identity' to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus. In some embodiments, the method of modifying a TRAC and a PD-1 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus. In some embodiments, the method of modifying a TRAC and a PD-1 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus. In some embodiments, the method of modifying a TRAC and a PD-1 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus. In some embodiments, the method of modifying a TRAC and a PD-1 locus in a 78 T cell comprises contacting to the 76 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus. In some embodiments, the method of modifying a TRAC and a PD-1 locus in a y5 T cell comprises contacting to the 78 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus. In some embodiments, the method of modifying a TRAC and a PD-1 locus in a 78 T cell comprises contacting to the 78 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus. In some embodiments, the method of modifying a TRAC and a PD-1 locus in a 78 T cell comprises contacting to the 78 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus. In some embodiments, the method of modifying a TRAC and a PD-1 locus in a y5 T cell comprises contacting to the yo T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus; and b) a second engineered sy stem comprising: (i) a second endonuclease encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus. In some embodiments, the method of modifying a TRAC and a PD-1 locus in a y5 T cell comprises contacting to the yo T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having 100% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having 100% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 70. 80%. 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 117-119, and 123. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80 sequence identify to any one of SEQ ID NOs: 117-119. and 123.

[0197] Further described herein are methods of modifying a TRAC and an IL- 17 locus in a y§ T cell comprising contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 70% sequence identify to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 70% sequence identify' to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL-17 locus. In some embodiments, the method of modifying a TRAC and an IL-17 locus in a y5 T cell comprises contacting to the 70 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL-17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus. In some embodiments, the method of modify ing a TRAC and an IL-17 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL-17 locus. In some embodiments, the method of modify ing a TRAC and an IL-17 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL-17 locus. In some embodiments, the method of modify ing a TRAC and an IL-17 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL-17 locus. In some embodiments, the method of modifying a TRAC and an IL-17 locus in a 78 T cell comprises contacting to the 76 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL-17 locus. In some embodiments, the method of modifying a TRAC and an IL-17 locus in a y5 T cell comprises contacting to the 78 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL-17 locus. In some embodiments, the method of modifying a TRAC and an IL-17 locus in a 78 T cell comprises contacting to the 78 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL-17 locus. In some embodiments, the method of modifying a TRAC and an IL-17 locus in a 78 T cell comprises contacting to the 78 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL-17 locus. In some embodiments, the method of modifying a TRAC and an IL-17 locus in a y5 T cell comprises contacting to the yo T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having 100% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL-17 locus. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 70. 80%. 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 117-119, and 124-126. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80 sequence identify to any one of SEQ ID NOs: 117-119. and 124-126.

[0198] Further described herein are methods of modifying a PD-1 and an IL-17 locus in a y6 T cell comprising contacting to the y8 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 70% sequence identify' to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 70% sequence identify' to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus. In some embodiments, the method of modifying a PD-1 and an IL- 17 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL-17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 80% sequence identify' to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus. In some embodiments, the method of modifying a PD-1 and an IL- 17 locus in a y5 T cell comprises contacting to the 70 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 85% sequence identify to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL-17 locus. In some embodiments, the method of modifying a PD-1 and an IL-17 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus. In some embodiments, the method of modifying a PD-1 and an IL- 17 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus. In some embodiments, the method of modifying a PD-1 and an IL- 17 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus. In some embodiments, the method of modifying a PD-1 and an IL- 17 locus in a 78 T cell comprises contacting to the 76 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus. In some embodiments, the method of modifying a PD-1 and an IL- 17 locus in a y5 T cell comprises contacting to the 78 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus. In some embodiments, the method of modifying a PD-1 and an IL- 17 locus in a 78 T cell comprises contacting to the 78 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus. In some embodiments, the method of modifying a PD-1 and an IL- 17 locus in a 78 T cell comprises contacting to the 78 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having 100% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL-17 locus. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 70, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 123-126. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80 sequence identity' to any one of SEQ ID NOs: 123-126.

[0199] Further described herein are methods of modifying a PD-1 and a TIM3 locus in a y5 T cell comprising contacting to the yo T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 70% sequence identify to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 70% sequence identify to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the T1M3 locus. In some embodiments, the method of modifying a PD-1 and an TIM3 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and a TIM3 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 85% sequence identify to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 85% sequence identify to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and an TIM3 locus in a y5 T cell comprises contacting to the 76 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 90% sequence identify to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and an TIM3 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and a TIM3 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 96% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and a TIM3 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 97% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and a TIM3 locus in a 78 T cell comprises contacting to the 76 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 98% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and a TIM3 locus in a y5 T cell comprises contacting to the 78 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 127, 128. and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and a TIM3 locus in a 78 T cell comprises contacting to the 78 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 100% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second endonuclease encoded by a sequence having 100% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and (ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 70, 80%, 85%, 90%. 95%. 97%. 98%. 99%. or 100% sequence identity to any one of SEQ ID NOs: 120-123. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80 sequence identity to any one of SEQ ID NOs: 120-123.

[0200] Further described herein are methods of modifying a PD-1 and a TIM3 locus in a y5 T cell comprising contacting to the y5 T cell: a) a first engineered system comprising: (i) a first base editor encoded by a sequence having at least 70% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a first engineered guide polynucleotide configured to form a complex with the first base editor and hybridize to a target nucleic acid sequence within the PD- 1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second base editor encoded by a sequence having at least 70% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a second engineered guide polynucleotide configured to form a complex with the second base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and an TIM3 locus in a y5 T cell comprises contacting to the yd T cell: a) a first engineered system comprising: (i) a first base editor encoded by a sequence having at least 80% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a first engineered guide polynucleotide configured to form a complex with the first base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second base editor encoded by a sequence having at least 80% sequence identify⁷ to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a second engineered guide polynucleotide configured to form a complex with the second base editor and hybridize to a target nucleic acid sequence within the PD- 1 or the TIM3 locus. In some embodiments, the method of modify ing a PD-1 and a TIM3 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first base editor encoded by a sequence having at least 85% sequence identify⁷ to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a first engineered guide polynucleotide configured to form a complex with the first base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second base editor encoded by a sequence having at least 85% sequence identify⁷ to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a second engineered guide polynucleotide configured to form a complex with the second base editor and hybridize to a target nucleic acid sequence within the PD- 1 or the TIM3 locus. In some embodiments, the method of modify ing a PD-1 and an TIM3 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first base editor encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a first engineered guide polynucleotide configured to form a complex with the first base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second base editor encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a second engineered guide polynucleotide configured to form a complex with the second base editor and hybridize to a target nucleic acid sequence within the PD- 1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and an TIM3 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first base editor encoded by a sequence having at least 95% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a first engineered guide polynucleotide configured to form a complex with the first base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second base editor encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a second engineered guide polynucleotide configured to form a complex with the second base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modify ing a PD-1 and a TIM3 locus in a y5 T cell comprises contacting to the y§ T cell: a) a first engineered system comprising: (i) a first base editor encoded by a sequence having at least 96% sequence identify’ to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a first engineered guide polynucleotide configured to form a complex with the first base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second base editor encoded by a sequence having at least 96% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a second engineered guide polynucleotide configured to form a complex with the second base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and a TIM3 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first base editor encoded by a sequence having at least 97% sequence identify’ to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a first engineered guide polynucleotide configured to form a complex with the first base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second base editor encoded by a sequence having at least 97% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a second engineered guide polynucleotide configured to form a complex with the second base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and a TIM3 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first base editor encoded by a sequence having at least 98% sequence identify’ to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a first engineered guide polynucleotide configured to form a complex with the first base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second base editor encoded by a sequence having at least 98% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a second engineered guide polynucleotide configured to form a complex with the second base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and a TIM3 locus in a y5 T cell comprises contacting to the yd T cell: a) a first engineered system comprising: (i) a first base editor encoded by a sequence having at least 99% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a first engineered guide polynucleotide configured to form a complex with the first base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered sy stem comprising: (i) a second base editor encoded by a sequence having at least 99% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a second engineered guide polynucleotide configured to form a complex with the second base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the method of modifying a PD-1 and a TIM3 locus in a y5 T cell comprises contacting to the y5 T cell: a) a first engineered system comprising: (i) a first base editor encoded by a sequence having at least 100% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a first engineered guide polynucleotide configured to form a complex with the first base editor and hy bridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising: (i) a second base editor encoded by a sequence having 100% sequence identify to SEQ ID NO: 175 or SEQ ID NO: 176; and (ii) a second engineered guide polynucleotide configured to form a complex with the second base editor and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 70, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identify to any one of SEQ ID NOs: 120-123. In some embodiments, the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80 sequence identify to any one of SEQ ID NOs: 120-123.

[0201] Aspects of the disclosure include methods of administering the engineered cells described herein for treating a subject in need of treatment, e.g., a subject having cancer. Administration is not limited to a particular site or method. The cells can be administered by any suitable means, such as parenteral administration. Methods of parenteral delivery⁷ include intramuscular, subcutaneous, intratumoral, intravenous, or intraperitoneal. In some embodiments, the engineered T cells are administered to the subject via intravenous injection in a physiologically acceptable medium. In some embodiments, the cells are injected directly into a tumor. [0202] A course of therapy may be a single dose or in multiple doses over a period of time. In some embodiments, the cells are administered in a single dose. In some embodiments, the cells are administered in two or more split doses. In some embodiments, the quantity of engineered cells administered in such split dosing protocols are the same in each administration or are provided at different levels. In some embodiments, multi-day dosing protocols over time periods are provided by a physician monitoring the administration of the cells taking into account the response of the subject to the treatment including adverse effects of the treatment. In some embodiments, engineered T cells described herein are administered before, during, or after the occurrence of a disease or condition, and the timing of administering a pharmaceutical composition containing an engineered cell can vary. For example, the engineered T cell are used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to decrease the likelihood of the occurrence of the disease or condition. In some embodiments, the engineered T ell are administered to a subject during or as soon as possible after the onset of the symptoms.

[0203] In some instances, the engineered T cells are autologous (“self) to the subject, i.e.. the cells are from the same subject. In some embodiments, cells are isolated from the subject organism, modified with the gene editing system described herein to generate engineered T cells, and the engineered T cells are re-infused back into the subject organism.

[0204] In some embodiments, the engineered T cells are non- autologous (“non-self,” e.g.. allogeneic, syngeneic or xenogeneic, i.e., the T cells are not derived from the subject who receives the treatment but from different individuals (donors) of the same species as the subject. In some embodiments, a donor is an individual who does not have or is not suspected of having the cancer being treated. In some embodiments, the donor is a healthy donor. In some embodiments, multiple donors, e.g., two or more donors, are used.

[0205] In some embodiments, the engineered cells of the disclosure are used in combination with one or more additional agents and therapies. For example, the engineered T cells disclosed herein are part of a combination therapy and co-used (e.g., concurrently, or sequentially) with other therapeutic agents, for treating the same indication, or for enhancing efficacy of the engineered T cells and/or reducing side effects of the engineered T cells. In some embodiments, the engineered T cells described herein are sued in a treatment regimen in combination with surgery, chemotherapy, and/or radiation. In some embodiments, the engineered T cells described herein are used in combination with immunosuppressive agents.

[0206] In some embodiments, the engineered cells and methods disclosed herein are used to treat patients with cancer and/or other diseases. In some embodiments, the engineered cells are used to treat patients with cancer, such as ovarian, endometrial, lung, breast, brain, kidney, and colon cancer. In some embodiments, the engineered cells are used to treat patients with ovarian cancer. In some embodiments, the engineered cells are used to treat patients with cancer that is characterized by tumor cells that exhibit cell surface expression of one or more tumor-associated antigens selected from the group consisting of MUC16, FOLR1, BCMA, CLDN6, SLC34A2, and TAG72. In some embodiments, the engineered cells are used to treat patients with cancer that is characterized by tumor cells that exhibit cell surface expression of FOLR1. In some embodiments, the engineered cells are used to treat patients with cancer that is characterized by tumor cells that exhibit cell surface expression of MUC16. In some embodiments, the engineered cells are used to treat patients with cancer that is characterized by tumor cells that exhibit cell surface expression of FOLR1 and MUC16.

[0207] In some embodiments, the engineered cells and methods disclosed herein are used to treat a condition that is characterized by cells that exhibit increased expression of one or more tumor- associated antigens selected from the group consisting of MUC16, FOLR1, BCMA, CLDN6, SLC34A2, and TAG72. In some embodiments, the engineered cells and methods disclosed herein are used to treat a condition that is characterized by cells that exhibit increased expression FOLR1. In some embodiments, the engineered cells and methods disclosed herein are used to treat endometriosis.

Delivery and Vectors

[0208] Disclosed herein, in some embodiments, are nucleic acid sequences encoding an engineered system described herein or components thereof (e.g., endonuclease, engineered guide polynucleotide, or donor nucleic acid). Further described herein, in certain embodiments, are methods and compositions comprising engineered y5 T cells comprising expression vectors comprising a nucleic acid encoding the engineered system.

[0209] In some embodiments, the nucleic acid encoding the engineered system described herein or components thereof is a DNA, for example a linear DNA, a plasmid DNA, or a minicircle DNA. In some embodiments, the nucleic acid encoding the engineered system described herein or components thereof is an RNA. for example a mRNA.

[0210] In some embodiments, the nucleic acid encoding the engineered system described herein or components thereof is delivered by a nucleic acid-based vector. In some embodiments, the nucleic acid-based vector is a plasmid (e.g, circular DNA molecules that can autonomously replicate inside a cell), cosmid (e.g., pWE or sCos vectors), artificial chromosome, human artificial chromosome (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosome (BAC), Pl -derived artificial chromosomes (PAC), phagemid, phage derivative, bacmid, or virus. In some embodiments, the nucleic acid-based vector is selected from the list consisting of: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NE0-C00H-3XFLAG, pSF- CMV-PUR0-NH2-GST-TEV. pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF la-mCherry-N 1 vector, pEFla-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), pSF-CMV-PURO-NH2- CMYC, pSF-OXB20-BetaGal,pSF-OXB20-Fluc, pSF-OXB20, pSF-Tac, pRI 101 -AN DNA, pCambia2301, pTYB21. pKLAC2, pAc5.1/V5-His A, and pDEST8.

[0211] In some embodiments, the nucleic acid-based vector comprises a promoter. In some embodiments, the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof. In some embodiments, the promoter is selected from the group consisting of CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6. lac. araBad, trp, Ptac, p5, pl9, p40, Synapsin, CaMKII, GRK1, and derivatives thereof. In some embodiments the promoter is a U6 promoter. In some embodiments, the promoter is a CAG promoter.

[0212] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus. a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a retrovirus. In some embodiments, the virus is an alphavirus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is an AAV. In some embodiments, the virus is a baculovirus. In some embodiments, the virus is a Dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a herpesvirus. In some embodiments, the virus is a poxvirus. In some embodiments, the virus is an anellovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is or a retrovirus.

[0213] In some embodiments, the AAV is AAV1. AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV- rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV- Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV- LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV- HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV- NP22, AAV-NP66, AAV-HSC16, or a derivative thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0214] In some embodiments, the virus is AAV1 or a derivative thereof. In some embodiments, the virus is AAV2 or a derivative thereof. In some embodiments, the virus is AAV3 or a derivative thereof. In some embodiments, the virus is AAV4 or a derivative thereof. In some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV 10 or a derivative thereof. In some embodiments, the virus is AAV11 or a derivative thereof. In some embodiments, the virus is AAV 12 or a derivative thereof. In some embodiments, the virus is AAV 13 or a derivative thereof. In some embodiments, the virus is AAV 14 or a derivative thereof. In some embodiments, the virus is AAV 15 or a derivative thereof. In some embodiments, the virus is AAV 16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rhlO or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof. In some embodiments, the virus is AAV-rhM4-l or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the virus is AAV-Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the virus is AAV-PHP-B or a derivative thereof. In some embodiments, the virus is AAV-PHP-EB or a derivative thereof. In some embodiments, the virus is AAV-2.5 or a derivative thereof. In some embodiments, the virus is AAV-2IYF or a derivative thereof. In some embodiments, the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof. In some embodiments, the virus is AAV-HSC8 or a derivative thereof. In some embodiments, the virus is AAV-HSC9 or a derivative thereof. In some embodiments, the virus is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV-HSC12 or a derivative thereof. In some embodiments, the virus is AAV-HSC13 or a derivative thereof. In some embodiments, the virus is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof. In some embodiments, the virus is AAV-DJ/8 or a derivative thereof. In some embodiments, the virus is AAV-Myo or a derivative thereof. In some embodiments, the virus is AAV-NP40 or a derivative thereof. In some embodiments, the vims is AAV-NP59 or a derivative thereof. In some embodiments, the vims is AAV-NP22 or a derivative thereof. In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof. [0215] In some embodiments, the virus is HSV-1 or a derivative thereof. In some embodiments, the vims is HSV-2 or a derivative thereof. In some embodiments, the virus is VZV or a derivative thereof. In some embodiments, the virus is EBV or a derivative thereof. In some embodiments, the virus is CMV or a derivative thereof. In some embodiments, the vims is HHV- 6 or a derivative thereof. In some embodiments, the virus is HHV-7 or a derivative thereof. In some embodiments, the vims is HHV-8 or a derivative thereof.

[0216] In some embodiments, the nucleic acid encoding the engineered system described herein or components thereof is delivered by a non-nucleic acid-based delivery system (e.g, a non-viral delivery system). In some embodiments, the non-viral delivery system is a liposome. In some embodiments, the nucleic acid is associated with a lipid. The nucleic acid associated with a lipid, in some embodiments, is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the nucleic acid, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. In some embodiments, the nucleic acid is comprised in a lipid nanoparticle (LNP).

[0217] In some embodiments, the engineered system described herein or components thereof is introduced into the cell in any suitable way, either stably or transiently. In some embodiments, the engineered system described herein or components thereof is transfected into the cell. In some embodiments, the cell is transduced or transfected with a nucleic acid construct that encodes the engineered system described herein or components thereof. For example, a cell is transduced (e.g., with a virus encoding the engineered system described herein or components thereof), or transfected (e.g., with a plasmid encoding the engineered system described herein or components thereof) with a nucleic acid that encodes the engineered system described herein or components thereof, or the translated the engineered system described herein or components thereof. In some embodiments, the transduction is a stable or transient transduction. In some embodiments, cells expressing the engineered system described herein or components thereof or containing the engineered system described herein or components thereof are transduced or transfected with one or more gRNA molecules, for example, when the engineered system described herein or components thereof comprises a CRISPR nuclease. In some embodiments, a plasmid expressing the engineered system described herein or components thereof is introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggyBac®) and viral transduction (for example lentivirus or AAV) or other methods known to those of skill in the art. In some embodiments, the gene editing system is introduced into the cell as one or more polypeptides. In some embodiments, delivery is achieved through the use of RNP complexes. Delivery methods to cells for polypeptides and/or RNPs are known in the art, for example by electroporation or by cell squeezing.

[0218] Exemplary methods of delivery of nucleic acids include lipofection, nucleofection, electroporation, stable genome integration (e.g., piggyBacK). microinjection, biolistics, virosomes, liposomes, immunoliposomes, poly cation or lipid nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5.049.386; 4,946.787; and 4,897.355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and SF Cell Line 4D-Nucleofector X Kit™ (Lonza)). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of WO 91/17424 and WO 91/16024. In some embodiments, the delivery is to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). In some embodiments, the nucleic acid is comprised in a liposome or a nanoparticle that specifically targets a host cell.

[0219] Additional methods for the delivery⁷ of nucleic acids to cells are known to those skilled in the art. See, for example, US 2003/0087817.

[0220] In some embodiments, the present disclosure provides a cell comprising a vector or a nucleic acid described herein. In some embodiments, the cell expresses a gene editing system or parts thereof. In some embodiments, the cell is a human cell. In some embodiments, the cell is genome edited ex vivo. In some embodiments, the cell is genome edited in vivo.

Kits

[0221] In some embodiments, this disclosure provides kits comprising one or more nucleic acid constructs encoding the various components of the gene editing system described herein. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of the gene editing system components.

[0222] In some embodiments, the engineered gene editing system or components thereof disclosed herein is assembled into a pharmaceutical, diagnostic, or research kit to facilitate its use in therapeutic, diagnostic, or research applications. A kit may include one or more containers housing any of the vectors disclosed herein and instructions for use.

[0223] The kit may be designed to facilitate use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g.. to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit. As used herein, "instructions" can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based communications, etc. The written instructions, in some embodiments, are in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use, or sale for animal administration.

EXAMPLES

[0224] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.

Example 1 - Isolation, expansion, and characterization of donor yb T cells

[0225] Isolation of yd T cells from healthy donor PBMCs and stimulation using IMMU510

[0226] PBMC samples were obtained by layering healthy donor leukapheresis product onto Ficoll-Paque Plus and centrifuging samples at 1200 rpm for 20 minutes at room temperature with the brake off. The mononuclear cell layer was collected and washed in PBS + 2% BSA prior to yo T cell negative selection. yb T cells were negatively selected by immunomagnetic selection methods prior to activation culture. The purify of yb T cells and frequencies of yb T cell Vbl+ and V52+ subsets was measured by flow cytometry pre- and post-isolation using PE anti- TCRy/5, APC anti-CD3, Vio Bright B515 anti-TCR V51 and VioBlue anti-TCR. Shown in FIGs. 1A-1B are representative flow cytometry plots of yb T cell purity pre- and post-isolation and yb T cell Vbl+ and Vb2+ subset frequencies post-isolation. The average frequency of yb T cells in PBMC samples prior to isolation was 2.5%, and post-isolation was 85.7% as shown in FIGs. 2A- 2B (n=12 donors). Isolated yb T cells were stimulated using immobilized IMMU510, a monoclonal antibody clone that specifically binds the yb TCR. Cell culture flasks were coated by overnight incubation with a 1 pg/mL solution of IMMU510 and isolated yb T cells were plated at a density of 1E+06 cells/mL in T cell expansion media containing 5% immune cell serum replacement and 2 mM L-glutamine and supplemented with 100 lU/mL IL-2 and 100 ng/mL IL- 15. y8 T cells were cultured on IMMU510-coated plates for 3 days, after which the cells were collected, washed, and further cultured in media containing 100 lU/mL IL-2 and 100 ng/mL IL- 15 for 4-17 additional days.

[0227] Culture and expansion of Delta One T cells

[0228] PBMC samples were obtained as above and depleted of a[3 T cells using positive selection. The remaining immune cells, enriched for y5 T cells, were stimulated with the anti- CD3 clone OKT3 and cultured in T cell expansion media containing 5% immune cell serum replacement and 2 mM L-glutamine supplemented with 100 ng/mL IL-4, 70 ng/mL IFN-y. 7 ng/mL IL-21, and 15 ng/mL IL- 1 . Cells were re-stimulated with 1 pg/mL OK.T3 on days 7. 11. and 15 of culture, supplemented with 13 ng/mL IL-21 and 70 ng/mL IL-15 on day 7 of culture, and further supplemented with 100 ng/mL IL-15 on days 11 and 15 of culture. Cells were cultured for a total of 21 days.

[0229 ] Culture and expansion of yd T cells activated using Zoledronate

[0230] PBMC samples or PBMC samples depleted of a[3 T cells were obtained as above, stimulated with 5 pM zoledronate, and cultured at a density of 0.5E+06 cells/cm² in T cell expansion media containing 10% FBS and 2 mM L-glutamine and supplemented with 1000 lU/mL IL-2. After 3 days, cells were diluted 2-fold in media supplemented with 1000 lU/mL IL- 2. Every 2-3 days additional media supplemented with 1000 lU/mL IL-2 was added to cultures to maintain a cell density between 0.5-2E+06 cells/mL. Cells were cultured for 13-20 days.

[0231] yd T cell phenotypic analysis

[0232] Surface staining with fluorescently labeled monoclonal antibodies was used to characterize the phenotype of cultured y5 T cells. As shown in FIGs. 3A-3B. the memory phenotype of V51+ and V62+ subsets were determined by staining with Vio Bright B515 anti- TCR V51, VioBlue anti-TCR V52, BV785 anti-CD45RA and AF700 anti-CD27. Cells were washed with FACs staining buffer (PBS + 0.5% BSA) and incubated with a 1 :20 dilution of stain for 10 minutes at room temperature. Appropriate volumes of monoclonal antibodies were diluted in FACs staining buffer and cells were stained in a total volume of 50 pL for 30 minutes at 4°C and then w ashed twice with FACs buffer. For further characterization of y3 T cell subsets, the following monoclonal antibody conjugates or equivalents were used: PE anti-TCRy/6, APC anti- CD3, APC anti-CD25, BV605 anti-CD69. APC anti-PD-1, BV650 anti-TIM3, AF700 anti- CXCR3, BV650 anti-CXCR4. BV421 anti-CD56. VioGreen anti-CD158e, BV711 anti-CD226 (DNAM-1), BV785 anti-NKp30, PerCP-Cy5.5 anti NKp44, and PE anti-NKG2D. Viable cells were identified, and dead cells excluded from analysis by staining. Gates were set to determine positive and negative cell populations based upon Fluorescence Minus One (FMO) controls. Data was acquired by flow cytometry and analyzed.

Example 2 - Assessment of anti-tumor y8T functionality in vitro in 2D

[0233] In vitro cytotoxicity assays were used to assess the anti-tumor capabilities of yb T-cells from Example 1 against various epithelial ovarian cancer (EOC) cell lines. Cytotoxicity assays were performed with multiple quantitative orthogonal readouts described below to evaluate y5 T cell functionality via liquid and solid tumor cytotoxicity' and effector cytokine secretion.

[0234] Liquid tumor cytotoxicity assay

[0235] To test the innate cytotoxicity of cultured y5 T cells, a co-culture assay was established using K562 cells, a model of chronic myelogenous leukemia. After 10-20 days of y5 T cell culture, K562s were stained (2.5 pM) for 20 minutes at room temperature and excess dye was quenched with FBS-containing media following incubation. Labeled K562 cells were co-cultured with y5 T cells at 10:1, 1: 1, and 0: 1 Effector: Target (E:T) ratios for 24 hours to determine innate cytotoxicity of cultured y5 T cells. After 24 hours, all cells were stained and the frequency of dead K562 cells was analyzed using flow cytometry. % fluorescent + CTV+ cells were reported as % Dead target cells. All donor-derived yb T cells tested (n=6) exhibited dose-dependent cytotoxic activity' against K562 cells (FIG. 5A).

[0236] Solid tumor cytotoxicity assays

[0237] Functionality of unmodified and engineered yb T cells against various EOC lines (ATCC) were assessed using a l) cytokine release assay, 2) luciferase-based assay, and 3) image-based assay. Target EOC lines were transduced using an EFla-GFP-Luciferase-IRES-Puro plasmid (SEQ ID NO: 34) according to standard lenti viral transduction methods. Various EOC-GFP- luciferase reporter lines including TOV112D, OVCAR-3. OV-90, and OVCAR-4 were seeded at 2x10⁴ cells/well in a 96-well flat-bottom plate in R10 media (RPMI-1640 + 2mM L-glutamine, 10% HI-FBS). After 24 hours, media was removed, and ybTs expanded for 13-20 days, as noted in Example 1, were added in R10 media at E:T ratios between 10: 1 and 1: 1. Cocultures were incubated at 37°C for 24 hours. Supernatants were collected for cytokine measurement and luminescence measurement using a luciferase assay. Coculture plates for image-based analysis were maintained separately for up to 72 hours.

[0238] Cytokine release measurement

[0239] Culture supernatants containing cytokines were collected after 24 hours of culture for measurement and diluted accordingly. 81 nM phorbol 12-myristate 13-acetate and 1.34 pM ionomycin (PMA/I) stimulation of effector cells was used as a positive control, yb T cells from a representative donor displayed a dose-dependent response to PMA/I stimulation (FIGs. 4A-4B). Production of cytokines including GMCSF, GZMA, GZMB, IFNy, IL-10, IL-12p70, IL-15, IL- 17/17A, IL-2, IL-4, IL-17. TNFa was quantified and data was analyzed. For single, nonmultiplexed analytes, supernatants were run on an automated enzyme-linked immunosorbent assay (ELISA).

[0240] Lucifei ■ase-based cytotoxicity’ assay

[0241] In vitro cytotoxicity assays were set up using luciferase-expressing EOC lines as described above in Example 2. After incubation at 37 °C for 24 hours, supernatants were removed for cytokine analysis. 150 pg/ml D-luciferin was added for total well volumes of 100 pl, incubated for 15 minutes at room temperature, then read on a microplate reader. % cytotoxicity⁷ was calculated by subtracting sample relative luminescence units (RLU) from target only RLU, divided by target only RLU. Donor-derived y6 T cells displayed dose-dependent cytotoxicity against a representative EOC reporter line TOV-1 12D (FIG. 5B).

Example 3 -y6 T Cell Cytotoxicity Assays (Prophetic)

[0242] Image-based cytotoxicity assay

[0243] Cytotoxicity assays for imaging are performed as described in Example 2. GFP- expressing EOC lines are seeded at l-2x!0⁴ cells/well in a 96-well flat-bottom tissue-culture treated optical plates. Effector cells are added at E:T ratios between 10:1-1 : 1 in phenol red-free RP MI-1640 + 2 mM L-glutamine + 10% HI-FBS in a total well volume of 100 pl. Images are collected every 2 hours from 0-72 hours with lOx or 20x objectives. Total fluorescence area was calculated to measure % cytotoxicity by subtracting sample fluorescence area from target only fluorescence area, divided by target only fluorescence area.

[0244] N on-cancer ous cell control for 2D assays

[0245] For the solid tumor cytotoxicity assay and cytokine release measurement described above, cell lines derived from normal (non-cancerous) human tissues, including human uterine fibroblast cell line (HUF) and fallopian tube cell lines (hTERT FT 194), will be used as normal (non- cancerous) cell controls.

Example 4 - Assessment of anti-tumor y6 T cell functionality in vitro using 3D disease model (Prophetic Example)

[0246] Fabrication of Ovarian Cancer Spheroids using EOC Lines

[0247] Spheroid Fabrication: GFP-luciferase reporter EOC lines (as described in Example 2) including OV-90, TOV-112D. OVCAR-3 are used to fabricate ovarian cancer spheroids. The collected EOC cells are suspended in spheroid forming media as explained below. After preparing cell suspensions, desired cell concentrations are pipetted into a 96-well plate at 200 pL/well to form spheroids within each microwell at the range of 150-600 microns. Next, the plates are centrifuged at 300 x g for 10 min and then cultured at 37 °C and 5% CO2. The fabricated spheroids are observed using a microscope over time up to Day 7 post-fabrication. [0248] Media Optimization: Spheroid fabrication media is optimized by combining at least one or more of following reagents including DMEM/F12 (1: 1), 1% B-27™ Supplement (50X) minus Vitamin A. 20 ng/mL of human heat stable bFGF. 20 ng/mL of human EGF recombinant protein, 0.10% of Insulin-Transferrin-Selenium and 10 pM of Rock-inhibitor supplements. Growth factors are added to spheroid media immediately prior to use.

[0249] Characterization and Validation of Ovarian Cancer Spheroids

[0250] Morphological Assessment: Fabricated spheroids are transferred to a 12-well chamber using wide bore pipette tips at various time points to determine morphological characteristics. The transferred spheroids are imaged under brightfield. Next, morphometric characterization of ovarian cancer spheroids are analyzed using an image analysis software, where the circularity of each spheroid is selected to be greater than 0.1, the spheroid size ranged between 150 to 600 micrometers.

[0251] Dead Staining: Fabricated ovarian cancer spheroids are evaluated for their viability at various time points. To visualize dead cells within the spheroids, a final concentration of 4 pM Ethidium Homodimer-1 (is added directly to each well for 30 min at 37 °C and 5% CO2 in the dark. Images are then taken under Alexa Fluor 568 nm for red fluorophores.

[0252] Proliferation Study: The cell proliferation assay is performed using a cell counting kit. Spheroids and the control group are treated with CCK-8 solution mixed into serum-free media at a mixing ratio of 1: 10 (v/v) for 4 hours and incubated at 37 °C and 5% CO2. After incubation, solution is placed into a new 96-well plate to determine the intensity using a microplate scanning spectrophotometer at 460/650 nm excitation.

[0253] rtPCR: rtPCR is used to quantify GOI mRNA transcripts in ovarian cancer spheroids. Briefly, spheroids are collected from plates using wide bore pipette tips into conical tubes. Thereafter, spheroids are spun down at 300 x g for 5 min and spheroid pellets are processed as explained in Example 5.

[0254] Immunofluorescence Staining: The fabricated spheroids are stained with Phalloidin, Hoechst and Hypoxia reagent dyes to visualize cytoskeletal organization, cell nuclei and hypoxic core of spheroids. In addition, tumor-associated antigens (TAA) are stained as explained in Example 5. The stained samples are imaged using a confocal microscope.

[0255] Cytokine Profiling: To identify secreted cytokines from ovarian cancer spheroids, we evaluated cytokine profiles including AECAM/CD166, Angiopoietin-2, CA125/MUC16, CCL2/JE/MCP-1, CCL4/MIP-1 beta, CCL22/MDC, CXCL1/GRO alpha/KC/CINC-1, EpCAM/TROPl, FGF basic/FGF2/bFGF, FIt-3 Ligand/FLT3L, ICAM-1/CD54, IL-6, IL- 10, Leptin/OB. LIF, MIF, MMP-2, MMP-9 and PDGF-BB. The cytokines are collected and processed as explained in Example 2.

[0256] Assessment of anti-tumor y5T functionality in 3D

[0257] Image-based cytotoxicity assay: Cytotoxic capability of both unmodified and engineered y6T cells against ovarian cancer spheroids are assessed using image-based cytotoxicity assays. Similarly to 2D co-culture assay, a human uterine fibroblast cell line (HUF) or fallopian tube cell line (hTERT FT 194) are used as negative controls. TritonX-114 is used as a lysis control for spheroids. At various time points, spheroids are fabricated and then expanded y5T cells as noted in Example 1 are added (20-40 pL/well) directly to spheroid wells at E:T ratios of 10: 1, 5: 1, 1: 1 and 0.5: 1. 3D co-cultures are incubated at 37 °C and 5% CO2 for up to 96 hours. Images are collected prior to adding y8 T cells and then after adding y8 T cells every 2-8 hours from 0-96 hours with both using lOx or 20x objectives using a confocal microscope. Changes in fluorescent intensity and the area to measure % specific killing and %reduction area for spheroids is determined.

[0258] Cytokine Release Measurement: Supernatants are collected from 3D co-cultures for cytokine measurement after 24 hours as described in Example 2.

Example 5 - Nomination of therapeutic genes of interest (GOIs) via Transcriptome Profiling

[0259] In-silico RNAseq disease characterization and establishment of analysis pipeline

[0260] An internal RNAseq patient-sample derived ovarian cancer database was generated from several databases. These samples were primarily derived from ovarian cancer (OVC), fallopian tube tissue adjacent to OVC tissue, and alveolar tissue. Potential therapeutic GOIs were identified based on differential gene expression across tissues.

[0261] scRNAseq for GOI Identification and Knock-Out (KO) Nomination

[0262] A transcriptome kit was used to process y8 T cell samples co-cultured 2: 1 with OVCAR-3 cells. Co-culture of OVCAR-3 and y5 T cells was initiated at D3 post-isolation. 1E6 cells each were collected at multiple time-points (D3, D4, D6, and D10) for processing with the transcriptome kit. Finished scRNAseq libraries were run for a final total of 6.7 billion reads. bcl2fastq, scanpy, and seurat were used to QC, process, and analyze the BCL and FASTQ files, as shown in FIG. 22. Python based implementation of scanpy was used for preprocessing, visualization, clustering, trajectory inference and differential expression testing. A combination of variations in the differential gene expression analysis were used to pool clusters of dysfunctional cells using cellxgene. Genes (IKZF2, RASA2, RASA3) differentially overexpressed in these clusters were analyzed for biological relevancy and nominated as potential candidates for KO.

[0263] Validation of nominated TAA expression in EOC cell lines

[0264] rtPCR GOI Transcriptomic Characterization & Validation: rtPCR was used to quantify GOI mRNA transcripts in EOC cell lines and y6 T cells under different culture methods to establish baselines for future technologies and characterize in-house cell lines (FIG. 23). Cells were pelleted and an RNA isolation was performed on each cell line in triplicate. RNA concentrations were quantified and normalized to 20 ng/pL. RIN scoring was performed (all samples below a RIN score of 7 were disregarded in future steps). 16 pL of RNA sample and 4 pL of CDNA synthesis master mix were added and processed in a thermocycler for 25 minutes to create a stock CDNA sample. CDNA samples were diluted 10: 1, and 4 pL of each sample were combined in 96 well plates in triplicate with 16 pL of rtPCR master-mix. A full rtPCR cycling protocol (50 °C - 2 minutes, 95 °C - 10 minutes, 40x [95 °C - 15 seconds, 60 °C - 60 seconds, Image capture]) was run a rtPCR thermocycler.

[0265] GOI expression characterization via Flow Cytometry: Flow cytometry was used to characterize EOC cell line protein expression and compare surface marker expression across the cell lines. Cells were stained with fluorescently labeled antibodies specific to the surface markers of interest (FIG. 23) (anti-MUC16, anti-HER2, anti-EPCAM, anti-CEACAM, anti-THY-1, anti- TAG72, anti-CLDN6, rb anti-SLC34A2, rb anti-WFDC2), then analyzed on a flow cytometer. Data was analyzed. The fluorescence intensity of the stained cells was compared to that of unstained cells and cells stained with isotype controls to confirm the specificity of the staining. The data were analyzed to determine the percentage of cells expressing the surface markers of interest and the mean fluorescent intensity of the stained cells.

[0266] GOI expression characterization via Immunocytochemistry staining: ICC was performed on the cell lines to allow for the identification and comparison of protein expression and localization between different cell lines for TAA’s of interest (FIG. 23). The cells were fixed with 2% paraformaldehyde solution to preserve the cellular structure and antigenicity. After fixation, the cells were permeabilized with a 0. 1% Triton-X in DPBS permeabilization buffer to allow access of the primary antibodies (anti-MUC 16, anti-HER2, anti-EPCAM, anti-CEACAM, anti-THY-1, anti-TAG72, anti-CLDN6, rb anti-SLC34A2, rb anti-WFDC2) to intracellular antigens. The cells were then incubated with primary antibodies specific to the proteins of interest, followed by appropriate secondary antibodies (goat anti-rabbit and goat anti-mouse) conjugated to Alexa Fluor-488. After the incubation, the coverslips were washed to remove any unbound antibodies and mounted onto microscope slides with mounting medium containing DAPI to visualize cell nuclei. Stained cells were imaged using a microscope and analyzed for either positive or negative signal.

Example 6 - Determination of therapeutic genes of interest (GOIs) via Transcriptome Profiling (Prophetic)

[0267] A process for genome-wide pooled functional genomics screening in y5 T cells includes expressing a pooled genome-wide guide RNA expression library into activated y5 T cells using lenti virus at low MOI such that the vast majority of cells receive only a single guide expression vector integration in their genome. Lentiviral delivery is optimized to achieve high enough transduction efficiency of yo T cells. Next, a CRISPR nuclease is introduced to y5 T cells to make the intended edits. Nuclease delivery- is optimized to reach high enough editing efficiencies. An antibiotic is used to enrich the library expressing y§ T cells. Antibiotic application is optimized to reach high enough expressing cell purities. y5 T cells are maintained in culture to amplify the functional effects of CRISPR edits (e.g., appreciable level of grow th difference betw een control guide edited and essential gene targeting guide edited cells, and/or to allow cellular proteins to reach new (high or low) levels that they are no longer exerting their intended biological effects). Next, cells are harvested and (1) directly put through a Next- Generation Sequencing (NGS) library preparation process from genomic DNA or (2) enriched for a desired phenotype (e.g., using fluorescence activated cell sorting) and then put through the NGS library preparation process. During library preparation, gDNA is extracted; a segment of the guide expression vector containing the guide spacer sequence is amplified using Polymerase Chain Reaction (PCR); and sequencing adapters are anchored to the amplicons from different samples while simultaneously they are differentially barcoded using sample indices. NGS libraries are then sequenced to obtain library- gRNA sequence counts. Guide RNA count frequencies are compared between treated and control samples using appropriate computational tools. Screen hits are those with low false discovery rates (q-values). Screen hits are then validated in downstream processes (e.g., arrayed screening, multiplex editing) using the same functional assay for repeatability or orthogonal assays to further evaluate the impact of the CRISPR edit(s) on y6 T cell function.

Example 7 - KO of therapeutic genes of interest

[0268] Engineering cells using electroporation (EP) reagent delivery

[0269] y5 T cells isolated from donor PBMCs w ere activated and expanded in culture as described in Example 1. 4 days post-isolation, activated y5 T cells were collected, counted, and washed multiple times using PBS. Cell pellets were resuspended at l e⁷ cells per mL in supplemented P3 nucleofection buffer. Cells w ere combined w-ith MG nuclease mRNA and/or MG nuclease RNP with one or more site-specific sgRNAs. Cell-RNP mixtures were distributed into 96-well nucleofection plates and/or nucleocuvettes (workflow in FIG. 6). The spacer sequences used for each GOI are represented by SEQ ID NO: 35-45.

[0270] On-target GOI knock-out efficiency was determined by NGS sample preparation using standard techniques. Briefly, genomic DNA was extracted from engineered samples on day 7 post-isolation (3 days post-engineering) and used as template for PCR amplification of relevant GOI amplicon (forward and reverse PCR primer sequences: SEQ ID NO: 65-86). Addition of NGS adapter sequence to 5’ and 3’ ends of PCR product was conducted during the first PCR amplification step. A subsequent PCR amplification using indexing primers enabled sequencing. Sequencing data was then analyzed to determine site-specific indel frequencies.

[0271] On-target GOI knock-out efficiency can also be evaluated at the protein level using antibody staining for target and flow cytometry readout using standard methods, as described in Example 9. For cytokine and chemokine targets, protein level knock-out can be assessed by flow cytometry and/or multiplexed cytokine assays, as described in Examples 1 and 2. As shown in FIGs. 8A-8B and 10, indel frequency was as high as 96% using PDCD1 guide H3 (SEQ ID NO: 42) in combination with MG3-6, as high as 86% using HAVCR2 guides Al and A4 (SEQ ID NO: 39, 40) with MG3-6, and as high as 42% using IL17A guide 1 (SEQ ID NO: 43).

[0272] Off-target sgRNA activity⁷ was predicted in silico for genome-wide evaluation of guide sequence homology. Screening for off-target sites allow up to 4 mismatched bases and 1 base pair insertions or deletions between genome sites and guide spacer sequence. Predicted off-target hits were then screened. Guides with high predicted off-target activity and/or predicted activity within exons were excluded from therapeutic programs.

[0273] Evaluation of off-target editing was performed to identify off-target cleavage sites within genomic DNA samples. In brief. gDNA was incubated with pre-complexed guide and nuclease (in the form of ribonucleoproteins, or RNPs), followed by 3’ adenylation and ligation of biotinylated adapters at nuclease cut sites. DNA was then fragmented and underwent biotin selection to enrich for fragments adjacent to nuclease cut sites. Resulting fragments were then ligated with 5' and 3’ adapters appropriate for NGS, and off-target sites were characterized.

[0274] To identify off-target cleavage sites in a cellular context, EP-based engineering workflows were conducted with the addition of double-stranded oligodeoxynucleotide (dsODNs), which incorporate into double stranded breaks (DSBs) across the genome. gDNA was then extracted. gDNA was then sheared and dsODN-specific amplification enriched samples for fragments containing cutsites. Resulting fragments were then ligated with 5’ and 3‘ adapters appropriate for NGS, and off-target sites were characterized.

[0275] Engineering cells using LNP reagent delivery [0276] Lipid nanoparticles (LNPs), encapsulating MG enzyme mRNA and sgRNAs, were formulated, concentrated, and cryopreserved prior to cell administration. LNPs were formulated in a lipid mixture consisting of a molar ratio of 16% DOPE, 1.5 % DSPE-PEG, and 47.5% cholesterol, along with 35% mRNA-capturing ionizable lipid, such as C 12-200. Lipids were resuspended in ethanol prior to formulation. mRNA and sgRNA were diluted in 100 mM sodium acetate (pH 4.0) prior to formulation at equal molar ratios to co-package within a single LNP. The NanoAssemblr Ignite was run at a 3: 1 Flow Rate Ratio of RNA to lipid. Formulated LNPs then underwent dialysis in 2L of PBS to remove residual ethanol and sodium acetate. LNPs were concentrated with a 100 kDa size cutoff. Upon achieving desired concentration, LNPs were then cryopreserved in 15% sucrose at -80 °C until use. T cells isolated from donor PBMCs were activated and expanded in culture as described in Example 1 . 16-24 hours after removal of activation, cells were collected, counted, and seeded in fresh media containing 1 pg/rnL of ApoE. Cryopreserved LNPs were thawed and added to wells at a range of 0.1 to 10 pg of encapsulated RNA per million cells. GOI knock-out efficiency and phenotyping of engineered cells were evaluated as described in Example 5 and 1. Indel frequency was up to 95% using LNP co-packaging TRAC guide 6 (SEQ ID NO: 36) and MG3-6, and up to 55% using LNPs co-packaging PDCD1 guide H3 (SEQ ID NO: 42) with MG3-6, as seen in FIG. 11.

Example 8 - Knockout of multiple therapeutic targets

[0278] Multiplex engineering cells using EP reagent delivery

[0279] Multiplex editing of multiple genomic sites in y5 T cells was conducted through simultaneous or sequential delivery of engineering reagents via EP. As described in Example 1,

T cells isolated from donor PBMCs were activated and expanded in culture. For simultaneous multiplexed editing, engineering reagents (guide(s) and nuclease(s)) targeting multiple genomic sites were delivered on D4 post-isolation and activation via EP in the same reaction. For sequential multiplexed editing, engineering reagents (guide and nuclease) for a single genomic site were delivered on D4 post-isolation. Subsequent EP reactions delivering engineering reagents targeting an additional site or GOI were conducted on D7 post-isolation. GOI knock-out efficiency and phenotyping of engineered cells were evaluated as described in Example 5 and 1. Multiplexed editing has been demonstrated using various combinations of guides targeting genes IL17A (SEQ ID NO: 43), HAVCR2 (SEQ ID NO: 39, 40), PDCD1 (SEQ ID NO: 42), TRAC (SEQ ID NO: 36), and B2M (SEQ ID NO: 35). As shown in FIG. 11D, simultaneous editing via EP can achieve up to 90% editing at two sites when delivering PDCD1 guide H3 (SEQ ID NO: 42) with TRAC guide 6 (SEQ ID NO: 36), in combination with MG3-6. [0280] Multiplex engineering cells using LNP reagent delivery

[0281] Multiplex editing of multiple genomic sites in y§ T cells was conducted through simultaneous or sequential delivery of engineering reagents via LNP. As described in Example 1, y5 T cells isolated from donor PBMCs were activated and expanded in culture. For simultaneous multiplexed editing, LNPs packaging engineering reagents (guide(s) and nuclease(s)) targeting multiple genomic sites were delivered on D4 post-isolation. Guides and nucleases targeting multiple sites may be packaged in the same LNP or in separate LNPs that were simultaneously delivered to cells. For sequential multiplexed editing, engineering reagents (guide and nuclease) for a single genomic site were packaged in an LNP and delivered on D4 post-isolation.

Subsequent administration of LNPs delivering engineering reagents targeting an additional site or GOI was conducted on D7 post-isolation. GOI knock-out efficiency and phenotyping of engineered cells were evaluated as described in Example 5 and 1 . Multiplexed editing using LNPs has been demonstrated using guides targeting genes PDCD1 (SEQ ID NO: 42) and TRAC (SEQ ID NO: 36). As shown in FIG. 11D, simultaneous editing via LNP can achieve up to 40% and 90% editing using PDCD1 guide H3 (SEQ ID NO: 42) with TRAC guide 6 (SEQ ID NO: 36), respectively, in combination with MG3-6.

Example 9 - Measurement of functional protein knockout

[0282] y5 T cells isolated from donor PBMCs were activated, expanded, and engineered as described in Example 1, 7. and 8 to knock out expression of various therapeutic targets. To validate protein knockout, protein expression was assessed via flow cytometry on D7 postisolation or Dll post-isolation. Cells were labeled and stained with antibodies as relevant to proteins of interest, such as APC anti-PD-1 or BV650 anti-TIM3. FIG. 9B shows a decrease of TIM3 protein expression from over 80% to less than 10% following the delivery of HAVCR2 guide Al (SEQ ID NO: 40) in combination with MG3-6.

Example 10 - Functional evaluation of engineered knockout cells

[0283] Repeated stimulation of cells to assess impact of gene knockout

[0284] Engineered y5 T cells were repeatedly challenged with TCR stimulation to assess the impact of engineering on cell growth and phenotype in response to chronic activation. Engineered cells (such as PD-1 or TIM3 knockout) were incubated for 24 hours on plates coated with IMMU510. Cells were placed in fresh media with no added cytokines for the duration of the 24 h stimulation period. Supernatants were collected at 24 h and analyzed for cytokine release. Post-stimulation, cells were then rested in the absence of stimulation for 6 days in cytokinecontaining media (IL-2 and IL- 15). Flow cytometry to measure surface expression of exhaustion and activation markers was conducted immediately prior and immediately following activation. Example surface markers include APC anti-PD-1 or BV650 anti-TIM3. As shown in FIGs. 9A- 9B, repeat stimulation of PD-1 knockout engineered y5 T cells demonstrates an increase in PD-1 expression with additional rounds of stimulation in unmodified and mock EP cells (to over 50% PD-1+ cells), while also illustrating that the PD-1 knockout in engineered y5 T cells persists throughout stimulation (maintaining below 15% PD+ cells).

[0285] Anti-tumor assessment of engineered yd T cells in vitro in 2D (Prophetic)

[0286] Engineered y5 T cells are evaluated using all assays enumerated in Example 2 and Example 4. Engineered cells are compared to unmodified and Mock EP or LNP control y5 T cells from the same donor.

Example 11 - Constructs for targeted insertion of transgene payload(s) into y8T cells [0287] For MGCT-100 chimeric antigen receptor (CAR) payloads, the general architecture is represented in FIG. 12A. The annotated sites (1-5) can be modified or changed in order to alter various characteristics of the CAR(s), or to exchange the entire transgene for an alternative integrating payload (cytokine/chemokine). Cloning sites are represented by arrows. The genomic locus of integration was specified by the flanking homology regions (denoted (1) in FIG. 12A). Specific sequences of the homology regions (SEQ ID NO: 46-53) were determined based on the spacer sequence of the sgRNA being used (SEQ ID NO: 35-45) to target a specific genetic locus, and the mechanism of double-strand break (DSB) formation by the specific enzyme being used. Expression of the MGCT-100 transgene payloads was driven by either the endogenous promoter (in-frame insertion) or by an exogenous promoter, depending on the application. The scFv domain (3) was variable based on the therapeutic target. scFv sequences used are represented by SEQ ID NO: 9-12 with their corresponding tumor-associated target antigen and represented in FIG. 12B. The costimulatory/signaling domain (denoted (4) in FIG. 12A) can be modified to tune the strength of CAR signaling in response to target recognition. Sequences for costimulatory domains used in MGCT-100 CAR include SEQ ID NO: 87-101, and ITAM-containing domains included SEQ ID NO: 102-111. In some iterations, the costimulatory domain and/or the CD3 domain was removed completely to generate a non-signaling CAR engager that functions to increase interactions of MGCT-100 with tumor targets. Some iterations of MGCT-100 utilized a co-expressed reporter domain for detection of engineered cells by flow cytometry' or immunofluorescence. This reporter (SEQ ID NO: 21-24) was expressed as a separate polypeptide by way of a 2A self-cleaving peptide (SEQ ID NO: 19 or 20). but driven from the same promoter as the CAR, and served as a surrogate marker of CAR expression, or means of purification of CAR+ populations. In some iterations, the reporter was fused directly to the costimulatoiy domain to generate a non-signaling CAR that can be assessed in terms of reporter clustering in response to antigen binding (SEQ ID NO: 112-115).

Example 12 - Modulation of transgene expression in y6T cells through exogenous promoter selection (Prophetic Example)

[0288] For exogenous promoters (denoted (2) in FIG. 12A), constructs utilize the MSCV promoter (SEQ ID NO: 1). EF 1 a full-length or EF la core promoter (SEQ ID NO: 2 and 3), CMV promoter (SEQ ID NO: 4), hPGK promoter (SEQ ID NO: 5), or a combination thereof.

Example 13 - Redirecting specificity of engineered y6T cells using CARs

[0289] The binding moiety used for redirecting specificity of y6T cells consisted of targetspecific VH/VL antibody domains joined by a short linker (SEQ ID NO: 13, 14, or 15), and was variable based on the therapeutic target. EOC tumor antigens targeted using the MGCT-100 CAR constructs include Mucin-16 ecto-domain (MUC16), Folate receptor alpha (FOLR1), Claudin-6 (CLDN6), SLC34A2, or TAG72. Upon binding and recognition of the target antigen by its corresponding binding moiety, the costimulatory domain and ITAM-containing domains generated an activating signal resulting in cytotoxic activity against the target (FIG. 13).

[0290] Some iterations of MGCT-100 utilized a co-expressed reporter domain for detection of engineered cells by flow cytometry or immunofluorescence. This reporter was expressed as a separate polypeptide due to the P2A self-cleaving peptide, but driven from the same promoter as the CAR, and served as a surrogate marker of CAR expression. In some iterations, the reporter was fused directly to the costimulatory domain to generate a non-signaling CAR that can be assessed in terms of reporter clustering in response to antigen binding (SEQ ID NO: 112-115).

Example 14 - Tunable signaling activity of CAR constructs with variable costimulatory and signaling domains (Prophetic Example)

[0291] The costimulatory/signaling domain (denoted (4) in FIG. 12A) can be modified to tune the strength of CAR signaling in response to target recognition. Sequences for costimulatory domains used in MGCT-100 CAR constructs are derived from endogenous TCR-signahng domains such as CD28 (SEQ ID NO: 89), 41BB (SEQ ID NO: 88), or 2B4 (SEQ ID NO: 93). The standard signaling domain used in MGCT-100 constructs utilizes the intracellular domain of CD3 (CD247; SEQ ID NO: 105). which contains three immunotyrosine activation motifs (IT AMs). In some constructs, one or more 1TAM domains may be replaced by other ITAM- contaming sequences such as CD3a (SEQ ID NO: 103), CD3y (SEQ ID NO: 104), CD35 (SEQ ID NO: 102), or DAP12 (SEQ ID NO: 108). The optimal combination of costimulatory domain(s) and ITAM-containing signaling domains is dependent on a number of factors including the binding strength of the targeting moiety, target antigen density, and y5T cell or another immune cell subtype.

Example 15 - Engineering y6T cells with non-signaling CAR “engager” (Prophetic Example)

[0292] In some MGCT-100 CAR constructs, the costimulatory domain and/or the CD3 domain is removed completely to generate a non-signaling CAR that functions to increase interactions of MGCT-100 endogenous TCR with tumor targets without signal transduction through the CAR.

Example 16 - Engineering of y6 T cells using transgene constructs

[0293] Delivery of KI cassette via AA V6

[0294] Donor-derived y5T cells were isolated and expanded as described in Example 1 and engineered using EP or LNP reagent delivery as described in Example 7. Following delivery of editing reagents, engineered cells were incubated in AAV-containing culture media at a concentration of le⁵ to 5e⁵ vg per cell for delivery of relevant transgene payloads. AAV was produced using constructs previously described in Example 11.

[0295] Delivery of KI cassette via msDNA

[0296] Donor-derived y5 T cells were isolated and expanded as described in Example 1. For msDNA delivery of relevant transgene payloads, previously cryopreserved msDNA was thawed and combined with MG enzyme mRNA or protein with site-specific sgRNA and delivered using the EP protocol previously described in Example 5 (FIG. 21). Alternatively, msDNA can be encapsulated in LNP and co-delivered with LNP containing the MG enzyme mRNA with sitespecific sgRNA, according to the described methods in Example 7.

[0297] Assessment of cassette integration and expression

[0298] Evaluation of knock-in gene cassette integration and expression w as performed at 3 days, 6 days, 7 days, 8 days, 9 days and/or 10 days post-engineering. CAR integration at the target GOI locus was determined using digital droplet PCR (ddPCR; FIG. 14). Following standard ddPCR protocols, primers and probes were designed to amplify from within the transgene cassette(s) into the genomic DNA, outside of the homology regions. Amplicons were only detected in genomic DNA of successfully CAR-engineered cells. An additional set of primers and probes can be used to quantify the levels of an amplicon internal to the transgene cassette. This allows for an estimate of integrated copies relative to ectopic copies present in the AAV genome and tracking of residual AAV in the engineered cells. Data w as collected and analyzed to determine transgene copy number. [0299] Surface staining with fluorescently labeled monoclonal antibodies was used to characterize standard phenotypic markers y6 T cells, as well as transgene reporter expression levels, and CAR expression levels by way of incubating cells with soluble protein target that was conjugated to biotin or fluorescent molecule. Cells were washed with FACs staining buffer (PBS + 0.5% BSA). Appropriate volumes of monoclonal antibodies and/or tagged soluble target proteins were diluted in FACs staining buffer and cells were stained in a total volume of 50 pL for 30 minutes at 4°C and then washed twice with FACs buffer. Antibodies and labeling reagents included those generally described in Example 1 and 7, as well as the following staining reagents and antibody conjugates (or equivalents): anti-Thyl. l, anti-CD271, FOLRl-biotin, BCMA- biotin, CLDN6-6x HIS (SEQ ID NO: 177). Viable cells were identified, and dead cells excluded from analysis by staining. Gates were set to determine positive and negative cell populations for reporter proteins (BFP, GFP) and antibody targets (FIGs. 15, 17, and 18). Data was acquired and analyzed.

Example 17 - Engineering of y3T cells with multiple transgene payloads

[0300] Co-delivery of transgene payloads, such as simultaneous delivery of two CAR constructs or simultaneous deliver}⁷ of a CAR construct with a cytokine/chemokine pay load were achieved by dual transduction with two separate AAV vectors that integrate at separate genetic loci (FIGs. 19A-19D and 20A-20B). Alternatively, a bicistronic or multicistronic architecture (FIG. 16) allows for expression of multiple components from a single construct. Depending on final construct size, the construct can be delivered via AAV or by msDNA or nanoplasmid according to the processes described in previous examples. y5T cells isolated and expanded as described in Example 1 were engineered using MG enzyme(s) with sgRNA specific to the target sites for DSB formation, as described in Example 7. Following delivery of engineering reagents by EP or LNP, the y8T cells were incubated in culture media containing the two relevant AAV supernatants.

Example 18 - Assessment of functional impact of transgene payload on engineered y6T cells [0301] CAR-mediated redirected specificity and cytotoxicity were evaluated using co-culture assays with TAA-overexpressing cell lines or TAA+ and TAA- EOC cell lines, as described in Example 2 (2D assays) and Example 4 (3D assays). Cytotoxicity⁷ assays were performed with multiple orthogonal readouts to evaluate y5 T Cell functionality via liquid and solid tumor cytotoxicity and effector cytokine secretion. Example 19 - Assessment of engineered Cytosine Base Editors to remove toxicity while maintaining high protein knockdown efficiency

[0302] Cytosine base editor (CBE) systems were modified using mutagenesis (such as point mutations) of cytosine deaminases as a strategy to increase desired activity while minimizing toxicity. With this strategy, two CBE systems were identified to have high editing efficiency and a promising cytotoxicity profile, namely CBE 139-52vl4 (SEQ ID NO: 175) and CBE 152-6vl3 (SEQ ID NO: 176). To this end, the two CBEs were tested for their protein knock-down (KO) efficiency profile and their genotoxicity in engineering T cell subsets to develop therapeutic applications.

[0303] Engineering cells using electroporation (EP) reagent delivery T cells isolated from donor PBMCs were activated and expanded in culture as previously described in Example 1. 4 days post-isolation, activated y8 T cells were collected, counted, and washed multiple times using PBS. Cell pellets were suspended at 1 x 10⁷ cells per mL in supplemented P3 nucleofection buffer. Cells were combined with MG CBE mRNA with one or more site-specific sgRNAs. Cell and mRNA mixtures were distributed into 96-well nucleofection plates (20 pl per well) and/or nucleocuvettes (100 pl per well), according to methods known in the art (an exemplary workflow is depicted in FIG. 6). The spacer sequences used for each target GOI (Gene Of Interest) are represented by SEQ ID NOs: 146-172.

[0305] Cell count and viability measurements w ere collected on the day of electroporation, and compared to measurements 72-hours post-electroporation, using a high-throughput cell counter. Briefly, cells were suspended in culture vessels prior to collecting a 30-50 pl aliquot and combined at a 1: 1 ratio with acridine orange and propidium iodide (AO/PI) solution for a readout of live and dead cells. In some instances, cell viability w as determined by staining the cells with Fixable Viability Dye APC-eFluor780 using standard flow cytometry workflows and gating on “Live” events (Dye-negative).

[0306] On-target GOI knock-out efficiency as determined by NGS sample preparation using standard techniques. Briefly, genomic DNA was extracted from engineered samples 72 hours post-electroporation using a DNA extraction solution, and then used as template for PCR amplification of relevant GOI amplicon (forward and reverse PCR primer sequences: SEQ ID NOs: 173-174). Addition of NGS adapter sequence to 5’ and 3’ ends of PCR product was conducted during the first PCR amplification step. A subsequent PCR amplification using indexing was performed. Sequencing data was then analyzed to determine indel frequencies, substitution rate, and frequency of base edits at each nucleotide within the targeted sequence. On- target knock-out efficiency can also be evaluated using antibody staining for target proteins and flow cytometry readout using methods known in the art, as described in Example 1. [0307] To evaluate the CBE Toxicity profile in primary y5 T cells, donor y5 T cells (n=3 donors) were electroporated with 3-6/8 CBE mRNA, isolated, and purified. These samples showed limited toxicity in terms of overall cell count and vi abil i ty measurements 72 hours postelectroporation, relative to control CBE and MG3-6 nuclease (FIG. 24). To compare with the toxicity/cell viability profile(s), single nuclease mRNA was also delivered to cells with sgRNA for MG3-6 TRAC site, with minimal impact on readouts relative to mRNA only conditions. [0308] From an arrayed screen of candidate sgRNAs targeting either PDCD1 or HAVCR2 genes, PDCDlgl and HAVCR2g2 were identified based on flow cytometry readouts for target-protein knock-out (KO) efficiency (FIG. 25C). Using purified y5 T cells from 2 donors, delivery of CBE-139 mRNA with PDCDlgl or HAVCR2g2 as seen in FIG. 25A results in an average 89% and 88% protein KO, respectively. Delivery of CBE-152 mRNA with PDCDlgl or HAVCR2g2 results in an average 41% and 23% protein KO, respectively. Delivery of CBE-139 mRNA or CBE-152 did not have a significant impact on cell viability, compared to controls (FIG. 25B).

Example 20 - Assessment of engineered Cytosine Base Editors to remove toxicity while maintaining high protein knockdown efficiency - Multiplex editing

[0309] Multiplex editing of multiple genomic sites in y5 T cells was conducted through simultaneous delivery' of engineering reagents via Electroporation (EP). As described herein, y5 T cells isolated from donor PBMCs were activated and expanded in culture. For multiplexed editing, engineering reagents (guide(s) and nuclease(s)) targeting multiple genomic sites were delivered on Day 4 post-isolation and activation via EP in the same reaction.

[0310] Multiplexed editing of HAVCR2 (TIM3), and PDCD1 using CBE-139 mRNA (SEQ ID NO: 175) resulted in highly-effective knock-out of both proteins with minimal impact on cell viability 72-hours post EP. This result was reproducible across the donors tested (n=4). Overall cell counts demonstrated some impact of the CBE+sgRNA delivery, though the result was comparable to that of MG3-6+sgRNA (FIG. 26A). As shown in FIG. 26C and FIG. 26D, single base-edit samples resulted in 80-90% protein KO for each target when delivering PDCDlgl sgRNA (SEQ ID NO: 157) or HAVCR2g2 sgRNA (SEQ ID NO: 147). in combination with CBE-139 mRNA. Protein KO levels were comparable to MG3-6 nuclease controls. Dual baseedited samples achieved up to 90% combined editing at both sites, demonstrating the feasibility of multiplexing multiple base edits w ith a single electroporation.

[0311] References

[0312] Silva-Santos, B., Serre, K. & Norell, H. y5 T cells in cancer. Nat Rev Immunol 15, 683- 691 (2015). [0313] Ribeiro, Sergio T., Julie C. Ribot, and Bruno Silva-Santos. Five Layers of Receptor Signaling in yoT-Cell Differentiation and Activation. Frontiers in Immunology (2015).

[0314] Sanchez Martinez D, Tirado N, Mensurado S, et al. Generation and proof-of-concept for allogeneic CD 123 CAR-Delta One T (DOT) cells in acute myeloid leukemia. Journal for ImmunoTherapy of Cancer 2022;10:e005400. doi: 10.1136/jitc-2022-005400.

[0315] Kondo, M.. Izumi, T., Fujieda. N., Kondo. A., Morishita, T., Matsushita, H., Kakimi. K. Expansion of Human Peripheral Blood y5 T Cells using Zoledronate. J. Vis. Exp. (55), e3182, DOI : 10.3791/3182 (2011).

[0316] Samuele Cancellieri, Matthew C Canver, Nicola Bombieri, Rosalba Giugno, Luca Pinello, CRISPRitz: rapid, high-throughput and variant-aware in silico off-target site identification for CRISPR genome editing, Bioinformatics, Volume 36. Issue 7, 1 April 2020, Pages 2001-2008.

[0317] Sievers, Nico M et al. “CARs: Beyond T Cells and T Cell-Derived Signaling Domains.” International Journal of Molecular Sciences 21 (2020): n. pag.

[0318] Zhao M, Kim P, Mitra R, Zhao J, Zhao Z. TSGene 2.0: an updated literature-based knowledgebase for tumor suppressor genes. Nucleic Acids Res. 2016 Jan 4;44(Dl):D1023-31. [0319] Andrew P. May, Peter Cameron, Alexander H. Settle et al. SITE-Seq: A Genome-wide Method to Measure Cas9 Cleavage, 02 May 2017, PROTOCOL (Version 1) available at Protocol Exchange [https :// doi. org/ 10.1038/protex.2017.043] .

[0320] D.Dimitrov,D. and Gu.Q. (2020) BingleSeq: a user-friendly R package for bulk and single-cell RNA-Seq data analysis. PeerJ, 8, el0469 [https://peeq.com/articles/10469/]

[0321] Wolf, F., Angerer, P. & Theis, F. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol 19, 15 (2018). https://doi.org/10.1186/sl3059-017-1382-0

[0322] While preferred embodiments of the present disclosure have been show n and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the disclosure be limited by the specific examples provided within the specification. While the disclosure has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. Furthermore, it shall be understood that all aspects of the disclosure are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is therefore contemplated that the disclosure shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby

SEQUENCE LISTING

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for engineering a y5 T cell, comprising contacting the y5 T cell using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M- locus.

2. A method for engineering a y5 T cell, comprising contacting the y5 T cell using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL-17, TRAC, and B2M locus.

3. The method of claim 1 or 2, wherein the engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 116-126 and 146- 172.

4. The method of any one of claims 1-3, wherein the engineered system further comprises one or more donor nucleic acids.

5. The method of claim 4, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR).

6. The method of claim 4 or 5, wherein the donor nucleic acid encodes a first cytokine.

7. The method of claim 5 or 6, w herein the donor nucleic acid further encodes a second CAR.

8. The method of claim 6 or 7, wherein the donor nucleic acid further encodes a second cytokine.

9. The method of any one of claims 5-8, wherein the first CAR and the second CAR comprises an extracellular antigen binding domain, and wherein the extracellular antigen binding domain binds to a tumor-associated antigen selected from the group consisting of MUC16.

FOLR1, BCMA. CLDN6, SLC34A2, and TAG72.

10. The method of claim 9, wherein the extracellular antigen binding domain of the first CAR binds to MUC16 or FOLR1.

1 1. The method of claim 10, wherein the extracellular antigen binding domain of the second CAR binds to MUC16 or FOLR1.

12. The method of any one of claims 9-11, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv).

13. The method of claim 12, wherein the scFv is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-12.

14. The method of claim 13, wherein the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 10.

15. The method of claim 13. wherein the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 11.

16. The method of any one of claims 5-15, wherein the first or the second CAR comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 28-31.

17. The method of any one of claims 5-16, wherein the first cytokine is IL-12 or IL-15.

18. The method of any one of claims 6-17, wherein the second cytokine is IL-12 or IL-15.

19. The method of any one of claims 4-18, wherein the donor nucleic acid comprises a first homology' arm and a second homology arm; and wherein the first homology arm comprises a sequence located on the 5‘ side of the target nucleic acid sequence and the second homology arm comprises a sequence located on the 3^? side of the target nucleic acid sequence.

20. The method of claim 19, wherein the first homology arm comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 46, 48, 50, and 52.

21. The method of any one of claims 19-20, wherein the second homology arm comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 47, 49, 51, and 53.

22. A pharmaceutical composition comprising the engineered y5 T cell produced by the method of any one of claims 1-21.

23. A gene editing system comprising: a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR).

24. A gene editing system comprising: a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL- 17, TRAC, and B2M locus; and c) one or more donor nucleic acids, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR).

25. The gene editing system of any one of claims 23-24, wherein the donor nucleic acid encodes a first cytokine.

26. The gene editing system of any one of claims 23-25, wherein the donor nucleic acid further encodes a second CAR.

27. The gene editing system of claim 25 or 26, wherein the donor nucleic acid further encodes a second cytokine.

28. The gene editing system of any one of claims 23-27, wherein the first CAR and the second CAR comprises an extracellular antigen binding domain, and wherein the extracellular antigen binding domain binds to a tumor-associated antigen selected from the group consisting of MUC16, FOLR1, BCMA, CLDN6, SLC34A2, and TAG72.

29. The gene editing system of claim 28, wherein the extracellular antigen binding domain of the first CAR binds to MUC16 or FOLR1.

30. The gene editing system of claim 29, wherein the extracellular antigen binding domain of the second CAR binds to MUC16 or FOLR1.

31. The gene editing system of any one of claims 28-30, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv).

32. The gene editing system of claim 31, wherein the scFv is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-12.

33. The gene editing system of claim 31, wherein the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 10.

34. The gene editing system of claim 31, wherein the scFv is encoded by a sequence having at least 80% sequence identity to SEQ ID NOs: 11.

35. The gene editing system of any one of claims 23-34, wherein the first or the second CAR comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 28-31.

36. The gene editing system of any one of claims 23-35, wherein the first cytokine is IL-12 or

IL- 15.

37. The gene editing system of any one of claims 25-36, wherein the second cytokine is IL- 12 or IL- 15.

38. A y5 T cell comprising the gene editing system of any one of claims 25-37.

39. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered y5 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus.

40. A method of killing a cancer cell comprising contacting the cancer cell with an engineered y5 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more endonucleases encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and b) one or more engineered guide polynucleotides configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL- 17, TRAC, and B2M locus.

41. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered y5 T cell, wherein the engineered y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3. IL-17, TRAC, and B2M locus.

42. A method of killing a cancer cell comprising contacting the cancer cell with an engineered y6 T cell, wherein the y5 T cell has been modified in one or more loci using an engineered system comprising: a) one or more base editors encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176, wherein the base editor comprises an endonuclease domain that is deficient in nuclease activity; and b) one or more engineered guide polynucleotides configured to form a complex with the base editor and hybridize to a target nucleic acid sequence within a locus selected from the group consisting of a PD-1, TIM3, IL- 17, TRAC, and B2M locus.

43. A method of modifying a TRAC and a PD-1 locus in a y5 T cell comprising contacting to the y§ T cell: a) a first engineered system comprising:

(i) a first endonuclease encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and

(ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus: and b) a second engineered system comprising:

(i) a second endonuclease encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127, 128, and 179; and

(ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the PD-1 locus.

44. A method of modifying a TRAC and an IL- 17 locus in a y5 T cell comprising contacting to the y5 T cell: a) a first engineered system comprising:

(i) a first endonuclease encoded by a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 127, 128, and 179; and

(ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL- 17 locus; and b) a second engineered system comprising:

(i) a second endonuclease encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127. 128, and 179; and

(ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the TRAC or the IL-17 locus.

45. A method of modifying a PD-1 and an IL-17 locus in a y5 T cell comprising contacting to the y6 T cell: a) a first engineered system comprising: (i) a first endonuclease encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 127. 128, and 179; and

(ii) a first engineered guide polynucleotide configured to form a complex wi th the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL-17 locus; and b) a second engineered system comprising:

(ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the IL- 17 locus.

46. A method of modifying a PD-1 and a TIM3 locus in a y5 T cell comprising contacting to the y5 T cell: a) a first engineered system comprising:

(ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising:

(i) a second endonuclease encoded by a sequence having at least 80% sequence identify to any one of SEQ ID NOs: 127, 128, and 179; and

(ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus.

47. A method of modifying a PD-1 and a TIM3 locus in a y5 T cell comprising contacting to the y§ T cell: a) a first engineered system comprising:

(i) a first base editor encoded by a sequence having at least 80% sequence identity to SEQ ID NO: 175 or SEQ ID NO: 176, wherein the first base editor comprises a first endonuclease domain that is deficient in nuclease activity; and (ii) a first engineered guide polynucleotide configured to form a complex with the first endonuclease domain and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus; and b) a second engineered system comprising:

(i) a second base editor encoded by a sequence having at least 80% sequence identity to any one SEQ ID NOs: 175-176. wherein the second base editor comprises a second endonuclease domain that is deficient in nuclease activity; and

(ii) a second engineered guide polynucleotide configured to form a complex with the second endonuclease domain and hybridize to a target nucleic acid sequence within the PD-1 or the TIM3 locus.

48. The method of claim 43, wherein the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 117-119, and 123.

49. The method of claim 44, wherein the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 117-119, and 124-126.

50. The method of claim 45, wherein the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 123-126.

51. The method of claim 46, wherein the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 120-123.

52. The method of claim 47, wherein the first engineered guide polynucleotide and the second engineered guide polynucleotide comprises a sequence having at least 80% sequence identity' to any one of SEQ ID NOs: 146-172.

53. The method of any one of claims 43-52, further comprising introducing to the y5 T cell one or more donor nucleic acids.

54. The method of claim 53, wherein the donor nucleic acid encodes a first Chimeric Antigen Receptor (CAR).

55. The method of claim 53 or 54, wherein the donor nucleic acid encodes a first cytokine.

56. The method of claim 54 or 55, wherein the donor nucleic acid further encodes a second CAR.

57. The method of any one of claims 54-56, wherein the donor nucleic acid further encodes a second cytokine.

58. The method of claim 54 or 55, wherein the first CAR and the second CAR comprises an extracellular antigen binding domain, and wherein the extracellular antigen binding domain binds to a tumor-associated antigen selected from the group consisting of MUC16, FOLR1, BCMA, CLDN6, SLC34A2, and TAG72.

59. The method of claim 58, wherein the extracellular antigen binding domain of the first CAR binds to MUC16 or FOLR1.

60. The method of claim 59, wherein the extracellular antigen binding domain of the second CAR binds to MUC16 or FOLR1.

61. The method of any one of claims 58-60, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv).

62. The method of claim 61, wherein the scFv is encoded by a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 9-12.

63. The method of any one of claims 54-62, wherein the first CAR or the second CAR comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 28-31.